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Modrich, Paul L.

Overview:

Paul Modrich, James B. Duke Professor of Medicine, was awarded the 2015 Nobel Prize for Chemistry along with Tomas Lindahl of the Francis Crick Institute and Clare Hall Laboratory in the UK, and Aziz Sancar of University of North Carolina, Chapel Hill, for mechanistic studies of DNA repair.

Mismatch repair is a mutation avoidance system that stabilizes the genome by correcting DNA biosynthetic errors, by blocking recombination between diverged DNA sequences, and in the case of human cells, by targeting for death cells that have suffered certain types of DNA chemical damage. We have reconstituted E. coli mismatch repair in a pure system comprised of ten activities, including the MutH, MutL, MutS, and MutU proteins. Current work on the bacterial pathway addresses the mechanism of this complex reaction and reagent applications of the repair proteins for physical manipulation of DNA sequences based on genetic differences. We have shown that mismatch repair in human cells occurs by a mechanism similar to that of the bacterial reaction and depends on heterodimeric complexes of homologs of E. coli MutS and MutL proteins. Genetic defects in human MutS and MutL homologs are the cause of hereditary nonpolyposis colon cancer, as well as a signficant fraction of sporadic tumors that occur in a variety of tissues. Genetic inactivation of the human MutS and MutL proteins also confers resistance to some chemotherapeutic agents that kill by inducing an apoptotic respose. We have shown that the cytotoxic DNA lesions produced by these drugs are recognized by the human mismatch repair system, and have concluded that this event is the initial step in the sequence of events that leads to cell death. The molecular nature of the human repair system and its role in the cellular response to DNA damage are currently under study in the lab.

Positions:

James B. Duke Professor of Medicine

Biochemistry
School of Medicine

Professor of Biochemistry

Biochemistry
School of Medicine

Professor of Chemistry

Chemistry
Trinity College of Arts & Sciences

Member of the Duke Cancer Institute

Duke Cancer Institute
School of Medicine

Education:

B.S. 1968

B.S. — Massachusetts Institute of Technology

Ph.D. 1973

Ph.D. — Stanford University

News:

Grants:

Occurrence and Functional Roles of High Energy Base Pairs in DNA

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Collaborator
Start Date
September 15, 2015
End Date
July 31, 2019

Enzymology Of Eukaryotic Mismatch Repair

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
January 01, 1991
End Date
February 28, 2019

Forces and Long-Distance Coupling along DNA in the Mismatch Repair (MMR) Pathway

Administered By
Mechanical Engineering and Materials Science
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
September 16, 2014
End Date
August 31, 2016

Structural Cell Biology of DNA Repair Machines Sub: Mismatch Repair Interactions

Administered By
Biochemistry
AwardedBy
Ernest Orlando Lawrence Berkeley National Laboratory
Role
Investigator
Start Date
December 13, 2001
End Date
August 31, 2016

Structural biology of human DNA mismatch repair machinery

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Collaborator
Start Date
September 30, 2009
End Date
August 31, 2012

Temodar Resistance in CNS Tumors

Administered By
Neurosurgery, Neuro-Oncology
AwardedBy
National Institutes of Health
Role
Collaborator
Start Date
June 22, 1992
End Date
April 30, 2010

Molecular Mechanisms of DNA-Protein Interaction

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
August 01, 1977
End Date
July 31, 2009

DNA repair in normal and hypermutable cancer cell lines

Administered By
Medicine, Medical Oncology
AwardedBy
National Institutes of Health
Role
Sponsor
Start Date
September 17, 1996
End Date
August 30, 2002

Molecular Mechanisms Of Dna-Protein Interaction

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
August 01, 1997
End Date
July 31, 1999

Molecular Mechanism Of Dna Protein Interaction

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
August 01, 1996
End Date
July 31, 1999

Enzymology Of Eukaryotic Dna Mismatch Repair

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
January 01, 1991
End Date
December 31, 1998

Comprehensive Cancer Center Core Support Grant

Administered By
Medicine, Medical Oncology
AwardedBy
National Institutes of Health
Role
Co-Principal Investigator
Start Date
September 01, 1976
End Date
December 31, 1998

Comprehensive Cancer Center Core Support Grant

Administered By
Medicine, Medical Oncology
AwardedBy
National Institutes of Health
Role
Co-Principal Investigator
Start Date
September 01, 1976
End Date
December 31, 1998

Dna Mismatch Repair Proteins In Human Cells

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
March 01, 1997
End Date
October 31, 1998

Molecular Mechanism Of Dna-Protein Interaction

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
August 01, 1992
End Date
July 31, 1996

Molecular Mechanisms Of Dna Protein Interaction

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
August 01, 1992
End Date
July 31, 1996

Genetics

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 1991
End Date
June 30, 1994

Genetics

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 1990
End Date
June 30, 1994

Molecular Mechanisms Of Dna - Protein Interaction

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
August 01, 1987
End Date
July 01, 1991
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Awards:

Nobel Prize in Chemistry for 2015. Royal Swedish Academy of Sciences.

Type
International
Awarded By
Royal Swedish Academy of Sciences
Date
October 07, 2015

Fellows. American Academy of Arts and Sciences.

Type
National
Awarded By
American Academy of Arts and Sciences
Date
January 01, 2004

Member. Institute of Medicine of The National Academies.

Type
National
Awarded By
Institute of Medicine of The National Academies
Date
January 01, 2003

Investigator/Alumni Investigator. Howard Hughes Medical Institute.

Type
National
Awarded By
Howard Hughes Medical Institute
Date
January 01, 1994

Members/ Foreign Associates. National Academy of Science.

Type
National
Awarded By
National Academy of Science
Date
January 01, 1993

Publications:

Mechanisms in E. coli and Human Mismatch Repair (Nobel Lecture)

Authors
Modrich, P
MLA Citation
Modrich, P. "Mechanisms in E. coli and Human Mismatch Repair (Nobel Lecture)." Angewandte Chemie International Edition 55.30 (July 18, 2016): 8490-8501.
Source
crossref
Published In
Angewandte Chemie International Edition
Volume
55
Issue
30
Publish Date
2016
Start Page
8490
End Page
8501
DOI
10.1002/anie.201601412

The C-terminal 20 Amino Acids of Drosophila Topoisomerase 2 Are Required for Binding to a BRCA1 C Terminus (BRCT) Domain-containing Protein, Mus101, and Fidelity of DNA Segregation

Authors
Chen, Y-TS; Wu, J; Modrich, P; Hsieh, T-S
MLA Citation
Chen, Y-TS, Wu, J, Modrich, P, and Hsieh, T-S. "The C-terminal 20 Amino Acids of Drosophila Topoisomerase 2 Are Required for Binding to a BRCA1 C Terminus (BRCT) Domain-containing Protein, Mus101, and Fidelity of DNA Segregation." Journal of Biological Chemistry 291.25 (June 17, 2016): 13216-13228.
Source
crossref
Published In
The Journal of biological chemistry
Volume
291
Issue
25
Publish Date
2016
Start Page
13216
End Page
13228
DOI
10.1074/jbc.M116.721357

MutL traps MutS at a DNA mismatch.

DNA mismatch repair (MMR) identifies and corrects errors made during replication. In all organisms except those expressing MutH, interactions between a DNA mismatch, MutS, MutL, and the replication processivity factor (β-clamp or PCNA) activate the latent MutL endonuclease to nick the error-containing daughter strand. This nick provides an entry point for downstream repair proteins. Despite the well-established significance of strand-specific nicking in MMR, the mechanism(s) by which MutS and MutL assemble on mismatch DNA to allow the subsequent activation of MutL's endonuclease activity by β-clamp/PCNA remains elusive. In both prokaryotes and eukaryotes, MutS homologs undergo conformational changes to a mobile clamp state that can move away from the mismatch. However, the function of this MutS mobile clamp is unknown. Furthermore, whether the interaction with MutL leads to a mobile MutS-MutL complex or a mismatch-localized complex is hotly debated. We used single molecule FRET to determine that Thermus aquaticus MutL traps MutS at a DNA mismatch after recognition but before its conversion to a sliding clamp. Rather than a clamp, a conformationally dynamic protein assembly typically containing more MutL than MutS is formed at the mismatch. This complex provides a local marker where interaction with β-clamp/PCNA could distinguish parent/daughter strand identity. Our finding that MutL fundamentally changes MutS actions following mismatch detection reframes current thinking on MMR signaling processes critical for genomic stability.

Authors
Qiu, R; Sakato, M; Sacho, EJ; Wilkins, H; Zhang, X; Modrich, P; Hingorani, MM; Erie, DA; Weninger, KR
MLA Citation
Qiu, R, Sakato, M, Sacho, EJ, Wilkins, H, Zhang, X, Modrich, P, Hingorani, MM, Erie, DA, and Weninger, KR. "MutL traps MutS at a DNA mismatch." Proceedings of the National Academy of Sciences of the United States of America 112.35 (September 2015): 10914-10919.
PMID
26283381
Source
epmc
Published In
Proceedings of the National Academy of Sciences of USA
Volume
112
Issue
35
Publish Date
2015
Start Page
10914
End Page
10919
DOI
10.1073/pnas.1505655112

Hydrolytic function of Exo1 in mammalian mismatch repair.

Genetic and biochemical studies have previously implicated exonuclease 1 (Exo1) in yeast and mammalian mismatch repair, with results suggesting that function of the protein in the reaction depends on both its hydrolytic activity and its ability to interact with other components of the repair system. However, recent analysis of an Exo1-E109K knockin mouse has concluded that Exo1 function in mammalian mismatch repair is restricted to a structural role, a conclusion based on a prior report that N-terminal His-tagged Exo1-E109K is hydrolytically defective. Because Glu-109 is distant from the nuclease hydrolytic center, we have compared the activity of untagged full-length Exo1-E109K with that of wild type Exo1 and the hydrolytically defective active site mutant Exo1-D173A. We show that the activity of Exo1-E109K is comparable to that of wild type enzyme in a conventional exonuclease assay and that in contrast to a D173A active site mutant, Exo1-E109K is fully functional in mismatch-provoked excision and repair. We conclude that the catalytic function of Exo1 is required for its participation in mismatch repair. We also consider the other phenotypes of the Exo1-E109K mouse in the context of Exo1 hydrolytic function.

Authors
Shao, H; Baitinger, C; Soderblom, EJ; Burdett, V; Modrich, P
MLA Citation
Shao, H, Baitinger, C, Soderblom, EJ, Burdett, V, and Modrich, P. "Hydrolytic function of Exo1 in mammalian mismatch repair." Nucleic acids research 42.11 (June 2014): 7104-7112.
PMID
24829455
Source
epmc
Published In
Nucleic Acids Research
Volume
42
Issue
11
Publish Date
2014
Start Page
7104
End Page
7112
DOI
10.1093/nar/gku420

Coupling of human DNA excision repair and the DNA damage checkpoint in a defined in vitro system.

DNA repair and DNA damage checkpoints work in concert to help maintain genomic integrity. In vivo data suggest that these two global responses to DNA damage are coupled. It has been proposed that the canonical 30 nucleotide single-stranded DNA gap generated by nucleotide excision repair is the signal that activates the ATR-mediated DNA damage checkpoint response and that the signal is enhanced by gap enlargement by EXO1 (exonuclease 1) 5' to 3' exonuclease activity. Here we have used purified core nucleotide excision repair factors (RPA, XPA, XPC, TFIIH, XPG, and XPF-ERCC1), core DNA damage checkpoint proteins (ATR-ATRIP, TopBP1, RPA), and DNA damaged by a UV-mimetic agent to analyze the basic steps of DNA damage checkpoint response in a biochemically defined system. We find that checkpoint signaling as measured by phosphorylation of target proteins by the ATR kinase requires enlargement of the excision gap generated by the excision repair system by the 5' to 3' exonuclease activity of EXO1. We conclude that, in addition to damaged DNA, RPA, XPA, XPC, TFIIH, XPG, XPF-ERCC1, ATR-ATRIP, TopBP1, and EXO1 constitute the minimum essential set of factors for ATR-mediated DNA damage checkpoint response.

Authors
Lindsey-Boltz, LA; Kemp, MG; Reardon, JT; DeRocco, V; Iyer, RR; Modrich, P; Sancar, A
MLA Citation
Lindsey-Boltz, LA, Kemp, MG, Reardon, JT, DeRocco, V, Iyer, RR, Modrich, P, and Sancar, A. "Coupling of human DNA excision repair and the DNA damage checkpoint in a defined in vitro system." The Journal of biological chemistry 289.8 (February 2014): 5074-5082.
PMID
24403078
Source
epmc
Published In
The Journal of biological chemistry
Volume
289
Issue
8
Publish Date
2014
Start Page
5074
End Page
5082
DOI
10.1074/jbc.m113.542787

Coupling of human DNA excision repair and the DNA damage checkpoint in a defined in vitro system

Background: Nucleotide excision repair and the ATR-mediated DNA damage checkpoint responses are genetically coupled. Results: We have analyzed the basic steps of ATR activation in a biochemically defined system. Conclusion: ATR signaling requires enlargement of the DNA excision gap by EXO1. Significance: The six excision repair factors, ATR-ATRIP, TopBP1, and EXO1 constitute the minimum essential set of proteins for ATR-activation upon UV-induced DNA damage. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Authors
Lindsey-Boltz, LA; Kemp, MG; Reardon, JT; DeRocco, V; Iyer, RR; Modrich, P; Sancar, A
MLA Citation
Lindsey-Boltz, LA, Kemp, MG, Reardon, JT, DeRocco, V, Iyer, RR, Modrich, P, and Sancar, A. "Coupling of human DNA excision repair and the DNA damage checkpoint in a defined in vitro system." Journal of Biological Chemistry 289.8 (2014): 5074-5082.
Source
scival
Published In
The Journal of biological chemistry
Volume
289
Issue
8
Publish Date
2014
Start Page
5074
End Page
5082
DOI
10.1074/jbc.M113.542787

Christian Raetz: scientist and friend extraordinaire.

Chris Raetz passed away on August 16, 2011, still at the height of his productive years. His seminal contributions to biomedical research were in the genetics, biochemistry, and structural biology of phospholipid and lipid A biosynthesis in Escherichia coli and other gram-negative bacteria. He defined the catalytic properties and structures of many of the enzymes responsible for the "Raetz pathway for lipid A biosynthesis." His deep understanding of chemistry, coupled with knowledge of medicine, biochemistry, genetics, and structural biology, formed the underpinnings for his contributions to the lipid field. He displayed an intense passion for science and a broad interest that came from a strong commitment to curiosity-driven research, a commitment he imparted to his mentees and colleagues. What follows is a testament to both Chris's science and humanity from his friends and colleagues.

Authors
Dowhan, W; Nikaido, H; Stubbe, J; Kozarich, JW; Wickner, WT; Russell, DW; Garrett, TA; Brozek, K; Modrich, P
MLA Citation
Dowhan, W, Nikaido, H, Stubbe, J, Kozarich, JW, Wickner, WT, Russell, DW, Garrett, TA, Brozek, K, and Modrich, P. "Christian Raetz: scientist and friend extraordinaire." Annual review of biochemistry 82 (2013): 1-24.
PMID
23472605
Source
scival
Published In
Annual Review of Biochemistry
Volume
82
Publish Date
2013
Start Page
1
End Page
24
DOI
10.1146/annurev-biochem-012512-091530

Extrahelical (CAG)/(CTG) triplet repeat elements support proliferating cell nuclear antigen loading and MutLa endonuclease activation

MutLa endonuclease can be activated on covalently continuous DNA that contains a MutSa- or MutSβ-recognizable lesion and a helix perturbation that supports proliferating cell nuclear antigen (PCNA) loading by replication factor C, providing a potential mechanism for triggering mismatch repair on nonreplicating DNA. Because mouse models for somatic expansion of disease-associated (CAG)n/(CTG)n triplet repeat sequences have implicated both MutSβ and MutLa and have suggested that expansions can occur in the absence of replication, we have asked whether an extrahelical (CAG)n or (CTG)n element is sufficient to trigger MutLa activation. (CAG)n and (CTG)n extrusions in relaxed closed circular DNA do in fact support MutSβ-, replication factor C-, and PCNA-dependent activation of MutLa endonuclease, which can incise either DNA strand. Extrahelical elements of two or three repeat units are the preferred substrates for MutLa activation, and extrusions of this size also serve as moderately effective sites for loading the PCNA clamp. Relaxed heteroduplex DNA containing a two or three-repeat unit extrusion also triggers MutSβ- and MutLa-endonuclease- dependent mismatch repair in nuclear extracts of human cells. This reaction occurs without obvious strand bias at about 10% the rate of that observed with otherwise identical nicked heteroduplex DNA. These findings provide a mechanism for initiation of triplet repeat processing in nonreplicating DNA that is consistent with several features of the model of Gomes-Pereira et al. [Gomes-PereiraM, FortuneMT, IngramL,McAbney JP,Monckton DG (2004) HumMol Genet 13(16):1815-1825]. They may also have implications for triplet repeat processing at a replication fork.

Authors
Pluciennik, A; Burdett, V; Baitinger, C; Iyer, RR; Shi, K; Modrich, P
MLA Citation
Pluciennik, A, Burdett, V, Baitinger, C, Iyer, RR, Shi, K, and Modrich, P. "Extrahelical (CAG)/(CTG) triplet repeat elements support proliferating cell nuclear antigen loading and MutLa endonuclease activation." Proceedings of the National Academy of Sciences of the United States of America 110.30 (2013): 12277-12282.
PMID
23840062
Source
scival
Published In
Proceedings of the National Academy of Sciences of USA
Volume
110
Issue
30
Publish Date
2013
Start Page
12277
End Page
12282
DOI
10.1073/pnas.1311325110

PARP-1 enhances the mismatch-dependence of 5'-directed excision in human mismatch repair in vitro.

End-directed mismatch-provoked excision has been reconstituted in several purified systems. While 3'-directed excision displays a mismatch dependence similar to that observed in nuclear extracts (≈20-fold), the mismatch dependence of 5'-directed excision is only 3-4-fold, significantly less than that in extracts (8-10-fold). Utilizing a fractionation-based approach, we have isolated a single polypeptide that enhances mismatch dependence of reconstituted 5'-directed excision and have shown it to be identical to poly[ADP-ribose] polymerase 1 (PARP-1). Titration of reconstituted excision reactions or PARP-1-depleted HeLa nuclear extract with purified PARP-1 showed that the protein specifically enhances mismatch dependence of 5'-directed excision. Analysis of a set of PARP-1 mutants revealed that the DNA binding domain and BRCT fold contribute to the regulation of excision specificity. Involvement of the catalytic domain is restricted to its ability to poly(ADP-ribosyl)ate PARP-1 in the presence of NAD(+), likely through interference with DNA binding. Analysis of protein-protein interactions demonstrated that PARP-1 interacts with mismatch repair proteins MutSα, exonuclease 1, replication protein A (RPA), and as previously shown by others, replication factor C (RFC) and proliferating cell nuclear antigen (PCNA) as well. The BRCT fold plays an important role in the interaction of PARP-1 with the former three proteins.

Authors
Liu, Y; Kadyrov, FA; Modrich, P
MLA Citation
Liu, Y, Kadyrov, FA, and Modrich, P. "PARP-1 enhances the mismatch-dependence of 5'-directed excision in human mismatch repair in vitro." DNA Repair (Amst) 10.11 (November 10, 2011): 1145-1153.
PMID
21945626
Source
pubmed
Published In
DNA Repair
Volume
10
Issue
11
Publish Date
2011
Start Page
1145
End Page
1153
DOI
10.1016/j.dnarep.2011.08.012

Purification, crystallization and preliminary X-ray diffraction analysis of the human mismatch repair protein MutSβ.

MutSβ is a eukaryotic mismatch repair protein that preferentially targets extrahelical unpaired nucleotides and shares partial functional redundancy with MutSα (MSH2-MSH6). Although mismatch recognition by MutSα has been shown to involve a conserved Phe-X-Glu motif, little is known about the lesion-binding mechanism of MutSβ. Combined MSH3/MSH6 deficiency triggers a strong predisposition to cancer in mice and defects in msh2 and msh6 account for roughly half of hereditary nonpolyposis colorectal cancer mutations. These three MutS homologs are also believed to play a role in trinucleotide repeat instability, which is a hallmark of many neurodegenerative disorders. The baculovirus overexpression and purification of recombinant human MutSβ and three truncation mutants are presented here. Binding assays with heteroduplex DNA were carried out for biochemical characterization. Crystallization and preliminary X-ray diffraction analysis of the protein bound to a heteroduplex DNA substrate are also reported.

Authors
Tseng, Q; Orans, J; Hast, MA; Iyer, RR; Changela, A; Modrich, PL; Beese, LS
MLA Citation
Tseng, Q, Orans, J, Hast, MA, Iyer, RR, Changela, A, Modrich, PL, and Beese, LS. "Purification, crystallization and preliminary X-ray diffraction analysis of the human mismatch repair protein MutSβ." Acta Crystallogr Sect F Struct Biol Cryst Commun 67.Pt 8 (August 1, 2011): 947-952.
PMID
21821902
Source
pubmed
Published In
Acta Crystallographica Section F
Volume
67
Issue
Pt 8
Publish Date
2011
Start Page
947
End Page
952
DOI
10.1107/S1744309111019300

Structures of human exonuclease 1 DNA complexes suggest a unified mechanism for nuclease family.

Human exonuclease 1 (hExo1) plays important roles in DNA repair and recombination processes that maintain genomic integrity. It is a member of the 5' structure-specific nuclease family of exonucleases and endonucleases that includes FEN-1, XPG, and GEN1. We present structures of hExo1 in complex with a DNA substrate, followed by mutagenesis studies, and propose a common mechanism by which this nuclease family recognizes and processes diverse DNA structures. hExo1 induces a sharp bend in the DNA at nicks or gaps. Frayed 5' ends of nicked duplexes resemble flap junctions, unifying the mechanisms of endo- and exonucleolytic processing. Conformational control of a mobile region in the catalytic site suggests a mechanism for allosteric regulation by binding to protein partners. The relative arrangement of substrate binding sites in these enzymes provides an elegant solution to a complex geometrical puzzle of substrate recognition and processing.

Authors
Orans, J; McSweeney, EA; Iyer, RR; Hast, MA; Hellinga, HW; Modrich, P; Beese, LS
MLA Citation
Orans, J, McSweeney, EA, Iyer, RR, Hast, MA, Hellinga, HW, Modrich, P, and Beese, LS. "Structures of human exonuclease 1 DNA complexes suggest a unified mechanism for nuclease family." Cell 145.2 (April 15, 2011): 212-223.
PMID
21496642
Source
pubmed
Published In
Cell
Volume
145
Issue
2
Publish Date
2011
Start Page
212
End Page
223
DOI
10.1016/j.cell.2011.03.005

BLM-DNA2-RPA-MRN and EXO1-BLM-RPA-MRN constitute two DNA end resection machineries for human DNA break repair

Repair of dsDNA breaks requires processing to produce 3′-terminated ssDNA. We biochemically reconstituted DNA end resection using purified human proteins: Bloom helicase (BLM); DNA2 helicase/nuclease; Exonuclease 1 (EXO1); the complex comprising MRE11, RAD50, and NBS1 (MRN); and Replication protein A (RPA). Resection occurs via two routes. In one, BLM and DNA2 physically and specifically interact to resect DNA in a process that is ATP-dependent and requires BLM helicase and DNA2 nuclease functions. RPA is essential for both DNA unwinding by BLM and enforcing 5′ → 3′ resection polarity by DNA2. MRN accelerates processing by recruiting BLM to the end. In the other, EXO1 resects the DNA and is stimulated by BLM, MRN, and RPA. BLM increases the affinity of EXO1 for ends, and MRN recruits and enhances the processivity of EXO1. Our results establish two of the core machineries that initiate recombinational DNA repair in human cells. © 2011 by Cold Spring Harbor Laboratory Press.

Authors
Nimonkar, AV; Genschel, J; Kinoshita, E; Polaczek, P; Campbell, JL; Wyman, C; Modrich, P; Kowalczykowski, SC
MLA Citation
Nimonkar, AV, Genschel, J, Kinoshita, E, Polaczek, P, Campbell, JL, Wyman, C, Modrich, P, and Kowalczykowski, SC. "BLM-DNA2-RPA-MRN and EXO1-BLM-RPA-MRN constitute two DNA end resection machineries for human DNA break repair." Genes and Development 25.4 (2011): 350-362.
PMID
21325134
Source
scival
Published In
Genes & development
Volume
25
Issue
4
Publish Date
2011
Start Page
350
End Page
362
DOI
10.1101/gad.2003811

MutLalpha and proliferating cell nuclear antigen share binding sites on MutSbeta.

MutSbeta (MSH2-MSH3) mediates repair of insertion-deletion heterologies but also triggers triplet repeat expansions that cause neurological diseases. Like other DNA metabolic activities, MutSbeta interacts with proliferating cell nuclear antigen (PCNA) via a conserved motif (QXX(L/I)XXFF). We demonstrate that MutSbeta-PCNA complex formation occurs with an affinity of approximately 0.1 microM and a preferred stoichiometry of 1:1. However, up to 20% of complexes are multivalent under conditions where MutSbeta is in molar excess over PCNA. Conformational studies indicate that the two proteins associate in an end-to-end fashion in solution. Surprisingly, mutation of the PCNA-binding motif of MutSbeta not only abolishes PCNA binding, but unlike MutSalpha, also dramatically attenuates MutSbeta-MutLalpha interaction, MutLalpha endonuclease activation, and bidirectional mismatch repair. As predicted by these findings, PCNA competes with MutLalpha for binding to MutSbeta, an effect that is blocked by the cell cycle regulator p21(CIP1). We propose that MutSbeta-MutLalpha interaction is mediated in part by residues ((L/I)SRFF) embedded within the MSH3 PCNA-binding motif. To our knowledge this is the first case where residues important for PCNA binding also mediate interaction with a second protein. These findings also indicate that MutSbeta- and MutSalpha-initiated repair events differ in fundamental ways.

Authors
Iyer, RR; Pluciennik, A; Genschel, J; Tsai, M-S; Beese, LS; Modrich, P
MLA Citation
Iyer, RR, Pluciennik, A, Genschel, J, Tsai, M-S, Beese, LS, and Modrich, P. "MutLalpha and proliferating cell nuclear antigen share binding sites on MutSbeta." J Biol Chem 285.15 (April 9, 2010): 11730-11739.
PMID
20154325
Source
pubmed
Published In
The Journal of biological chemistry
Volume
285
Issue
15
Publish Date
2010
Start Page
11730
End Page
11739
DOI
10.1074/jbc.M110.104125

Interactions of human mismatch repair proteins MutSalpha and MutLalpha with proteins of the ATR-Chk1 pathway.

At clinically relevant doses, chemotherapeutic S(N)1 DNA methylating agents induce an ATR-mediated checkpoint response in human cells that is dependent on functional MutSalpha and MutLalpha. Deficiency of either mismatch repair activity renders cells highly resistant to this class of drug, but the mechanisms linking mismatch repair to checkpoint activation have remained elusive. In this study we have systematically examined the interactions of human MutSalpha and MutLalpha with proteins of the ATR-Chk1 pathway using both nuclear extracts and purified proteins. Using nuclear co-immunoprecipitation, we have detected interaction of MutSalpha with ATR, TopBP1, Claspin, and Chk1 and interaction of MutLalpha with TopBP1 and Claspin. We were unable to detect interaction of MutSalpha or MutLalpha with Rad17, Rad9, or replication protein A in the extract system. Use of purified proteins confirmed direct interaction of MutSalpha with ATR, TopBP1, and Chk1 and of MutLalpha with TopBP1. MutSalpha-Claspin and MutLalpha-Claspin interactions were not demonstrable with purified proteins, suggesting that extract interactions are indirect or depend on post-translational modification. Use of a modified chromatin immunoprecipitation assay showed that proliferating cell nuclear antigen, ATR, TopBP1, and Chk1 are recruited to chromatin in a MutLalpha- and MutSalpha-dependent fashion after N-methyl-N'-nitro-N-nitrosoguanidine treatment. However, chromatin enrichment of replication protein A, Claspin, Rad17-RFC, and Rad9-Rad1-Hus1 was not detected in these experiments. Although our failure to observe enrichment of the latter activities could be due to sensitivity limitations, these observations may indicate a novel mechanism for ATR activation.

Authors
Liu, Y; Fang, Y; Shao, H; Lindsey-Boltz, L; Sancar, A; Modrich, P
MLA Citation
Liu, Y, Fang, Y, Shao, H, Lindsey-Boltz, L, Sancar, A, and Modrich, P. "Interactions of human mismatch repair proteins MutSalpha and MutLalpha with proteins of the ATR-Chk1 pathway." J Biol Chem 285.8 (February 19, 2010): 5974-5982.
PMID
20029092
Source
pubmed
Published In
The Journal of biological chemistry
Volume
285
Issue
8
Publish Date
2010
Start Page
5974
End Page
5982
DOI
10.1074/jbc.M109.076109

PCNA function in the activation and strand direction of MutLα endonuclease in mismatch repair

MutLα (MLH1-PMS2) is a latent endonuclease that is activated in a mismatch-, MutSα-, proliferating cell nuclear antigen (PCNA)-, replication factor C (RFC)-, and ATP-dependent manner, with nuclease action directed to the heteroduplex strand that contains a preexisting break. RFC depletion experiments and use of linear DNAs indicate that RFC function in endonuclease activation is limited to PCNA loading. Whereas nicked circular heteroduplex DNA is a good substrate for PCNA loading and for endonuclease activation on the incised strand, covalently closed, relaxed circular DNA is a poor substrate for both reactions. However, covalently closed supercoiled or bubble-containing relaxed heteroduplexes, which do support PCNA loading, also support MutLα activation, but in this case cleavage strand bias is largely abolished. Based on these findings we suggest that PCNA has two roles in MutLα function: The clamp is required for endonuclease activation, an effect that apparently involves interaction of the two proteins, and by virtue of its loading orientation, PCNA determines the strand direction of MutLα incision. These results also provide a potential mechanism for activation of mismatch repair on nonreplicating DNA, an effect that may have implications for the somatic phase of triplet repeat expansion.

Authors
Pluciennik, A; Dzantiev, L; Iyer, RR; Constantin, N; Kadyrov, FA; Modrich, P
MLA Citation
Pluciennik, A, Dzantiev, L, Iyer, RR, Constantin, N, Kadyrov, FA, and Modrich, P. "PCNA function in the activation and strand direction of MutLα endonuclease in mismatch repair." Proceedings of the National Academy of Sciences of the United States of America 107.37 (2010): 16066-16071.
PMID
20713735
Source
scival
Published In
Proceedings of the National Academy of Sciences of USA
Volume
107
Issue
37
Publish Date
2010
Start Page
16066
End Page
16071
DOI
10.1073/pnas.1010662107

PMS2 endonuclease activity has distinct biological functions and is essential for genome maintenance

The DNA mismatch repair protein PMS2 was recently found to encode a novel endonuclease activity. To determine the biological functions of this activity in mammals, we generated endonuclease-deficient Pms2E702K knock-in mice. Pms2EK/EK mice displayed increased genomic mutation rates and a strong cancer predisposition. In addition, class switch recombination, but not somatic hypermutation,was impaired in Pms2EK/EK B cells, indicating a specific role in Ig diversity. In contrast to Pms2-/- mice, Pms2EK/EK male mice were fertile, indicating that this activity is dispensable in spermatogenesis. Therefore, the PMS2 endonuclease activity has distinct biological functions and is essential for genome maintenance and tumor suppression.

Authors
Oers, JMMV; Roa, S; Werling, U; Liu, Y; Genschel, J; Jr, HH; Sellers, RS; Modrich, P; Scharff, MD; Edelmann, W
MLA Citation
Oers, JMMV, Roa, S, Werling, U, Liu, Y, Genschel, J, Jr, HH, Sellers, RS, Modrich, P, Scharff, MD, and Edelmann, W. "PMS2 endonuclease activity has distinct biological functions and is essential for genome maintenance." Proceedings of the National Academy of Sciences of the United States of America 107.30 (2010): 13384-13389.
PMID
20624957
Source
scival
Published In
Proceedings of the National Academy of Sciences of USA
Volume
107
Issue
30
Publish Date
2010
Start Page
13384
End Page
13389
DOI
10.1073/pnas.1008589107

Structure of the Endonuclease Domain of MutL: Unlicensed to Cut

DNA mismatch repair corrects errors that have escaped polymerase proofreading, increasing replication fidelity 100- to 1000-fold in organisms ranging from bacteria to humans. The MutL protein plays a central role in mismatch repair by coordinating multiple protein-protein interactions that signal strand removal upon mismatch recognition by MutS. Here we report the crystal structure of the endonuclease domain of Bacillus subtilis MutL. The structure is organized in dimerization and regulatory subdomains connected by a helical lever spanning the conserved endonuclease motif. Additional conserved motifs cluster around the lever and define a Zn2+-binding site that is critical for MutL function in vivo. The structure unveils a powerful inhibitory mechanism to prevent undesired nicking of newly replicated DNA and allows us to propose a model describing how the interaction with MutS and the processivity clamp could license the endonuclease activity of MutL. The structure also provides a molecular framework to propose and test additional roles of MutL in mismatch repair. © 2010 Elsevier Inc.

Authors
Pillon, MC; Lorenowicz, JJ; Uckelmann, M; Klocko, AD; Mitchell, RR; Chung, YS; Modrich, P; Walker, GC; Simmons, LA; Friedhoff, P; Guarné, A
MLA Citation
Pillon, MC, Lorenowicz, JJ, Uckelmann, M, Klocko, AD, Mitchell, RR, Chung, YS, Modrich, P, Walker, GC, Simmons, LA, Friedhoff, P, and Guarné, A. "Structure of the Endonuclease Domain of MutL: Unlicensed to Cut." Molecular Cell 39.1 (2010): 145-151.
PMID
20603082
Source
scival
Published In
Molecular Cell
Volume
39
Issue
1
Publish Date
2010
Start Page
145
End Page
151
DOI
10.1016/j.molcel.2010.06.027

Mismatch repair and nucleotide excision repair proteins cooperate in the recognition of DNA interstrand crosslinks.

DNA interstrand crosslinks (ICLs) are among the most cytotoxic types of DNA damage, thus ICL-inducing agents such as psoralen, are clinically useful chemotherapeutics. Psoralen-modified triplex-forming oligonucleotides (TFOs) have been used to target ICLs to specific genomic sites to increase the selectivity of these agents. However, how TFO-directed psoralen ICLs (Tdp-ICLs) are recognized and processed in human cells is unclear. Previously, we reported that two essential nucleotide excision repair (NER) protein complexes, XPA-RPA and XPC-RAD23B, recognized ICLs in vitro, and that cells deficient in the DNA mismatch repair (MMR) complex MutSbeta were sensitive to psoralen ICLs. To further investigate the role of MutSbeta in ICL repair and the potential interaction between proteins from the MMR and NER pathways on these lesions, we performed electrophoretic mobility-shift assays and chromatin immunoprecipitation analysis of MutSbeta and NER proteins with Tdp-ICLs. We found that MutSbeta bound to Tdp-ICLs with high affinity and specificity in vitro and in vivo, and that MutSbeta interacted with XPA-RPA or XPC-RAD23B in recognizing Tdp-ICLs. These data suggest that proteins from the MMR and NER pathways interact in the recognition of ICLs, and provide a mechanistic link by which proteins from multiple repair pathways contribute to ICL repair.

Authors
Zhao, J; Jain, A; Iyer, RR; Modrich, PL; Vasquez, KM
MLA Citation
Zhao, J, Jain, A, Iyer, RR, Modrich, PL, and Vasquez, KM. "Mismatch repair and nucleotide excision repair proteins cooperate in the recognition of DNA interstrand crosslinks." Nucleic Acids Res 37.13 (July 2009): 4420-4429.
PMID
19468048
Source
pubmed
Published In
Nucleic Acids Research
Volume
37
Issue
13
Publish Date
2009
Start Page
4420
End Page
4429
DOI
10.1093/nar/gkp399

Involvement of the β clamp in methyl-directed mismatch repair in vitro

We have examined function of the bacterial β replication clamp in the different steps of methyl-directed DNA mismatch repair. The mismatch-, MutS-, and MutL-dependent activation of MutH is unaffected by the presence or orientation of loaded β clamp on either 3′ or 5′ heteroduplexes. Similarly, β is not required for 3′ or 5′ mismatch-provoked excision when scored in the presence of γ complex or in the presence of γ complex and DNA polymerase III core components. However, mismatch repair does not occur in the absence of β, an effect we attribute to a requirement for the clamp in the repair DNA synthesis step of the reaction. We have confirmed previous findings that β clamp interacts specifically with MutS and MutL (López de Saro, F. J., Marinus, M. G., Modrich, P., and O'Donnell, M. (2006) J. Biol. Chem. 281, 14340-14349) and show that the mutator phenotype conferred by amino acid substitution within the MutS N-terminal β-interaction motif is the probable result of instability coupled with reduced activity in multiple steps of the repair reaction. In addition, we have found that the DNA polymerase III α catalytic subunit interacts strongly and specifically with both MutS and MutL. Because interactions of polymerase III holoenzyme components with MutS and MutL appear to be of limited import during the initiation and excision steps of mismatch correction, we suggest that their significance might lie in the control of replication fork events in response to the sensing of DNA lesions by the repair system. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

Authors
Pluciennik, A; Burdett, V; Lukianova, O; O'Donnell, M; Modrich, P
MLA Citation
Pluciennik, A, Burdett, V, Lukianova, O, O'Donnell, M, and Modrich, P. "Involvement of the β clamp in methyl-directed mismatch repair in vitro." Journal of Biological Chemistry 284.47 (2009): 32782-32791.
PMID
19783657
Source
scival
Published In
The Journal of biological chemistry
Volume
284
Issue
47
Publish Date
2009
Start Page
32782
End Page
32791
DOI
10.1074/jbc.M109.054528

Functions of MutLα, replication protein A (RPA), and HMGB1 in 5′-directed mismatch repair

A purified system comprised of MutSα, MutLα, exonuclease 1 (Exo1), and replication protein A (RPA) (in the absence or presence of HMGB1) supports 5′-directed mismatch-provoked excision that terminates after mismatch removal. MutLα is not essential for this reaction but enhances excision termination, although the basis of this effect has been uncertain. One model attributes the primary termination function in this system to RPA, with MutLα functioning in a secondary capacity by suppressing Exo1 hydrolysis of mismatch-free DNA (Genschel, J., and Modrich, P. (2003) Mol. Cell 12, 1077-1086). A second invokes MutLα as the primary effector of excision termination (Zhang, Y., Yuan, F., Presnell, S. R., Tian, K., Gao, Y., Tomkinson, A. E., Gu, L., and Li, G. M. (2005) Cell 122, 693-705). In the latter model, RPA provides a secondary termination function, but together with HMGB1, also participates in earlier steps of the reaction. To distinguish between these models, we have reanalyzed the functions of MutLα, RPA, and HMGB1 in 5′-directed mismatch-provoked excision using purified components as well as mammalian cell extracts. Analysis of extracts derived from A2780/AD cells, which are devoid of MutLα but nevertheless support 5′-directed mismatch repair, has demonstrated that 5′-directed excision terminates normally in the absence of MutLα. Experiments using purified components confirm a primary role for RPA in terminating excision by MutSα-activated Exo1 but are inconsistent with direct participation of MutLα in this process. While HMGB1 attenuates excision by activated Exo1, this effect is distinct from that mediated by RPA. Assay of extracts derived from HMGB1+/+ and HMGB1-/- mouse embryo fibroblast cells indicates that HMGB1 is not essential for mismatch repair. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

Authors
Genschel, J; Modrich, P
MLA Citation
Genschel, J, and Modrich, P. "Functions of MutLα, replication protein A (RPA), and HMGB1 in 5′-directed mismatch repair." Journal of Biological Chemistry 284.32 (2009): 21536-21544.
PMID
19515846
Source
scival
Published In
The Journal of biological chemistry
Volume
284
Issue
32
Publish Date
2009
Start Page
21536
End Page
21544
DOI
10.1074/jbc.M109.021287

A possible mechanism for exonuclease 1-independent eukaryotic mismatch repair

Mismatch repair contributes to genetic stability, and inactivation of the mammalian pathway leads to tumor development. Mismatch correction occurs by an excision-repair mechanism and has been shown to depend on the 5′ to 3′ hydrolytic activity exonuclease 1 (Exo1) in eukaryotic cells. However, genetic and biochemical studies have indicated that one or more Exo1-independent modes of mismatch repair also exist. We have analyzed repair of nicked circular heteroduplex DNA in extracts of Exo1-deficient mouse embryo fibroblast cells. Exo1-independent repair under these conditions is MutLα-dependent and requires functional integrity of the MutLα endonuclease metal-binding motif. In contrast to the Exo1-dependent reaction, we have been unable to detect a gapped excision intermediate in Exo1-deficient extracts when repair DNA synthesis is blocked. A possible explanation for this finding has been provided by analysis of a purified system comprised of MutSα, MutLα, replication factor C, proliferating cell nuclear antigen, replication protein A, and DNA polymerase δ that supports Exo1-independent repair in vitro. Repair in this system depends on MutLα incision of the nicked heteroduplex strand and dNTP-dependent synthesis-driven displacement of a DNA segment spanning the mismatch. Such a mechanism may account, at least in part, for the Exo1-independent repair that occurs in eukaryotic cells, and hence the modest cancer predisposition of Exo1-deficient mammalian cells.

Authors
Kadyrov, FA; Genschel, J; Fang, Y; Penland, E; Edelmann, W; Modrich, P
MLA Citation
Kadyrov, FA, Genschel, J, Fang, Y, Penland, E, Edelmann, W, and Modrich, P. "A possible mechanism for exonuclease 1-independent eukaryotic mismatch repair." Proceedings of the National Academy of Sciences of the United States of America 106.21 (2009): 8495-8500.
PMID
19420220
Source
scival
Published In
Proceedings of the National Academy of Sciences of USA
Volume
106
Issue
21
Publish Date
2009
Start Page
8495
End Page
8500
DOI
10.1073/pnas.0903654106

Mismatch repair deficiency does not mediate clinical resistance to temozolomide in malignant glioma.

PURPOSE: A major mechanism of resistance to methylating agents, including temozolomide, is the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT). Preclinical data indicates that defective DNA mismatch repair (MMR) results in tolerance to temozolomide regardless of AGT activity. The purpose of this study was to determine the role of MMR deficiency in mediating resistance in samples from patients with both newly diagnosed malignant gliomas and those who have failed temozolomide therapy. EXPERIMENTAL DESIGN: The roles of AGT and MMR deficiency in mediating resistance in glioblastoma multiforme were assessed by immunohistochemistry and microsatellite instability (MSI), respectively. The mutation status of the MSH6 gene, a proposed correlate of temozolomide resistance, was determined by direct sequencing and compared with data from immunofluorescent detection of MSH6 protein and reverse transcription-PCR amplification of MSH6 RNA. RESULTS: Seventy percent of newly diagnosed and 78% of failed-therapy glioblastoma multiforme samples expressed nuclear AGT protein in > or = 20% of cells analyzed, suggesting alternate means of resistance in 20% to 30% of cases. Single loci MSI was observed in 3% of patient samples; no sample showed the presence of high MSI. MSI was not shown to correlate with MSH6 mutation or loss of MSH6 protein expression. CONCLUSIONS: Although high AGT levels may mediate resistance in a portion of these samples, MMR deficiency does not seem to be responsible for mediating temozolomide resistance in adult malignant glioma. Accordingly, the presence of a fraction of samples exhibiting both low AGT expression and MMR proficiency suggests that additional mechanisms of temozolomide resistance are operational in the clinic.

Authors
Maxwell, JA; Johnson, SP; McLendon, RE; Lister, DW; Horne, KS; Rasheed, A; Quinn, JA; Ali-Osman, F; Friedman, AH; Modrich, PL; Bigner, DD; Friedman, HS
MLA Citation
Maxwell, JA, Johnson, SP, McLendon, RE, Lister, DW, Horne, KS, Rasheed, A, Quinn, JA, Ali-Osman, F, Friedman, AH, Modrich, PL, Bigner, DD, and Friedman, HS. "Mismatch repair deficiency does not mediate clinical resistance to temozolomide in malignant glioma." Clin Cancer Res 14.15 (August 1, 2008): 4859-4868.
PMID
18676759
Source
pubmed
Published In
Clinical cancer research : an official journal of the American Association for Cancer Research
Volume
14
Issue
15
Publish Date
2008
Start Page
4859
End Page
4868
DOI
10.1158/1078-0432.CCR-07-4807

The MutSalpha-proliferating cell nuclear antigen interaction in human DNA mismatch repair.

We have examined the interaction parameters, conformation, and functional significance of the human MutSalpha(.) proliferating cell nuclear antigen (PCNA) complex in mismatch repair. The two proteins associate with a 1:1 stoichiometry and a K(D) of 0.7 microm in the absence or presence of heteroduplex DNA. PCNA does not influence the affinity of MutSalpha for a mismatch, and mismatch-bound MutSalpha binds PCNA. Small angle x-ray scattering studies have established the molecular parameters of the complex, which are consistent with an elongated conformation in which the two proteins associate in an end-to-end fashion in a manner that does not involve an extended unstructured tether, as has been proposed for yeast MutSalpha and PCNA ( Shell, S. S., Putnam, C. D., and Kolodner, R. D. (2007) Mol. Cell 26, 565-578 ). MutSalpha variants lacking the PCNA interaction motif are functional in 3'- or 5'-directed mismatch-provoked excision, but display a partial defect in 5'-directed mismatch repair. This finding is consistent with the modest mutability conferred by inactivation of the MutSalpha PCNA interaction motif and suggests that interaction of the replication clamp with other repair protein(s) accounts for the essential role of PCNA in MutSalpha-dependent mismatch repair.

Authors
Iyer, RR; Pohlhaus, TJ; Chen, S; Hura, GL; Dzantiev, L; Beese, LS; Modrich, P
MLA Citation
Iyer, RR, Pohlhaus, TJ, Chen, S, Hura, GL, Dzantiev, L, Beese, LS, and Modrich, P. "The MutSalpha-proliferating cell nuclear antigen interaction in human DNA mismatch repair." J Biol Chem 283.19 (May 9, 2008): 13310-13319.
PMID
18326858
Source
pubmed
Published In
The Journal of biological chemistry
Volume
283
Issue
19
Publish Date
2008
Start Page
13310
End Page
13319
DOI
10.1074/jbc.M800606200

Human exonuclease 1 and BLM helicase interact to resect DNA and initiate DNA repair

The error-free repair of double-stranded DNA breaks by homologous recombination requires processing of broken ends. These processed ends are substrates for assembly of DNA strand exchange proteins that mediate DNA strand invasion. Here, we establish that human BLM helicase, a member of the RecQ family, stimulates the nucleolytic activity of human exonuclease 1 (hExo1), a 5′→3′ double-stranded DNA exonuclease. The stimulation is specific because other RecQ homologs fail to stimulate hExo1. Stimulation of DNA resection by hExo1 is independent of BLM helicase activity and is, instead, mediated by an interaction between the 2 proteins. Finally, we show that DNA ends resected by hExo1 and BLM are used by human Rad51, but not its yeast or bacterial counterparts, to promote homologous DNA pairing. This in vitro system recapitulates initial steps of homologous recombination and provides biochemical evidence for a role of BLM and Exo1 in the initiation of recombinational DNA repair. © 2008 by The National Academy of Sciences of the USA.

Authors
Nimonkar, AV; Özsoy, AZ; Genschel, J; Modrich, P; Kowalczykowski, SC
MLA Citation
Nimonkar, AV, Özsoy, AZ, Genschel, J, Modrich, P, and Kowalczykowski, SC. "Human exonuclease 1 and BLM helicase interact to resect DNA and initiate DNA repair." Proceedings of the National Academy of Sciences of the United States of America 105.44 (2008): 16906-16911.
PMID
18971343
Source
scival
Published In
Proceedings of the National Academy of Sciences of USA
Volume
105
Issue
44
Publish Date
2008
Start Page
16906
End Page
16911
DOI
10.1073/pnas.0809380105

Direct Visualization of Asymmetric Adenine Nucleotide-Induced Conformational Changes in MutLα

MutLα, the heterodimeric eukaryotic MutL homolog, is required for DNA mismatch repair (MMR) in vivo. It has been suggested that conformational changes, modulated by adenine nucleotides, mediate the interactions of MutLα with other proteins in the MMR pathway, coordinating the recognition of DNA mismatches by MutSα and the activation of MutLα with the downstream events that lead to repair. Thus far, the only evidence for these conformational changes has come from X-ray crystallography of isolated domains, indirect biochemical analyses, and comparison to other members of the GHL ATPase family to which MutLα belongs. Using atomic force microscopy (AFM), coupled with biochemical techniques, we demonstrate that adenine nucleotides induce large asymmetric conformational changes in full-length yeast and human MutLα and that these changes are associated with significant increases in secondary structure. These data reveal an ATPase cycle in which sequential nucleotide binding, hydrolysis, and release modulate the conformational states of MutLα. © 2008 Elsevier Inc. All rights reserved.

Authors
Sacho, EJ; Kadyrov, FA; Modrich, P; Kunkel, TA; Erie, DA
MLA Citation
Sacho, EJ, Kadyrov, FA, Modrich, P, Kunkel, TA, and Erie, DA. "Direct Visualization of Asymmetric Adenine Nucleotide-Induced Conformational Changes in MutLα." Molecular Cell 29.1 (2008): 112-121.
PMID
18206974
Source
scival
Published In
Molecular Cell
Volume
29
Issue
1
Publish Date
2008
Start Page
112
End Page
121
DOI
10.1016/j.molcel.2007.10.030

Structure of the human MutSalpha DNA lesion recognition complex.

Mismatch repair (MMR) ensures the fidelity of DNA replication, initiates the cellular response to certain classes of DNA damage, and has been implicated in the generation of immune diversity. Each of these functions depends on MutSalpha (MSH2*MSH6 heterodimer). Inactivation of this protein complex is responsible for tumor development in about half of known hereditary nonpolyposis colorectal cancer kindreds and also occurs in sporadic tumors in a variety of tissues. Here, we describe a series of crystal structures of human MutSalpha bound to different DNA substrates, each known to elicit one of the diverse biological responses of the MMR pathway. All lesions are recognized in a similar manner, indicating that diversity of MutSalpha-dependent responses to DNA lesions is generated in events downstream of this lesion recognition step. This study also allows rigorous mapping of cancer-causing mutations and furthermore suggests structural pathways for allosteric communication between different regions within the heterodimer.

Authors
Warren, JJ; Pohlhaus, TJ; Changela, A; Iyer, RR; Modrich, PL; Beese, LS
MLA Citation
Warren, JJ, Pohlhaus, TJ, Changela, A, Iyer, RR, Modrich, PL, and Beese, LS. "Structure of the human MutSalpha DNA lesion recognition complex." Mol Cell 26.4 (May 25, 2007): 579-592.
PMID
17531815
Source
pubmed
Published In
Molecular Cell
Volume
26
Issue
4
Publish Date
2007
Start Page
579
End Page
592
DOI
10.1016/j.molcel.2007.04.018

Saccharomyces cerevisiae MutLα is a mismatch repair endonuclease

MutL homologs are crucial for mismatch repair and genetic stability, but their function is not well understood. Human MutLα(MLH1-PMS2 heterodimer) harbors a latent endonuclease that is dependent on the integrity of a PMS2 DQHA(X)2E(X)4E motif (Kadyrov, F. A., Dzantiev, L., Constantin, N., and Modrich, P. (2006) Cell 126, 297-308). This sequence element is conserved in many MutL homologs, including the PMS1 subunit of Saccharomyces cerevisiae MutLα, but is absent in MutL proteins from bacteria like Escherichia coli that rely on d(GATC) methylation for strand directionality. We show that yeast MutLα is a strand-directed endonuclease that incises DNA in a reaction that depends on a mismatch, yMutSα, yRFC, yPCNA, ATP, and a pre-existing strand break, whereas E. coli MutL is not. Amino acid substitution within the PMS1 DQHA(X)2E(X)4E motif abolishes yMutLα endonuclease activity in vitro and confers strong genetic instability in vivo, but does not affect yMutLα ATPase activity or the ability of the protein to support assembly of the yMutLα· yMutSα·heteroduplex ternary complex. The loaded form of yPCNA may play an important effector role in directing yMutLα incision to the discontinuous strand of a nicked heteroduplex.

Authors
Kadyrov, FA; Holmes, SF; Arana, ME; Lukianova, OA; O'Donnell, M; Kunkel, TA; Modrich, P
MLA Citation
Kadyrov, FA, Holmes, SF, Arana, ME, Lukianova, OA, O'Donnell, M, Kunkel, TA, and Modrich, P. "Saccharomyces cerevisiae MutLα is a mismatch repair endonuclease." Journal of Biological Chemistry 282.51 (2007): 37181-37190.
PMID
17951253
Source
scival
Published In
The Journal of biological chemistry
Volume
282
Issue
51
Publish Date
2007
Start Page
37181
End Page
37190
DOI
10.1074/jbc.M707617200

Protein roadblocks and helix discontinuities are barriers to the initiation of mismatch repair

The hemimethylated d(GATC) sequence that directs Escherichia coli mismatch repair can reside on either side of a mismatch at a separation distance of 1,000 bp or more. Initiation of repair involves the mismatch-, MutS-, and MutL-dependent activation of MutH endonuclease, which incises the unmethylated strand at the d(GATC) sequence, with the ensuing strand break serving as the loading site for the appropriate 3′-to-5′ or 5′-to-3′ excision system. However, the mechanism responsible for the coordinated recognition of the mismatch and a hemimodified d(GATC) site is uncertain. We show that a protein roadblock (EcoRlEIIIQ, a hydrolytically defective form of EcoRI endonuclease) placed on the helix between the two DNA sites inhibits MutH activation by 70-80% and that events that escape inhibition are attributable, at least in part, to diffusion of EcoRIEIIIQ away from its recognition site. We also demonstrate that a double-strand break located within the shorter path linking the mismatch and a d(GATC) site in a circular heteroduplex abolishes MutH activation, whereas a double-strand break within the longer path is without effect. These findings support the idea that initiation of mismatch repair involves signaling along the helix contour. © 2007 by The National Academy of Sciences of the USA.

Authors
Pluciennik, A; Modrich, P
MLA Citation
Pluciennik, A, and Modrich, P. "Protein roadblocks and helix discontinuities are barriers to the initiation of mismatch repair." Proceedings of the National Academy of Sciences of the United States of America 104.31 (2007): 12709-12713.
PMID
17620611
Source
scival
Published In
Proceedings of the National Academy of Sciences of USA
Volume
104
Issue
31
Publish Date
2007
Start Page
12709
End Page
12713
DOI
10.1073/pnas.0705129104

A phase II window trial of procarbazine and topotecan in children with high-grade glioma: a report from the children's oncology group.

The role of chemotherapy in the treatment of high-grade gliomas in children is unclear. Early reports were suggestive of improved outcome in children with high-grade glioma with the addition of chemotherapy after surgery and radiation therapy. Subsequent studies did not show similar favorable contribution of chemotherapy to the outcome of these children. Further efforts to identify active chemotherapy agents in children include use of agents that have shown efficacy in adult patients with high-grade glioma and agents that have shown promise in mice bearing human xenografts of brain tumors. A Pediatric Oncology Group (POG 9431) trial tested the activity of two such agents, procarbazine and topotecan in newly diagnosed patients with high-grade glioma who had measurable disease after diagnostic surgery. Neither agent showed efficacy within the confines of the statistical design of the study. This study showed that children with high-grade glioma have an innate resistance to alkylating agents based on mismatch repair deficiency and high levels of alkyguanine transferase (AGT). Future trials should consider strategies to overcome the resistance mechanisms in children with high-grade glioma.

Authors
Chintagumpala, MM; Friedman, HS; Stewart, CF; Kepner, J; McLendon, RE; Modrich, PL; McCluggage, C; Burger, P; Holmes, E; Thompson, S; Rutka, J; Michalski, J; Woo, S; Blaney, SM; Kun, LE; Horowitz, ME; Pediatric Oncology Group Study,
MLA Citation
Chintagumpala, MM, Friedman, HS, Stewart, CF, Kepner, J, McLendon, RE, Modrich, PL, McCluggage, C, Burger, P, Holmes, E, Thompson, S, Rutka, J, Michalski, J, Woo, S, Blaney, SM, Kun, LE, Horowitz, ME, and Pediatric Oncology Group Study, . "A phase II window trial of procarbazine and topotecan in children with high-grade glioma: a report from the children's oncology group." J Neurooncol 77.2 (April 2006): 193-198.
PMID
16314955
Source
pubmed
Published In
Journal of Neuro-Oncology
Volume
77
Issue
2
Publish Date
2006
Start Page
193
End Page
198
DOI
10.1007/s11060-005-9024-x

DNA mismatch repair: functions and mechanisms.

Authors
Iyer, RR; Pluciennik, A; Burdett, V; Modrich, PL
MLA Citation
Iyer, RR, Pluciennik, A, Burdett, V, and Modrich, PL. "DNA mismatch repair: functions and mechanisms." Chem Rev 106.2 (February 2006): 302-323. (Review)
PMID
16464007
Source
pubmed
Published In
Chemical Reviews
Volume
106
Issue
2
Publish Date
2006
Start Page
302
End Page
323
DOI
10.1021/cr0404794

Mechanisms in eukaryotic mismatch repair

Authors
Modrich, P
MLA Citation
Modrich, P. "Mechanisms in eukaryotic mismatch repair." Journal of Biological Chemistry 281.41 (2006): 30305-30309.
PMID
16905530
Source
scival
Published In
The Journal of biological chemistry
Volume
281
Issue
41
Publish Date
2006
Start Page
30305
End Page
30309
DOI
10.1074/jbc.R600022200

Endonucleolytic Function of MutLα in Human Mismatch Repair

Half of hereditary nonpolyposis colon cancer kindreds harbor mutations that inactivate MutLα (MLH1•PMS2 heterodimer). MutLα is required for mismatch repair, but its function in this process is unclear. We show that human MutLα is a latent endonuclease that is activated in a mismatch-, MutSα-, RFC-, PCNA-, and ATP-dependent manner. Incision of a nicked mismatch-containing DNA heteroduplex by this four-protein system is strongly biased to the nicked strand. A mismatch-containing DNA segment spanned by two strand breaks is removed by the 5′-to-3′ activity of MutSα-activated exonuclease I. The probable endonuclease active site has been localized to a PMS2 DQHA(X)2E(X)4E motif. This motif is conserved in eukaryotic PMS2 homologs and in MutL proteins from a number of bacterial species but is lacking in MutL proteins from bacteria that rely on d(GATC) methylation for strand discrimination in mismatch repair. Therefore, the mode of excision initiation may differ in these organisms. © 2006 Elsevier Inc. All rights reserved.

Authors
Kadyrov, FA; Dzantiev, L; Constantin, N; Modrich, P
MLA Citation
Kadyrov, FA, Dzantiev, L, Constantin, N, and Modrich, P. "Endonucleolytic Function of MutLα in Human Mismatch Repair." Cell 126.2 (2006): 297-308.
PMID
16873062
Source
scival
Published In
Cell
Volume
126
Issue
2
Publish Date
2006
Start Page
297
End Page
308
DOI
10.1016/j.cell.2006.05.039

Engineering life: Building a FAB for biology

DNA methods are now mainstays of modern research in genetic engineering. One reason is that the tools available for building with biological parts have yet to reach a level of standardization. Standardization of methods and components in biological engineering could give rise to design libraries of comparable parts and make outsourcing of fabrication possible. In living organisms, biological machinery composed of enzymes such as polymerase is able to manufacture and repair DNA molecules at speeds of up to 500 bases a second, with error rates of about one base in a billion. The most striking thing about synthetic biological circuits that are being build is that they are identical in function to the first types of circuits that electrical engineers build when they want to test a new process for manufacturing semiconductor chips.

Authors
Baker, D; Church, G; Collins, J; Endy, D; Jacobson, J; Keasling, J; Modrich, P; Smolke, C; Weiss, R
MLA Citation
Baker, D, Church, G, Collins, J, Endy, D, Jacobson, J, Keasling, J, Modrich, P, Smolke, C, and Weiss, R. "Engineering life: Building a FAB for biology." Scientific American 294.6 (2006): 44-51.
PMID
16711359
Source
scival
Published In
Scientific American
Volume
294
Issue
6
Publish Date
2006
Start Page
44
End Page
51

Mismatch repair-dependent iterative excision at irreparable O 6-methylguanine lesions in human nuclear extracts

The response of mammalian cells to Sn1 DNA methylators depends on functional MutSα and MutLα. Cells deficient in either of these activities are resistant to the cytotoxic effects of this class of chemotherapeutic drug. Because killing by Sn1 methylators has been attributed to O6-methylguanine (MeG), we have constructed nicked circular heteroduplexes that contain a single MeG-T mispair, and we have examined processing of these molecules by mismatch repair in nuclear extracts of human cells. Excision provoked by MeG-T is restricted to the incised heteroduplex strand, leading to removal of the MeG when it resides on this strand. However, when the MeG is located on the continuous strand, the heteroduplex is irreparable. MeG-T-dependent repair DNA synthesis is observed on both reparable and irreparable 3′ and 5′ heteroduplexes as judged by [32P]dAMP incorporation. Labeling with [α- 32P]dATP followed by a cold dATP chase has demonstrated that newly synthesized DNA on irreparable molecules is subject to re-excision in a reaction that is MutLα-dependent, an effect attributable to the presence of MeG on the template strand. Processing of the irreparable 3′ heteroduplex is also associated with incision of the discontinuous strand of a few percent of molecules near the thymidylate of the MeG-T base pair. These results provide the first direct evidence for mismatch repair-mediated iterative processing of DNA methylator damage, an effect that may be relevant to damage signaling events triggered by this class of chemotherapeutic agent. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

Authors
York, SJ; Modrich, P
MLA Citation
York, SJ, and Modrich, P. "Mismatch repair-dependent iterative excision at irreparable O 6-methylguanine lesions in human nuclear extracts." Journal of Biological Chemistry 281.32 (2006): 22674-22683.
PMID
16772289
Source
scival
Published In
The Journal of biological chemistry
Volume
281
Issue
32
Publish Date
2006
Start Page
22674
End Page
22683
DOI
10.1074/jbc.M603667200

The β sliding clamp binds to multiple sites within MutL and MutS

The MutL and MutS proteins are the central components of the DNArepair machinery that corrects mismatches generated by DNA polymerases during synthesis. We find that MutL interacts directly with the β sliding clamp, a ring-shaped dimeric protein that confers processivity to DNA polymerases by tethering them to their substrates. Interestingly, the interaction of MutL with β only occurs in the presence of single-stranded DNA. We find that the interaction occurs via a loop in MutL near the ATP-binding site. The binding site of MutL on β locates to the hydrophobic pocket between domains two and three of the clamp. Site-specific replacement of two residues in MutL diminished interaction with β without disrupting MutL function with helicase II. In vivo studies reveal that this mutant MutL is no longer functional in mismatch repair. In addition, the human MLH1 has a close match to the proliferating cell nuclear antigen clamp binding motif in the region that corresponds to the β interaction site in Escherichia coli MutL, and a peptide corresponding to this site binds proliferating cell nuclear antigen. The current report also examines in detail the interaction of β with MutS. We find that two distinct regions of MutS interact with β. One is located near the C terminus and the other is close to the N terminus, within the mismatch binding domain. Complementation studies using genes encoding different MutS mutants reveal that the N-terminal β interaction motif on MutS is essential for activity in vivo, but the C-terminal interaction site for β is not. In light of these results, we propose roles for the β clamp in orchestrating the sequence of events that lead to mismatch repair in the cell. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

Authors
Saro, FJLD; Marinus, MG; Modrich, P; O'Donnell, M
MLA Citation
Saro, FJLD, Marinus, MG, Modrich, P, and O'Donnell, M. "The β sliding clamp binds to multiple sites within MutL and MutS." Journal of Biological Chemistry 281.20 (2006): 14340-14349.
PMID
16546997
Source
scival
Published In
The Journal of biological chemistry
Volume
281
Issue
20
Publish Date
2006
Start Page
14340
End Page
14349
DOI
10.1074/jbc.M601264200

Analysis of the Excision Step in Human DNA Mismatch Repair

The reaction responsible for replication error correction by mismatch repair proceeds via several steps: mismatch recognition, mismatch-provoked excision, repair DNA synthesis, and ligation. Key steps in this process are the recognition and subsequent exonucleolytic removal of the mispair. A minimal system comprised of human MutSα (MSH2•MSH6), MutLα (MLH1•PMS2), exonuclease I (EXOI), replication protein A (RPA), proliferating cell nuclear antigen (PCNA), and replication factor C (RFC) is sufficient to support mismatch-provoked excision in vitro. This chapter describes methods for analysis of the reconstituted excision reaction. © 2006 Elsevier Inc. All rights reserved.

Authors
Genschel, J; Modrich, P
MLA Citation
Genschel, J, and Modrich, P. "Analysis of the Excision Step in Human DNA Mismatch Repair." Methods in Enzymology 408 (2006): 273-284.
PMID
16793375
Source
scival
Published In
Methods in Enzymology
Volume
408
Publish Date
2006
Start Page
273
End Page
284
DOI
10.1016/S0076-6879(06)08017-7

Poly(ADP-ribose) polymerase-1 inhibition reverses temozolomide resistance in a DNA mismatch repair-deficient malignant glioma xenograft.

Temozolomide is a DNA-methylating agent used in the treatment of malignant gliomas. In this study, we have examined if inhibition of poly(ADP-ribose) polymerase (PARP) could increase the cytotoxicity of temozolomide, particularly in cells deficient in DNA mismatch repair. Athymic mice, transplanted with mismatch repair-proficient [D-245 MG] or deficient [D-245 MG (PR)] xenografts, were treated with a combination of temozolomide and the PARP inhibitor, INO-1001. For the tumors deficient in mismatch repair, the most effective dose of INO-1001 was found to be 150 mg/kg, given i.p. thrice at 4-hour intervals with the first injection in combination with 262.5 mg/kg temozolomide (0.75 LD(10)). This dose of temozolomide by itself induced no partial regressions and a 4-day growth delay. In two separate experiments, the combination therapy increased the growth delay by 21.6 and 9.7 days with partial regressions observed in four of eight and three of nine mice, respectively. The addition of INO-1001 had a more modest, yet statistically significant, increase in tumor growth delay in the mismatch repair-proficient xenografts. In these experiments, mice were treated with a lower amount of temozolomide (88 mg/kg), which resulted in growth delays of 43.1 and 39.2 days. When the temozolomide treatment was in combination with 200 mg/kg INO-1001, there was an increase in growth delay to 48.9 and 45.7 days, respectively. These results suggest that inhibition of PARP may increase the efficacy of temozolomide in the treatment of malignant gliomas, particularly in tumors deficient in DNA mismatch repair.

Authors
Cheng, CL; Johnson, SP; Keir, ST; Quinn, JA; Ali-Osman, F; Szabo, C; Li, H; Salzman, AL; Dolan, ME; Modrich, P; Bigner, DD; Friedman, HS
MLA Citation
Cheng, CL, Johnson, SP, Keir, ST, Quinn, JA, Ali-Osman, F, Szabo, C, Li, H, Salzman, AL, Dolan, ME, Modrich, P, Bigner, DD, and Friedman, HS. "Poly(ADP-ribose) polymerase-1 inhibition reverses temozolomide resistance in a DNA mismatch repair-deficient malignant glioma xenograft." Mol Cancer Ther 4.9 (September 2005): 1364-1368.
PMID
16170028
Source
pubmed
Published In
Molecular cancer therapeutics
Volume
4
Issue
9
Publish Date
2005
Start Page
1364
End Page
1368
DOI
10.1158/1535-7163.MCT-05-0128

HIF-1alpha induces genetic instability by transcriptionally downregulating MutSalpha expression.

Hypoxia promotes genetic instability by undefined mechanisms. The transcription factor HIF-1alpha is crucial for the cellular response to hypoxia and is frequently overexpressed in human cancers, resulting in the activation of genes essential for cell survival. Here, we demonstrate that HIF-1alpha is responsible for genetic instability at the nucleotide level by inhibiting MSH2 and MSH6, thereby decreasing levels of the MSH2-MSH6 complex, MutSalpha, which recognizes base mismatches. HIF-1alpha displaces the transcriptional activator Myc from Sp1 binding to repress MutSalpha expression in a p53-dependent manner; Sp1 serves as a molecular switch by recruiting HIF-1alpha to the gene promoter under hypoxia. Furthermore, in human sporadic colon cancers, HIF-1alpha overexpression is statistically associated with the loss of MSH2 expression, especially when p53 is immunochemically undetectable. These findings indicate that the regulation of DNA repair is an integral part of the hypoxic response, providing molecular insights into the mechanisms underlying hypoxia-induced genetic instability.

Authors
Koshiji, M; To, KK-W; Hammer, S; Kumamoto, K; Harris, AL; Modrich, P; Huang, LE
MLA Citation
Koshiji, M, To, KK-W, Hammer, S, Kumamoto, K, Harris, AL, Modrich, P, and Huang, LE. "HIF-1alpha induces genetic instability by transcriptionally downregulating MutSalpha expression." Mol Cell 17.6 (March 18, 2005): 793-803.
PMID
15780936
Source
pubmed
Published In
Molecular Cell
Volume
17
Issue
6
Publish Date
2005
Start Page
793
End Page
803
DOI
10.1016/j.molcel.2005.02.015

Methyl-directed repair of DNA base-pair mismatches in vitro. 1983.

Authors
Lu, A-L; Clark, S; Modrich, P
MLA Citation
Lu, A-L, Clark, S, and Modrich, P. "Methyl-directed repair of DNA base-pair mismatches in vitro. 1983." DNA Repair (Amst) 4.1 (January 2, 2005): 134-138.
PMID
15533843
Source
pubmed
Published In
DNA Repair
Volume
4
Issue
1
Publish Date
2005
Start Page
134
End Page
138
DOI
10.1016/j.dnarep.2004.07.003

Human mismatch repair: Reconstitution of a nick-directed bidirectional reaction

Bidirectional mismatch repair directed by a strand break located 3′ or 5′ to the mispair has been reconstituted using seven purified human activities: MutSα, MutLα, EXOI, replication protein A (RPA), proliferating cell nuclear antigen (PCNA), replication factor C (RFC) and DNA polymerase δ. In addition to DNA polymerase δ, PCNA, RFC, and RPA, 5′-directed repair depends on MutSα and EXOI, whereas 3′-directed mismatch correction also requires MutLα. The repair reaction displays specificity for DNA polymerase δ, an effect that presumably reflects interactions with other repair activities. Because previous studies have suggested potential involvement of the editing function of a replicative polymerase in mismatch-provoked excision, we have evaluated possible participation of DNA polymerase δ in the excision step of repair. RFC and PCNA dramatically activate polymerase δ-mediated hydrolysis of a primer-template. Nevertheless, the contribution of the polymerase to mismatch-provoked excision is very limited, both in the purified system and in HeLa extracts, as judged by in vitro assay using nicked circular heteroplex DNAs. Thus, excision and repair in the purified system containing polymerase δ are reduced 10-fold upon omission of EXOI or by substitution of a catalytically dead form of the exonuclease. Furthermore, aphidicolin inhibits both 3′- and 5′-directed excision in HeLa nuclear extracts by only 20-30%. Although this modest inhibition could be because of nonspecific effects, it may indicate limited dependence of bidirectional excision on an aphidicolin-sensitive DNA polymerase. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

Authors
Constantin, N; Dzantiev, L; Kadyrov, FA; Modrich, P
MLA Citation
Constantin, N, Dzantiev, L, Kadyrov, FA, and Modrich, P. "Human mismatch repair: Reconstitution of a nick-directed bidirectional reaction." Journal of Biological Chemistry 280.48 (2005): 39752-39761.
PMID
16188885
Source
scival
Published In
The Journal of biological chemistry
Volume
280
Issue
48
Publish Date
2005
Start Page
39752
End Page
39761
DOI
10.1074/jbc.M509701200

Early thinking on the nature of mismatch repair

Replication error correction by mismatch repair relies on the strand-directed nature of the reaction. Ephrussi-Taylor and Gray attributed the marker-dependent recovery of pneumoccocal transformants to a mismatch-provoked repair reaction characterized by a strong strand bias and long excision tracts. This idea was extended by Wagner and Meselson in the context of a study on mismatch repair of multiply marked lambda heteroduplexes, with the explicit proposal that strand-directed mismatch repair could provide a mechanism for correcting DNA biosynthetic errors. © 2004 Elsevier B.V. All rights reserved.

Authors
Modrich, P
MLA Citation
Modrich, P. "Early thinking on the nature of mismatch repair." DNA Repair 4.1 (2005): 103-131.
Source
scival
Published In
DNA Repair
Volume
4
Issue
1
Publish Date
2005
Start Page
103
End Page
131
DOI
10.1016/j.dnarep.2004.07.002

Brain tumor cell lines resistant to O6-benzylguanine/1,3-bis(2-chloroethyl)-1-nitrosourea chemotherapy have O6-alkylguanine-DNA alkyltransferase mutations.

The chemotherapeutic activity of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU or carmustine) may be improved by the addition of O6-benzylguanine (O6-BG). The reaction of O6-BG with O6-alkylguanine-DNA alkyltransferase (AGT) prevents the repair of O6-chloroethyl lesions caused by BCNU. In clinics, the combination of O6-BG and BCNU is now being tested for the treatment of brain tumors. However, the effectiveness of this drug regimen may be limited by drug resistance acquired during treatment. To understand the possible mechanisms of resistance of brain tumor cells to the O6-BG/BCNU combination, we generated medulloblastoma cell lines (D283 MED, D341 MED, and Daoy) resistant to the combination of O6-BG and BCNU [O6-BG/BCNU resistant (OBR)]. DNA sequencing showed that all of the parent cell lines express wild-type AGTs, whereas every OBR cell line exhibited mutations that potentially affected the binding of O6-BG to the protein as evidenced previously by in vitro mutagenesis and structural studies of AGT. The D283 MED (OBR), Daoy (OBR), and D341 MED (OBR) cell lines expressed G156C, Y114F, and K165T AGT mutations, respectively. We reported previously that rhabdomyosarcoma TE-671 (OBR) also expresses a G156C mutation. These data suggest that the clonal selection of AGT mutants during treatment with O6-BG plus an alkylator may produce resistance to this intervention in clinical settings.

Authors
Bacolod, MD; Johnson, SP; Pegg, AE; Dolan, ME; Moschel, RC; Bullock, NS; Fang, Q; Colvin, OM; Modrich, P; Bigner, DD; Friedman, HS
MLA Citation
Bacolod, MD, Johnson, SP, Pegg, AE, Dolan, ME, Moschel, RC, Bullock, NS, Fang, Q, Colvin, OM, Modrich, P, Bigner, DD, and Friedman, HS. "Brain tumor cell lines resistant to O6-benzylguanine/1,3-bis(2-chloroethyl)-1-nitrosourea chemotherapy have O6-alkylguanine-DNA alkyltransferase mutations." Mol Cancer Ther 3.9 (September 2004): 1127-1135.
PMID
15367707
Source
pubmed
Published In
Molecular cancer therapeutics
Volume
3
Issue
9
Publish Date
2004
Start Page
1127
End Page
1135

Differential specificities and simultaneous occupancy of human MutSalpha nucleotide binding sites.

We have examined the permissible nucleotide occupancy states of human MutSalpha. The MSH2.MSH6 heterodimer binds 1 mol of ADP and 1 mol of adenosine 5'-O-(thiotriphosphate) (ATPgammaS), with a K(d) for each nucleotide of about 1 microm. Anisotropy measurements using BODIPY TR and BODIPY FL fluorescent derivatives of ADP and 5'-adenylyl-beta,gamma-imidodiphosphate (AMPPNP) also indicate an interaction stoichiometry of 1 mol of ADP and 1 mol of triphosphate analogue per MutSalpha heterodimer. Di- and triphosphate sites can be simultaneously occupied as judged by sequential filling of the two binding site classes with differentially radiolabeled ADP and ATPgammaS and by fluorescence resonance energy transfer between BODIPY TR- and BODIPY FL-labeled ADP and AMPPNP. ATP hydrolysis by MutSalpha is accompanied by a pre-steady-state burst of ADP formation, and analysis of MutSalpha-bound nucleotide during the first turnover has demonstrated the presence of both ADP and ATP. Simultaneous presence of ADP and a nonhydrolyzable ATP analogue modulates MutSalpha.heteroduplex interaction in a manner that is distinct from that observed in the presence of ADP or nonhydrolyzable triphosphate alone, and it is unlikely that this effect is due to the presence of a mixed population of binary complexes between MutSalpha and ADP or a triphosphate analogue. These findings imply that MutSalpha has two nucleotide binding sites with differential specificities for ADP and ATP and suggest that the ADP.MutSalpha.ATP ternary complex has an important role in mismatch repair.

Authors
Martik, D; Baitinger, C; Modrich, P
MLA Citation
Martik, D, Baitinger, C, and Modrich, P. "Differential specificities and simultaneous occupancy of human MutSalpha nucleotide binding sites." J Biol Chem 279.27 (July 2, 2004): 28402-28410.
PMID
15105434
Source
pubmed
Published In
The Journal of biological chemistry
Volume
279
Issue
27
Publish Date
2004
Start Page
28402
End Page
28410
DOI
10.1074/jbc.M312108200

A defined human system that supports bidirectional mismatch-provoked excision.

Mismatch-provoked excision directed by a strand break located 3' or 5' to the mispair has been reconstituted using purified human proteins. While MutSalpha, EXOI, and RPA are sufficient to support hydrolysis directed by a 5' strand break, 3' directed excision also requires MutLalpha, PCNA, and RFC. EXOI interacts with PCNA. RFC and PCNA suppress EXOI-mediated 5' to 3' hydrolysis when the nick that directs excision is located 3' to the mispair and activate 3' to 5' excision, which is dependent on loaded PCNA and apparently mediated by a cryptic EXOI 3' to 5' hydrolytic function. By contrast, RFC and PCNA have only a limited effect on 5' to 3' excision directed by a 5' strand break.

Authors
Dzantiev, L; Constantin, N; Genschel, J; Iyer, RR; Burgers, PM; Modrich, P
MLA Citation
Dzantiev, L, Constantin, N, Genschel, J, Iyer, RR, Burgers, PM, and Modrich, P. "A defined human system that supports bidirectional mismatch-provoked excision." Mol Cell 15.1 (July 2, 2004): 31-41.
PMID
15225546
Source
pubmed
Published In
Molecular Cell
Volume
15
Issue
1
Publish Date
2004
Start Page
31
End Page
41
DOI
10.1016/j.molcel.2004.06.016

Targeting wide-range oncogenic transformation via PU24FCl, a specific inhibitor of tumor Hsp90.

Agents that inhibit Hsp90 function hold significant promise in cancer therapy. Here we present PU24FCl, a representative of the first class of designed Hsp90 inhibitors. By specifically and potently inhibiting tumor Hsp90, PU24FCl exhibits wide-ranging anti-cancer activities that occur at similar doses in all tested tumor types. Normal cells are 10- to 50-fold more resistant to these effects. Its Hsp90 inhibition results in multiple anti-tumor-specific effects, such as degradation of Hsp90-client proteins involved in cell growth, survival, and specific transformation, inhibition of cancer cell growth, delay of cell cycle progression, induction of morphological and functional changes, and apoptosis. In concordance with its higher affinity for tumor Hsp90, in vivo PU24FCl accumulates in tumors while being rapidly cleared from normal tissue. Concentrations achieved in vivo in tumors lead to single-agent anti-tumor activity at non-toxic doses.

Authors
Vilenchik, M; Solit, D; Basso, A; Huezo, H; Lucas, B; He, H; Rosen, N; Spampinato, C; Modrich, P; Chiosis, G
MLA Citation
Vilenchik, M, Solit, D, Basso, A, Huezo, H, Lucas, B, He, H, Rosen, N, Spampinato, C, Modrich, P, and Chiosis, G. "Targeting wide-range oncogenic transformation via PU24FCl, a specific inhibitor of tumor Hsp90." Chem Biol 11.6 (June 2004): 787-797.
PMID
15217612
Source
pubmed
Published In
Chemistry & Biology
Volume
11
Issue
6
Publish Date
2004
Start Page
787
End Page
797
DOI
10.1016/j.chembiol.2004.04.008

The mismatch DNA repair heterodimer, hMSH2/6, regulates BLM helicase.

The human MSH2/6 complex is essential for mismatch recognition during the repair of replication errors. Although mismatch repair components have been implicated in DNA homologous recombination repair, the exact function of hMSH2/6 in this pathway is unclear. Here, we show that the recombinant hMSH2/6 protein complex stimulated the ability of the Bloom's syndrome gene product, BLM, to process Holliday junctions in vitro, an activity that could also be regulated by p53. Consistent with these observations, hMSH6 colocalized with BLM and phospho-ser15-p53 in hydroxyurea-induced RAD51 nuclear foci that may correspond to the sites of presumed stalled DNA replication forks and more likely the resultant DNA double-stranded breaks. In addition, we show that hMSH2 and hMSH6 coimmunoprecipitated with BLM, p53, and RAD51. Both the number of RAD51 foci and the amount of the BLM-p53-RAD51 complex are increased in hMSH2- or hMSH6-deficient cells. These data suggest that hMSH2/6 formed a complex with BLM-p53-RAD51 in response to the damaged DNA forks during double-stranded break repair.

Authors
Yang, Q; Zhang, R; Wang, XW; Linke, SP; Sengupta, S; Hickson, ID; Pedrazzi, G; Perrera, C; Stagljar, I; Littman, SJ; Modrich, P; Harris, CC
MLA Citation
Yang, Q, Zhang, R, Wang, XW, Linke, SP, Sengupta, S, Hickson, ID, Pedrazzi, G, Perrera, C, Stagljar, I, Littman, SJ, Modrich, P, and Harris, CC. "The mismatch DNA repair heterodimer, hMSH2/6, regulates BLM helicase." Oncogene 23.21 (May 6, 2004): 3749-3756.
PMID
15064730
Source
pubmed
Published In
Oncogene: Including Oncogene Reviews
Volume
23
Issue
21
Publish Date
2004
Start Page
3749
End Page
3756
DOI
10.1038/sj.onc.1207462

Hydrolytically deficient MutS E694A is defective in the MutL-dependent activation of MutH and in the mismatch-dependent assembly of the MutS.MutL.heteroduplex complex.

The roles of ATP binding and hydrolysis by MutS in mismatch repair are poorly understood. MutS E694A, in which Glu-694 of the Walker B motif is substituted with alanine, is defective in hydrolysis of bound ATP and has been reported to support MutL-dependent activation of the MutH d(GATC) endonuclease in a trans DNA activation assay (Junop, M. S., Obmolova, G., Rausch, K., Hsieh, P., and Yang, W. (2001) Mol. Cell 7, 1-12). Because the MutH trans activation assay used in these previous studies was characterized by high background and low efficiency, we have re-evaluated the activities of MutS E694A. In contrast to native MutS, which can be isolated in a nucleotide-free form, purified MutS E694A contains 1.0 mol of bound ATP per dimer equivalent, and substoichiometric levels of bound ADP (0.08-0.58 mol/dimer), consistent with the suggestion that the ADP.MutS.ATP complex comprises a significant fraction of the protein in solution (Bjornson, K. P. and Modrich, P. (2003) J. Biol. Chem. 278, 18557-18562). In the presence of Mg2+, endogenous ATP is hydrolyzed with a rate constant of 0.12 min-1 at 30 degrees C, and hydrolysis yields a protein that displays increased specificity for heteroduplex DNA. As observed with wild type MutS, ATP can promote release of MutS E694A from a mismatch. However, the mutant protein is defective in the methyl-directed, mismatch- and MutL-dependent cis activation of MutH endonuclease on a 6.4-kilobase pair heteroduplex, displaying only 1 to 2% of the activity of wild type MutS. The mutant protein also fails to support normal assembly of the MutS.MutL.DNA ternary complex. Although a putative ternary complex can be observed in the presence of MutS E694A, assembly of this structure displays little if any dependence on a mismatched base pair.

Authors
Baitinger, C; Burdett, V; Modrich, P
MLA Citation
Baitinger, C, Burdett, V, and Modrich, P. "Hydrolytically deficient MutS E694A is defective in the MutL-dependent activation of MutH and in the mismatch-dependent assembly of the MutS.MutL.heteroduplex complex." J Biol Chem 278.49 (December 5, 2003): 49505-49511.
PMID
14506224
Source
pubmed
Published In
The Journal of biological chemistry
Volume
278
Issue
49
Publish Date
2003
Start Page
49505
End Page
49511
DOI
10.1074/jbc.M308738200

Mechanism of 5'-directed excision in human mismatch repair.

We have developed a purified system that supports mismatch-dependent 5'-->3' excision. In the presence of RPA, ATP, and a mismatch, MutSalpha activates 5'-->3' excision by EXOI, and excision terminates after removal of the mispair. MutSalpha confers high processivity on EXOI, and termination is due to RPA-dependent displacement of this processive complex from the helix and a weak ability of EXOI to reload at the RPA-bound gap in the product, as well as MutSalpha- and MutLalpha-dependent suppression of EXOI activity in the absence of a mismatch cofactor. As observed in the purified system, excision directed by a 5' strand break in HeLa nuclear extract can proceed in the absence of MutLalpha or PCNA, although 3' excision in the extract system requires both proteins.

Authors
Genschel, J; Modrich, P
MLA Citation
Genschel, J, and Modrich, P. "Mechanism of 5'-directed excision in human mismatch repair." Mol Cell 12.5 (November 2003): 1077-1086.
PMID
14636568
Source
pubmed
Published In
Molecular Cell
Volume
12
Issue
5
Publish Date
2003
Start Page
1077
End Page
1086

Assembly and molecular activities of the MutS tetramer.

Analytical equilibrium ultracentrifugation indicates that Escherichia coli MutS exists as an equilibrating mixture of dimers and tetramers. The association constant for the dimer-to-tetramer transition is 2.1 x 10(7) M-1, indicating that the protein would consist of both dimers and tetramers at physiological concentrations. The carboxyl terminus of MutS is required for tetramer assembly because a previously described 53-amino acid carboxyl-terminal truncation (MutS800) forms a limiting species of a dimer (Obmolova, G., Ban, C., Hsieh, P., and Yang, W. (2000) Nature 407, 703-710; Lamers, M. H., Perrakis, A., Enzlin, J. H., Winterwerp, H. H., de Wind, N., and Sixma, T. K. (2000) Nature 407, 711-717). MutS800 binds a 20-base pair heteroduplex an order of magnitude more weakly than full-length MutS, and at saturating protein concentrations, the heteroduplex-bound mass observed with MutS800 is only half that observed with the full length protein, indicating that the subunit copy number of heteroduplex-bound MutS is twice that of MutS800. Analytical equilibrium ultracentrifugation using a fluorescein-tagged 20-base pair heteroduplex demonstrated that native MutS forms a tetramer on this single site-sized heteroduplex DNA. Equilibrium fluorescence experiments indicated that dimer-to-tetramer assembly promotes mismatch binding by MutS and that the tetramer can bind only a single heteroduplex molecule, implying nonequivalence of the two dimers within the tetramer. Compared with native MutS, the ability of MutS800 to promote MutL-dependent activation of MutH is substantially reduced.

Authors
Bjornson, KP; Blackwell, LJ; Sage, H; Baitinger, C; Allen, D; Modrich, P
MLA Citation
Bjornson, KP, Blackwell, LJ, Sage, H, Baitinger, C, Allen, D, and Modrich, P. "Assembly and molecular activities of the MutS tetramer." J Biol Chem 278.36 (September 5, 2003): 34667-34673.
PMID
12829697
Source
pubmed
Published In
The Journal of biological chemistry
Volume
278
Issue
36
Publish Date
2003
Start Page
34667
End Page
34673
DOI
10.1074/jbc.M305513200

Differential and simultaneous adenosine di- and triphosphate binding by MutS.

The roles of ATP binding and hydrolysis in the function of MutS in mismatch repair are poorly understood. As one means of addressing this question, we have determined the affinities and number of adenosine di- and triphosphate binding sites within MutS. Nitrocellulose filter binding assay and equilibrium fluorescence anisotropy measurements have demonstrated that MutS has one high affinity binding site for ADP and one high affinity site for nonhydrolyzable ATP analogues per dimer equivalent. Low concentrations of 5'-adenylylimidodiphosphate (AMPPNP) promote ADP binding and a large excess of AMPPNP is required to displace ADP from the protein. Fluorescence energy transfer and filter binding assays indicate that ADP and nonhydrolyzable ATP analogues can bind simultaneously to adjacent subunits within the MutS oligomer with affinities in the low micromolar range. These findings suggest that the protein exists primarily as the ATP.MutS.ADP ternary complex in solution and that this may be the form of the protein that is involved in DNA encounters in vivo.

Authors
Bjornson, KP; Modrich, P
MLA Citation
Bjornson, KP, and Modrich, P. "Differential and simultaneous adenosine di- and triphosphate binding by MutS." J Biol Chem 278.20 (May 16, 2003): 18557-18562.
PMID
12624105
Source
pubmed
Published In
The Journal of biological chemistry
Volume
278
Issue
20
Publish Date
2003
Start Page
18557
End Page
18562
DOI
10.1074/jbc.M301101200

Mechanisms of resistance to 1,3-bis(2-chloroethyl)-1-nitrosourea in human medulloblastoma and rhabdomyosarcoma.

Medulloblastoma (D-341 MED) and rhabdomyosarcoma (TE-671) cell lines, which are resistant to either 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or the combination of BCNU and O6-benzylguanine (O6-BG), were generated by serial escalation of BCNU. The activities of O6-alkylguanine-DNA alkyltransferase (AGT), glutathione-S-transferase (GST), and total glutathione (GSH) of the parental, BCNU-resistant (BR), and BCNU + O6-BG-resistant (OBR) cells were measured. No significant differences in GST activity or total GSH were seen between the parental, BR, and OBR cells of both TE-671 and D-341 MED. The AGT activities of D-341 MED (BR) and TE-671 (BR) were twice those of D-341 MED and TE-671, respectively, confirming the importance of this enzyme for BCNU resistance. The D-341 MED (OBR) cells did not exhibit any AGT activity, suggesting that another mechanism must play a role in the drug resistance. Fewer DNA interstrand cross-links (ICLs) were observed in D-341 MED (OBR) than in D-341 MED after 8 h BCNU (100-400 microM) treatment. However, the amounts of DNA ICLs observed in D-341 MED and D-341 MED (OBR) were stable after 24 h. Microarray analysis showed the increased expressions of several metallothionein genes and down-regulation of several proapoptotic genes. The AGT activity of TE-671 (OBR) was 223 fmol/mg when the cells were grown in 10 microM O6-BG and decreased to about half this value when the O6-BG concentration was increased 60 microM. The AGT cDNA of TE-671 (OBR) cells was cloned and found to contain a G-to-T transversion at codon 156, resulting in conversion of glycine to cysteine (G156C). In vitro mutagenesis has shown that the G156C AGT mutant is resistant to inactivation by O6-BG. Thus, the selection of a mutant AGT with decreased sensitivity to O6-BG is a significant contributing factor to BCNU + O6-BG resistance.

Authors
Bacolod, MD; Johnson, SP; Ali-Osman, F; Modrich, P; Bullock, NS; Colvin, OM; Bigner, DD; Friedman, HS
MLA Citation
Bacolod, MD, Johnson, SP, Ali-Osman, F, Modrich, P, Bullock, NS, Colvin, OM, Bigner, DD, and Friedman, HS. "Mechanisms of resistance to 1,3-bis(2-chloroethyl)-1-nitrosourea in human medulloblastoma and rhabdomyosarcoma." Mol Cancer Ther 1.9 (July 2002): 727-736.
PMID
12479369
Source
pubmed
Published In
Molecular cancer therapeutics
Volume
1
Issue
9
Publish Date
2002
Start Page
727
End Page
736

Human exonuclease I is required for 5' and 3' mismatch repair.

We have partially purified a human activity that restores mismatch-dependent, bi-directional excision to a human nuclear extract fraction depleted for one or more mismatch repair excision activities. Human EXOI co-purifies with the excision activity, and the purified activity can be replaced by near homogeneous recombinant hEXOI. Despite the reported 5' to 3' hydrolytic polarity of this activity, hEXOI participates in mismatch-provoked excision directed by a strand break located either 5' or 3' to the mispair. When the strand break that directs repair is located 3' to the mispair, hEXOI- and mismatch-dependent gap formation in excision-depleted extracts requires both hMutSalpha and hMutLalpha. However, excision directed by a 5' strand break requires hMutSalpha but can occur in absence of hMutLalpha. In systems comprised of pure components, the 5' to 3' hydrolytic activity of hEXOI is activated by hMutSalpha in a mismatch-dependent manner. These observations indicate a hydrolytic function for hEXOI in 5'-heteroduplex correction. The involvement of hEXOI in 3'-heteroduplex repair suggests that it has a regulatory/structural role in assembly of the 3'-excision complex or that the protein possesses a cryptic 3' to 5' hydrolytic activity.

Authors
Genschel, J; Bazemore, LR; Modrich, P
MLA Citation
Genschel, J, Bazemore, LR, and Modrich, P. "Human exonuclease I is required for 5' and 3' mismatch repair." J Biol Chem 277.15 (April 12, 2002): 13302-13311.
PMID
11809771
Source
pubmed
Published In
The Journal of biological chemistry
Volume
277
Issue
15
Publish Date
2002
Start Page
13302
End Page
13311
DOI
10.1074/jbc.M111854200

High rate of CAD gene amplification in human cells deficient in MLH1 or MSH6

MutS and MutL homologs have been implicated in multiple genetic stabilization pathways. The activities participate in the correction of DNA biosynthetic errors, are involved in cellular responses to certain types of DNA damage, and serve to ensure the fidelity of genetic recombination. We show here that the rate of CAD (carbamyl-P synthetase/aspartate transcarbamylase/dihydroorotase) gene amplification is elevated 50- to 100-fold in human cell lines deficient in MLH1 or MSH6, as compared with mismatch repair-proficient control cells. Fluorescence in situ hybridization indicates that these amplification events are the probable consequence of unequal sister chromatid exchanges involving chromosome 2, as well as translocation events involving other chromosomes. These results implicate MutSα and MutLα in the suppression of gene amplification and suggest that defects in this genetic stabilization function may contribute to the cancer predisposition associated with mismatch repair deficiency.

Authors
Chen, S; Bigner, SH; Modrich, P
MLA Citation
Chen, S, Bigner, SH, and Modrich, P. "High rate of CAD gene amplification in human cells deficient in MLH1 or MSH6." Proceedings of the National Academy of Sciences of the United States of America 98.24 (2001): 13802-13807.
PMID
11717437
Source
scival
Published In
Proceedings of the National Academy of Sciences of USA
Volume
98
Issue
24
Publish Date
2001
Start Page
13802
End Page
13807
DOI
10.1073/pnas.241508098

DNA Chain Length Dependence of Formation and Dynamics of hMutSα·hMutLα·Heteroduplex Complexes

Formation of a ternary complex between human MutSα, MutLα, and heteroduplex DNA has been demonstrated by surface plasmon resonance spectroscopy and electrophoretic gel shift methods. Formation of the hMutLα·hMutSα·heteroduplex complex requires a mismatch and ATP hydrolysis, and depends on DNA chain length. Ternary complex formation was supported by a 200-base pair G-T heteroduplex, a 100-base pair substrate was somewhat less effective, and a 41-base pair heteroduplex was inactive. As judged by surface plasmon resonance spectroscopy, ternary complexes produced with the 200-base pair G-T DNA contained ∼0.8 mol of hMutLα/mol of heteroduplex-bound hMutSα. Although the steady-state levels of the hMutLα·hMutSα· heteroduplex were substantial, this complex was found to turn over, as judged by surface plasmon resonance spectroscopy and electrophoretic gel shift analysis. With the former method, the majority of the complexes dissociated rapidly upon termination of protein flow, and dissociation occurred in the latter case upon challenge with competitor DNA. However, ternary complex dissociation as monitored by gel shift assay was prevented if both ends of the heteroduplex were physically blocked with streptavidin·biotin complexes. This observation suggests that, like hMutSα, the hMutLα·hMutSα complex can migrate along the helix contour to dissociate at DNA ends.

Authors
Blackwell, LJ; Wang, S; Modrich, P
MLA Citation
Blackwell, LJ, Wang, S, and Modrich, P. "DNA Chain Length Dependence of Formation and Dynamics of hMutSα·hMutLα·Heteroduplex Complexes." Journal of Biological Chemistry 276.35 (2001): 33233-33240.
PMID
11441019
Source
scival
Published In
Journal of Biological Chemistry
Volume
276
Issue
35
Publish Date
2001
Start Page
33233
End Page
33240
DOI
10.1074/jbc.M105076200

Distinct MutS DNA-binding Modes That Are Differentially Modulated by ATP Binding and Hydrolysis

The role of MutS ATPase in mismatch repair is controversial. To clarify further the function of this activity, we have examined adenine nucleotide effects on interactions of Escherichia coli MutS with homoduplex and heteroduplex DNAs. In contrast to previous results with human MutSα, we find that a physical block at one end of a linear heteroduplex is sufficient to support stable MutS complex formation in the presence of ATP·Mg 2+. Surface plasmon resonance analysis at low ionic strength indicates that the lifetime of MutS complexes with heteroduplex DNA depends on the nature of the nucleotide present when MutS binds. Whereas complexes prepared in the absence of nucleotide or in the presence of ADP undergo rapid dissociation upon challenge with ATP·Mg2+, complexes produced in the presence of ATP·Mg2+, adenosine 5′-(β ,γ-imino)triphosphate (AMPPNP)·Mg2+, or ATP (no Mg 2+) are resistant to dissociation upon ATP challenge. AMPPNP·Mg2+ and ATP (no Mg2+) reduce MutS affinity for heteroduplex but have little effect on homoduplex affinity, resulting in abolition of specificity for mispaired DNA at physiological salt concentrations. Conversely, the highest mismatch specificity is observed in the absence of nucleotide or in the presence of ADP. ADP has only a limited effect on heteroduplex affinity but reduces MutS affinity for homoduplex DNA.

Authors
Blackwell, LJ; Bjornson, KP; Allen, DJ; Modrich, P
MLA Citation
Blackwell, LJ, Bjornson, KP, Allen, DJ, and Modrich, P. "Distinct MutS DNA-binding Modes That Are Differentially Modulated by ATP Binding and Hydrolysis." Journal of Biological Chemistry 276.36 (2001): 34339-34347.
PMID
11454861
Source
scival
Published In
Journal of Biological Chemistry
Volume
276
Issue
36
Publish Date
2001
Start Page
34339
End Page
34347
DOI
10.1074/jbc.M104256200

In vivo requirement for RecJ, ExoVII, ExoI, and ExoX in methyl-directed mismatch repair

Biochemical studies with model DNA heteroduplexes have implicated Red exonuclease, exonuclease VII, exonuclease I, and exonuclease X in Escherichia coli methyl-directed mismatch correction. However, strains deficient in the four exonucleases display only a modest increase in mutation rate, raising questions concerning involvement of these activities in mismatch repair in vivo. The quadruple mutant deficient in the four exonucleases, as well as the triple mutant deficient in RecJ exonuclease, exonuclease VII, and exonuclease I, grow poorly in the presence of the base analogue 2-aminopurine, and exposure to the base analogue results in filament formation, indicative of induction of SOS DNA damage response. The growth defect and filamentation phenotypes associated with 2-aminopurine exposure are effectively suppressed by null mutations in mutH, mutL, mutS, or uvrD/mutU, which encode activities that act upstream of the four exonucleases in the mechanism for the methyl-directed reaction that has been proposed based on in vitro studies. The quadruple exonuclease mutant is also cold-sensitive, having a severe growth defect at 30°C. This phenotype is suppressed by a uvrD/mutU defect, and partially suppressed by mutH, mutL, or muts mutations. These observations confirm involvement of the four exonucleases in methyl-directed mismatch repair in vivo and suggest that the low mutability of exonuclease-deficient strains is a consequence of under recovery of mutants due to a reduction in viability and/or chromosome loss associated with activation of the mismatch repair system in the absence of Red exonuclease, exonuclease VII, exonuclease I, and exonuclease X.

Authors
Burdett, V; Baitinger, C; Viswanathan, M; Lovett, ST; Modrich, P
MLA Citation
Burdett, V, Baitinger, C, Viswanathan, M, Lovett, ST, and Modrich, P. "In vivo requirement for RecJ, ExoVII, ExoI, and ExoX in methyl-directed mismatch repair." Proceedings of the National Academy of Sciences of the United States of America 98.12 (2001): 6765-6770.
PMID
11381137
Source
scival
Published In
Proceedings of the National Academy of Sciences of USA
Volume
98
Issue
12
Publish Date
2001
Start Page
6765
End Page
6770
DOI
10.1073/pnas.121183298

Redundant Exonuclease Involvement in Escherichia coli Methyl-directed Mismatch Repair

Previous biochemical analysis of Escherichia coli methyl-directed mismatch repair implicates three redundant single-strand DNA-specific exonucleases (RecJ, ExoI, and ExoVII) and at least one additional unknown exonuclease in the excision reaction (Cooper, D. L., Lahue, R. S., and Modrich, P. (1993) J. Biol. Chem 268, 11823-11829). We show here that ExoX also participates in methyl-directed mismatch repair. Analysis of the reaction with crude extracts and purified components demonstrated that ExoX can mediate repair directed from a strand signal 3′ of a mismatch. Whereas extracts of all possible single, double, and triple exonuclease mutants displayed significant residual mismatch repair, extracts deficient in RecJ, ExoI, ExoVII, and ExoX exonucleases were devoid of normal repair activity. The RecJ- ExoVII- ExoI- ExoX- strain displayed a 7-fold increase in mutation rate, a significant increase, but less than that observed for other blocks of the mismatch repair pathway. This elevation is epistatic to deficiency for MutS, suggesting an effect via the mismatch repair pathway. Our other work (Burdett, V., Baitinger, C., Viswanathan, M., Lovett, S. T., and Modrich, P. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 6765-6770) suggests that mutants are under-recovered in the exonuclease-deficient strain due to loss of viability that is triggered by mismatched base pairs in this genetic background. The availability of any one exonuclease is enough to support full mismatch correction, as evident from the normal mutation rates of all triple mutants. Because three of these exonucleases possess a strict polarity of digestion, this suggests that mismatch repair can occur exclusively from a 3′ or a 5′ direction to the mismatch, if necessary.

Authors
Viswanathan, M; Burdett, V; Baitinger, C; Modrich, P; Lovett, ST
MLA Citation
Viswanathan, M, Burdett, V, Baitinger, C, Modrich, P, and Lovett, ST. "Redundant Exonuclease Involvement in Escherichia coli Methyl-directed Mismatch Repair." Journal of Biological Chemistry 276.33 (2001): 31053-31058.
PMID
11418610
Source
scival
Published In
Journal of Biological Chemistry
Volume
276
Issue
33
Publish Date
2001
Start Page
31053
End Page
31058
DOI
10.1074/jbc.M105481200

The MutL ATPase is required for mismatch repair

Members of the MutL family contain a novel nucleotide binding motif near their amino terminus, and the Escherichia coli protein has been found to be a weak ATPase (Ban, C., and Yang, W. (1998) Cell 95, 541-552). Genetic analysis has indicated that substitution of Lys for Glu-32 within this motif of bacterial MutL results in a strong dominant negative phenotype (Aronshtam, A., and Marinus, M. G. (1996) Nucleic Acids Res. 24, 2498-2504). By in vitro comparison of MutL-E32K with the wild type protein, we show the mutant protein to be defective in DNA-activated ATP hydrolysis, as well as MutS- and MutL-dependent activation of the MutH d(GATC) endonuclease and the mismatch repair excision system. MutL-E32K also acts in dominant negative manner in the presence of wild type MutL in vitro, inhibiting the overall mismatch repair reaction, as well as MutH activation. As judged by protein affinity chromatography, MutL and MutL-E32K both support formation of ternary complexes that also contain MutS and MutH or MutS and DNA helicase II. These findings imply that the MutL nucleotide binding center is required for mismatch repair and suggest that the dominant negative behavior of the MutL- E32K mutation is due to the formation of dead-end complexes in which the MutL-E32K protein is unable to transduce a signal from MutS that otherwise results in mismatch-dependent activation of the MutH d(GATC) endonuclease or the unwinding activity of helicase II.

Authors
Spampinato, C; Modrich, P
MLA Citation
Spampinato, C, and Modrich, P. "The MutL ATPase is required for mismatch repair." Journal of Biological Chemistry 275.13 (2000): 9863-9869.
PMID
10734142
Source
scival
Published In
Journal of Biological Chemistry
Volume
275
Issue
13
Publish Date
2000
Start Page
9863
End Page
9869
DOI
10.1074/jbc.275.13.9863

Somatic mutation of hPMS2 as a possible cause of sporadic human colon cancer with microsatellite instability

Inactivation of DNA-mismatch repair underlies the genesis of microsatellite unstable (MSI) colon cancers. hPMS2 is one of several genes encoding components of the DNA-mismatch repair complex, and germline hPMS2 mutations have been found in a few kindreds with hereditary nonpolyposis colorectal carcinoma (HNPCC), in whom hereditary MSI colon cancers develop. However, mice bearing null hPMS2 genes do not develop colon cancers and hPMS2 mutations in sporadic human colon cancers have not been described. Here we report that in Vaco481 colon cancer the hPMS2 gene is inactivated by somatic mutations of both hPMS2 alleles. The cell line derived from this tumor is functionally deficient in DNA mismatch repair. This deficiency can be biochemically complemented by addition of a purified hMLH1-hPMS2 (hMutLα) complex. The hPMS2 deficient Vaco481 cancer cell line demonstrates microsatellite instability, an elevated HPRT gene mutation rate, and resistance to the cytotoxicity of the alkylator MNNG. We conclude that somatic inactivation of hPMS2 can play a role in development of sporadic MSI colon cancer expressing the full range of cancer phenotypes associated with inactivation of the mismatch repair system.

Authors
Ma, A-H; Xia, L; Littman, SJ; Swinler, S; Lader, G; Polinkovsky, A; Olechnowicz, J; Kasturi, L; Lutterbaugh, J; Modrich, P; Veigl, ML; Markowitz, SD; Sedwick, WD
MLA Citation
Ma, A-H, Xia, L, Littman, SJ, Swinler, S, Lader, G, Polinkovsky, A, Olechnowicz, J, Kasturi, L, Lutterbaugh, J, Modrich, P, Veigl, ML, Markowitz, SD, and Sedwick, WD. "Somatic mutation of hPMS2 as a possible cause of sporadic human colon cancer with microsatellite instability." Oncogene 19.18 (2000): 2249-2256.
PMID
10822375
Source
scival
Published In
Oncogene
Volume
19
Issue
18
Publish Date
2000
Start Page
2249
End Page
2256

Modulation of MutS ATP hydrolysis by DNA cofactors

Escherichia coli MutS protein, which is required for mismatch repair, has a slow ATPase activity that obeys Michalelis-Menten kinetics. At 37 °C, the steady-state turnover rate for ATP hydrolysis is 1.0 ± 0.3 min-1 per monomer equivalent with a K(m) of 33 ± 6 μM. Hydrolysis is competitively inhibited by the ATP analogues AMPPNP and ATPγS, with K(i) values of 4 μM in both cases, and by ADP with a K(i) of 40 μM. The rate of ATP hydrolysis is stimulated 2-5-fold by short hetero- and homoduplex DNAs. The concentration of DNA cofactor that yields half-maximal stimulation is lowest for oligodeoxynucleotide duplexes that contain a mismatched base pair. Pre- steady-state chemical quench analysis has demonstrated a substoichiometric initial burst of ADP formation by free MutS that is governed by a rate constant of 78 min-1, indicating that the rate-limiting step for the steady-state reaction occurs after hydrolysis. Prebinding of MutS to homoduplex DNA does not alter the burst kinetics or amplitude but only increases the steady-state rate. In contrast, binding of the protein to heteroduplex DNA abolishes the burst of ADP formation, indicating that the rate-limiting step now occurs before hydrolysis. Gel filtration analysis indicates that the MutS dimer assembles into higher order oligomers in a concentration-dependent manner, and that ATP binding shifts this equilibrium to favor assembly. These results, together with kinetic findings, indicate nonequivalence of subunits within a MutS oligomer with respect to ATP hydrolysis and DNA binding.

Authors
Bjornson, KP; Allen, DJ; Modrich, P
MLA Citation
Bjornson, KP, Allen, DJ, and Modrich, P. "Modulation of MutS ATP hydrolysis by DNA cofactors." Biochemistry 39.11 (2000): 3176-3183.
PMID
10715140
Source
scival
Published In
Biochemistry
Volume
39
Issue
11
Publish Date
2000
Start Page
3176
End Page
3183
DOI
10.1021/bi992286u

Identifying sequence similarities between DNA molecules

An atomic force microscope (AFM) imaging technique is described to compare sequences between two different DNA molecules and precisely locate nonhomologies in DNA strands. Sequence comparisons are made by forming heteroduplexes between the two molecules and, by AFM imaging the intact molecules formed, identifying both homologous and nonhomologous regions. By forming heteroduplexes between linearized wildtype pSV-β-galactosidase plasmid (6821 bp) and a series of deletion mutants we have identified nonhomologies (deletions) as small as 22 bp and as large as 418 bp. Furthermore, by incorporating our technique for AFM-mediated restriction mapping of DNA these mutations can be positioned relative to EcoRI restriction sites. These results suggest AFM can be useful in identifying molecular level similarities and differences in DNA. (C) 2000 Elsevier Science B.V.

Authors
Hoyt, PR; Doktycz, MJ; Modrich, P; Warmack, RJ; Allison, DP
MLA Citation
Hoyt, PR, Doktycz, MJ, Modrich, P, Warmack, RJ, and Allison, DP. "Identifying sequence similarities between DNA molecules." Ultramicroscopy 82.1-4 (2000): 237-244.
PMID
10741675
Source
scival
Published In
Ultramicroscopy
Volume
82
Issue
1-4
Publish Date
2000
Start Page
237
End Page
244
DOI
10.1016/S0304-3991(99)00153-9

Modulation of cyclophosphamide activity by O6-alkylguanine-DNA alkyltransferase.

PURPOSE: The human medulloblastoma cell line D283 Med (4-HCR), a line resistant to 4-hydroperoxycyclophosphamide (4-HC), displays enhanced repair of DNA interstrand crosslinks induced by phosphoramide mustard. D283 Med (4-HCR) cells are cross-resistant to 1,3-bis(2-chloroethyl)- -nitrosourea, but partial sensitivity is restored after elevated levels of O6-alkylguanine-DNA alkyltransferase (AGT) are depleted by O6-benzylguanine (O6-BG). Studies were conducted to define the activity of 4-HC and 4-hydroperoxydidechlorocyclophosphamide against D283 Med (4-HCR) after AGT is depleted by O6-BG. METHODS: Limiting dilution and xenograft studies were conducted to define the activity of 4-HC and 4-hydroperoxydidechlorocyclophosphamide with or without O6-BG. RESULTS: The activity of 4-HC and 4-hydroperoxydidechlorocyclophosphamide against D283 Med (4-HCR) was increased after AGT depletion by O6-BG preincubation. Similar studies with Chinese hamster ovary cells, with or without stable transfection with a plasmid expressing the human AGT protein, revealed that the AGT-expressing cells were significantly less sensitive to 4-HC and 4-hydroperoxydidechlorocyclophosphamide. Reaction of DNA with 4-HC, phosphoramide mustard, or acrolein revealed that only 4-HC and acrolein caused a decrease in AGT levels. CONCLUSIONS: We propose that a small but potentially significant part of the cellular toxicity of cyclophosphamide in these cells is due to acrolein, and that this toxicity is abrogated by removal of the acrolein adduct from DNA by AGT.

Authors
Friedman, HS; Pegg, AE; Johnson, SP; Loktionova, NA; Dolan, ME; Modrich, P; Moschel, RC; Struck, R; Brent, TP; Ludeman, S; Bullock, N; Kilborn, C; Keir, S; Dong, Q; Bigner, DD; Colvin, OM
MLA Citation
Friedman, HS, Pegg, AE, Johnson, SP, Loktionova, NA, Dolan, ME, Modrich, P, Moschel, RC, Struck, R, Brent, TP, Ludeman, S, Bullock, N, Kilborn, C, Keir, S, Dong, Q, Bigner, DD, and Colvin, OM. "Modulation of cyclophosphamide activity by O6-alkylguanine-DNA alkyltransferase." Cancer Chemother Pharmacol 43.1 (1999): 80-85.
PMID
9923545
Source
pubmed
Published In
Cancer Chemotherapy and Pharmacology
Volume
43
Issue
1
Publish Date
1999
Start Page
80
End Page
85
DOI
10.1007/s002800050866

Multiple DNA repair mechanisms and alkylator resistance in the human medulloblastoma cell line D-283 Med (4-HCR).

PURPOSE: We have previously reported preferential repair of DNA interstrand crosslinks in the 4-hydroperoxycyclophosphamide-resistant human medulloblastoma cell line D-283 Med (4-HCR). We now report further studies that explored the potential mechanisms underlying this repair. METHODS: Limiting dilution assays and Western, Southern, and Northern blots were used to compare specific differences between D-283 Med (4-HCR) and its parental line D-283 Med. RESULTS: D-283 Med (4-HCR) was cross-resistant to melphalan and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), with O6-alkylguanine-DNA alkyltransferase (AGT) levels of 466+/-164 fmol/mg protein; AGT levels in the parental line, D-283 Med, were 76+/-96 fmol/mg. The increase in AGT activity was not a result of gene amplification. Depleting AGT with O6-benzylguanine partially restored sensitivity to BCNU. Both cell lines were deficient in the human mismatch protein MutLalpha. ERCC4 mRNA and poly(ADP-ribose) polymerase levels were similar in both cell lines, and ERCC1 mRNA levels were 2- to 2.5-fold lower in D-283 Med (4-HCR). Topoisomerase I levels were 2- to 2.5-fold higher in D-283 Med compared with D-283 Med (4-HCR). CONCLUSION: These results, while illustrating the multiple differences between D-283 Med and D-283 Med (4-HCR), do not explain the enhanced DNA interstrand crosslink repair seen in D-283 Med (4-HCR).

Authors
Dong, Q; Johnson, SP; Colvin, OM; Bullock, N; Kilborn, C; Runyon, G; Sullivan, DM; Easton, J; Bigner, DD; Nahta, R; Marks, J; Modrich, P; Friedman, HS
MLA Citation
Dong, Q, Johnson, SP, Colvin, OM, Bullock, N, Kilborn, C, Runyon, G, Sullivan, DM, Easton, J, Bigner, DD, Nahta, R, Marks, J, Modrich, P, and Friedman, HS. "Multiple DNA repair mechanisms and alkylator resistance in the human medulloblastoma cell line D-283 Med (4-HCR)." Cancer Chemother Pharmacol 43.1 (1999): 73-79.
PMID
9923544
Source
pubmed
Published In
Cancer Chemotherapy and Pharmacology
Volume
43
Issue
1
Publish Date
1999
Start Page
73
End Page
79
DOI
10.1007/s002800050865

Nucleotide-promoted release of hMutSα from heteroduplex DNA is consistent with an ATP-dependent translocation mechanism

ATP hydrolysis by bacterial and eukaryotic MutS activities is required for their function in mismatch correction, and two different models for the role of ATP in MutS function have been proposed. In the translocation model, based on study of bacterial MutS, ATP binding reduces affinity of the protein for a mismatch and activates secondary DNA binding sites that are subsequently used for movement of the protein along the helix contour in a reaction dependent on nucleotide hydrolysis (Allen, D. J., Makhov, A., Grilley, M., Taylor, J., Thresher, R., Modrich, P., and Griffith, J. D. (1997) EMBO J. 16, 4467-4476). The molecular switch model, based on study of human MutSα, invokes mismatch recognition by the MutSα·ADP complex. After recruitment of downstream repair activities to the MutSα·mismatch complex, ATP binding results in release of MutSα from the heteroduplex (Gradia, S., Acharya, S., and Fishel, R.(1997) Cell 91, 995-1005). To further clarify the function of ATP binding and hydrolysis in human MutSα action, we evaluated the effects of ATP, ADP, and non-hydrolyzable ATP analogs on the lifetime of protein. DNA complexes. All of these nucleotides were found to increase the rate of dissociation of MutSα from oligonucleotide heteroduplexes. These experiments also showed that ADP is not required for mismatch recognition by MutSα, but that the nucleotide alters the dynamics of formation and dissociation of specific complexes. Analysis of the mechanism of ATP-promoted dissociation of MutSα from a 200-base pair heteroduplex demonstrated that dissociation occurs at DNA ends in a reaction dependent on ATP hydrolysis, implying that release from this molecule involves movement of the protein along the helix contour as predicted for a translocation mechanism. In order to reconcile the relatively large rate of movement of MutS homologs along the helix with their modest rate of ATP hydrolysis, we propose a novel mechanism for protein translocation along DNA that supports directional movement over long distances with minimal energy input.

Authors
Blackwell, LJ; Martik, D; Bjornson, KP; Bjornson, ES; Modrich, P
MLA Citation
Blackwell, LJ, Martik, D, Bjornson, KP, Bjornson, ES, and Modrich, P. "Nucleotide-promoted release of hMutSα from heteroduplex DNA is consistent with an ATP-dependent translocation mechanism." Journal of Biological Chemistry 273.48 (1999): 32055-32062.
PMID
9822680
Source
scival
Published In
The Journal of biological chemistry
Volume
273
Issue
48
Publish Date
1999
Start Page
32055
End Page
32062
DOI
10.1074/jbc.273.48.32055

DNA-dependent activation of the hMutSα ATPase

ATP hydrolysis by MutS homologs is required for function of these proteins in mismatch repair. However, the function of ATP hydrolysis in the repair reaction is controversial. In this paper we describe a steady-state kinetic analysis of the DNA-activated ATPase of human MutSα. Comparison of salt concentration effects on mismatch repair and mismatch-provoked excision in HeLa nuclear extracts with salt effects on the DNA-activated ATPase suggests that ATP hydrolysis by MutSα is involved in the rate determining step in the repair pathway. While the ATPase is activated by homoduplex and heteroduplex DNA, the half-maximal concentration for activation by heteroduplex DNA is significantly lower under physiological salt concentrations. Furthermore, at optimal salt concentration, heteroduplex DNA increases the heat for ATP hydrolysis to a greater extent than does homoduplex DNA. We also demonstrate that the degree of ATPase activation is dependent on DNA chain length, with the k(cat) for hydrolysis increasing significantly with chain length of the DNA cofactor. These results are discussed in terms of the translocation (Allen, D.J., Makhov, A., Grilley, M., Taylor, J., Thresher, R., Modrich, P., and Griffith, J. D. (1997) EMBO J. 16, 4467-4476) and the molecular switch (Gradia, S., Acharya, S., and Fishel, R. (1997) Cell 91, 995-1005) models that invoke distinct roles for ATP hydrolysis in MutS homolog function.

Authors
Blackwell, LJ; Bjornson, KP; Modrich, P
MLA Citation
Blackwell, LJ, Bjornson, KP, and Modrich, P. "DNA-dependent activation of the hMutSα ATPase." Journal of Biological Chemistry 273.48 (1999): 32049-32054.
PMID
9822679
Source
scival
Published In
The Journal of biological chemistry
Volume
273
Issue
48
Publish Date
1999
Start Page
32049
End Page
32054
DOI
10.1074/jbc.273.48.32049

The kinetic mechanism of EcoRI endonuclease

Steady-state parameters governing cleavage of pBR322 DNA by EcoRI endonuclease are highly sensitive to ionic environment, with K(m) and k(cat) increasing 1,000-fold and 15-fold, respectively, when ionic strength is increased from 0.059 to 0.23 M. By contrast, pre-steady-state analysis has shown that recognition, as well as first and second strand cleavage events that occur once the enzyme has arrived at the EcoRI site, are essentially insensitive to ionic strength, and has demonstrated that the rate-limiting step for endonuclease turnover occurs after double-strand cleavage under all conditions tested. Furthermore, processive cleavage of a pBR322 variant bearing two closely spaced EcoRI sites is governed by the same turnover number as hydrolysis of parental pBR322, which contains only a single EcoRI sequence, ruling out slow release of the enzyme from the cleaved site or a slow conformational change subsequent to double-strand cleavage. We attribute the effects of ionic strength on steady-state parameters to nonspecific endonuclease. DNA interactions, reflecting facilitated diffusion processes, that occur prior to EcoRI sequence recognition and subsequent to DNA cleavage.

Authors
Wright, DJ; Jack, WE; Modrich, P
MLA Citation
Wright, DJ, Jack, WE, and Modrich, P. "The kinetic mechanism of EcoRI endonuclease." Journal of Biological Chemistry 274.45 (1999): 31896-31902.
PMID
10542216
Source
scival
Published In
The Journal of biological chemistry
Volume
274
Issue
45
Publish Date
1999
Start Page
31896
End Page
31902
DOI
10.1074/jbc.274.45.31896

Repair of large insertion/deletion heterologies in human nuclear extracts is directed by a 5' single-strand break and is independent of the mismatch repair system

The repair of 12-, 27-, 62-, and 216-nucleotide unpaired insertion/deletion heterologies has been demonstrated in nuclear extracts of human cells. When present in covalently closed circular heteroduplexes or heteroduplexes containing a single-strand break 3' to the heterology, such structures are subject to a low level repair reaction that occurs with little strand bias. However, the presence of a single-strand break 5' to the insertion/deletion heterology greatly increases the efficiency of rectification and directs repair to the incised DNA strand. Because nick direction of repair is independent of the strand in which a particular heterology is placed, the observed strand bias is not due to asymmetry imposed on the heteroduplex by the extrahelical DNA segment. Strand-specific repair by this system requires ATP and the four dNTPs and is inhibited by aphidicolin. Repair is independent of the mismatch repair proteins MSh2, MSH6, MLH1, and PMS2 and occurs by a mechanism that is distinct from that of the conventional mismatch repair system. Large heterology repair in nuclear extracts of human cells is also independent of the XPF gene product, and extracts of Chinese hamster ovary cells deficient in the ERCC1 and ERCC4 gene products also support the reaction.

Authors
Littman, SJ; Fang, W-H; Modrich, P
MLA Citation
Littman, SJ, Fang, W-H, and Modrich, P. "Repair of large insertion/deletion heterologies in human nuclear extracts is directed by a 5' single-strand break and is independent of the mismatch repair system." Journal of Biological Chemistry 274.11 (1999): 7474-7481.
PMID
10066813
Source
scival
Published In
The Journal of biological chemistry
Volume
274
Issue
11
Publish Date
1999
Start Page
7474
End Page
7481
DOI
10.1074/jbc.274.11.7474

hMutSα- and hMutLα-dependent phosphorylation of p53 in response to DNA methylator damage

hMSH2 · hMSH6 heterodimer (hMutSα) and hMLH1 · hPMS2 complex (hMutLα) have been implicated in the cytotoxic response of mammalian cells to a number of DNA-damaging compounds, including methylating agents that produce O6-methylguanine (O6MeG) adducts. This study demonstrates that O6MeG lesions, in which the damaged base is paired with either T or C, are subject to excision repair in a reaction that depends on a functional mismatch repair system. Furthermore, treatment of human cells with the S(N)1 DNA methylators N-methyl-N-nitrosourea or N-methyl-N'-nitro-N- nitrosoguanidine results in p53 phosphorylation on serine residues 15 and 392, and these phosphorylation events depend on the presence of functional hMutSα and hMutLα. Coupled with the previous demonstration that O6MeG · T and O6MeG · C pairs are recognized by hMutSα, these results implicate action of the mismatch repair system in the initial step of a damage- signaling cascade that can lead to cell-cycle checkpoint activation or cell death in response to DNA methylator damage.

Authors
Duckett, DR; Bronstein, SM; Taya, Y; Modrich, P
MLA Citation
Duckett, DR, Bronstein, SM, Taya, Y, and Modrich, P. "hMutSα- and hMutLα-dependent phosphorylation of p53 in response to DNA methylator damage." Proceedings of the National Academy of Sciences of the United States of America 96.22 (1999): 12384-12388.
PMID
10535931
Source
scival
Published In
Proceedings of the National Academy of Sciences of the United States of America
Volume
96
Issue
22
Publish Date
1999
Start Page
12384
End Page
12388
DOI
10.1073/pnas.96.22.12384

DNA mismatch repair and O6-alkylguanine-DNA alkyltransferase analysis and response to Temodal in newly diagnosed malignant glioma.

PURPOSE: We evaluated the response to Temodal (Schering-Plough Research Institute, Kenilworth, NJ) of patients with newly diagnosed malignant glioma, as well as the predictive value of quantifying tumor DNA mismatch repair activity and O6-alkylguanine-DNA alkyltransferase (AGT). PATIENTS AND METHODS: Thirty-three patients with newly diagnosed glioblastoma multiforme (GBM) and five patients with newly diagnosed anaplastic astrocytoma (AA) were treated with Temodal at a starting dose of 200 mg/m2 daily for 5 consecutive days with repeat dosing every 28 days after the first daily dose. Immunochemistry for the detection of the human DNA mismatch repair proteins MSH2 and MLH1 and the DNA repair protein AGT was performed with monoclonal antibodies and characterized with respect to percent positive staining. RESULTS: Of the 33 patients with GBM, complete responses (CRs) occurred in three patients, partial responses (PRs) occurred in 14 patients, stable disease (SD) was seen in four patients, and 12 patients developed progressive disease (PD). Toxicity included infrequent grades 3 and 4 myelosuppression, constipation, nausea, and headache. Thirty tumors showed greater than 60% cells that stained for MSH2 and MLH1, with three CRs, 12 PRs, three SDs, and 12 PDs. Eight tumors showed 60% or less cells that stained with antibodies to MSH2 and/or MLH1, with 3 PRs, 3 SDs, and 2 PDs. Eleven tumors showed 20% or greater cells that stained with an antibody to AGT, with 1 PR, 2 SDs, and 8 PDs. Twenty-five tumors showed less than 20% cells that stained for AGT, with 3 CRs, 12 PRs, 4 SDs, and 6 PDs. CONCLUSION: These results suggest that Temodal has activity against newly diagnosed GBM and AA and warrants continued evaluation of this agent. Furthermore, pretherapy analysis of tumor DNA mismatch repair and, particularly, AGT protein expression may identify patients in whom tumors are resistant to Temodal.

Authors
Friedman, HS; McLendon, RE; Kerby, T; Dugan, M; Bigner, SH; Henry, AJ; Ashley, DM; Krischer, J; Lovell, S; Rasheed, K; Marchev, F; Seman, AJ; Cokgor, I; Rich, J; Stewart, E; Colvin, OM; Provenzale, JM; Bigner, DD; Haglund, MM; Friedman, AH; Modrich, PL
MLA Citation
Friedman, HS, McLendon, RE, Kerby, T, Dugan, M, Bigner, SH, Henry, AJ, Ashley, DM, Krischer, J, Lovell, S, Rasheed, K, Marchev, F, Seman, AJ, Cokgor, I, Rich, J, Stewart, E, Colvin, OM, Provenzale, JM, Bigner, DD, Haglund, MM, Friedman, AH, and Modrich, PL. "DNA mismatch repair and O6-alkylguanine-DNA alkyltransferase analysis and response to Temodal in newly diagnosed malignant glioma." J Clin Oncol 16.12 (December 1998): 3851-3857.
PMID
9850030
Source
pubmed
Published In
Journal of Clinical Oncology
Volume
16
Issue
12
Publish Date
1998
Start Page
3851
End Page
3857
DOI
10.1200/JCO.1998.16.12.3851

Biallelic inactivation of hMLH1 by epigenetic gene silencing, a novel mechanism causing human MSI cancers.

Mutations of DNA mismatch repair genes, including the hMLH1 gene, have been linked to human colon and other cancers in which defective DNA repair is evidenced by the associated instability of DNA microsatellite sequences (MSI). Germ-line hMLH1 mutations are causally associated with inherited MSI colon cancer, and somatic mutations are causally associated with sporadic MSI colon cancer. Previously however, we demonstrated that in many sporadic MSI colon cancers hMLH1 and all other DNA mismatch repair genes are wild type. To investigate this class of tumors further, we examined a group of MSI cancer cell lines, most of which were documented as established from antecedent MSI-positive malignant tumors. In five of six such cases we found that hMLH1 protein was absent, even though hMLH1-coding sequences were wild type. In each such case, absence of hMLH1 protein was associated with the methylation of the hMLH1 gene promoter. Furthermore, in each case, treatment with the demethylating agent 5-azacytidine induced expression of the absent hMLH1 protein. Moreover, in single cell clones, hMLH1 expression could be turned on, off, and on again by 5-azacytidine exposure, washout, and reexposure. This epigenetic inactivation of hMLH1 additionally accounted for the silencing of both maternal and paternal tumor hMLH1 alleles, both of which could be reactivated by 5-azacytidine. In summary, substantial numbers of human MSI cancers appear to arise by hMLH1 silencing via an epigenetic mechanism that can inactivate both of the hMLH1 alleles. Promoter methylation is intimately associated with this epigenetic silencing mechanism.

Authors
Veigl, ML; Kasturi, L; Olechnowicz, J; Ma, AH; Lutterbaugh, JD; Periyasamy, S; Li, GM; Drummond, J; Modrich, PL; Sedwick, WD; Markowitz, SD
MLA Citation
Veigl, ML, Kasturi, L, Olechnowicz, J, Ma, AH, Lutterbaugh, JD, Periyasamy, S, Li, GM, Drummond, J, Modrich, PL, Sedwick, WD, and Markowitz, SD. "Biallelic inactivation of hMLH1 by epigenetic gene silencing, a novel mechanism causing human MSI cancers." Proc Natl Acad Sci U S A 95.15 (July 21, 1998): 8698-8702.
PMID
9671741
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
95
Issue
15
Publish Date
1998
Start Page
8698
End Page
8702

Analysis of DNA mismatch repair proteins in human medulloblastoma.

During replication, the primary function of the eukaryotic DNA mismatch repair (MMR) system is to recognize and correct mismatched base pairs within the DNA helix. Deficiencies in MMR have been reported previously in cases of hereditary nonpolyposis colorectal cancer and sporadic tumors occurring in a variety of tissues including gliomas. Furthermore, recent evidence indicates that the MMR system may be involved in mediating therapeutic sensitivity to alkylating agents. In this study, 22 neoplastic tissue samples from 22 patients who underwent surgical resection for medulloblastoma, a common cerebellar tumor of childhood, were assayed for the presence or absence of MMR polypeptides using Western blot and immunohistochemical techniques. Results from these experiments indicate that the MMR system is not commonly deficient in medulloblastoma.

Authors
Lee, SE; Johnson, SP; Hale, LP; Li, J; Bullock, N; Fuchs, H; Friedman, A; McLendon, R; Bigner, DD; Modrich, P; Friedman, HS
MLA Citation
Lee, SE, Johnson, SP, Hale, LP, Li, J, Bullock, N, Fuchs, H, Friedman, A, McLendon, R, Bigner, DD, Modrich, P, and Friedman, HS. "Analysis of DNA mismatch repair proteins in human medulloblastoma." Clin Cancer Res 4.6 (June 1998): 1415-1419.
PMID
9626457
Source
pubmed
Published In
Clinical cancer research : an official journal of the American Association for Cancer Research
Volume
4
Issue
6
Publish Date
1998
Start Page
1415
End Page
1419

Therapeutic efficacy of vinorelbine against pediatric and adult central nervous system tumors.

PURPOSE: The activity of vinorellbine, a new semisynthetic vinca alkaloid, was evaluated against a battery of human tumor xenografts derived from adult and pediatric CNS malignancies. METHODS: Tumors included adult high-grade gliomas (D-54 MG, D-245 MG), childhood high-grade gliomas (D-212 MG, D-456 MG), medulloblastomas (D-341 MED, D-487 MED), ependymomas (D-612 EP, D-528 EP), and a mismatch repair-deficient procarbazine-resistant glioma [D-245 MG (PR)]. Tumors were grown subcutaneously in athymic nude mice and vinorelbine was administered at a dose of 11 mg/kg on days 1, 5, and 9. Additionally, vinorelbine was also administered in combination with BCNU against D-54 MG. RESULTS: Vinorelbine produced statistically significant growth delays in D-456 MG, D-245 MG, and D-245 MG (PR). No statistically significant growth delays were observed in D-54 MG, D-487 MED, D-212 MG, D-528 EP, D-341 MED or D-612 EP. The antitumor effects of the vinorelbine/BCNU combination were additive. Growth delays observed in the procarbazine-resistant line [D-245 MG (PR)] were greater than twofold the delays seen in the parent line (D-245 MG). Vincristine was equally potent against D-245 MG and D-245 MG (PR). Taxol demonstrated little activity against D-245 MG but produced 32- and 18-day growth delays in D245 MG (PR). CONCLUSIONS: These studies indicate that vinorelbine possesses antitumor activity against several glioma tumor xenografts with marked activity in a mismatch repair deficient-tumor.

Authors
Hanley, ML; Elion, GB; Colvin, OM; Modrich, PL; Keir, S; Adams, DJ; Bigner, DD; Friedman, HS
MLA Citation
Hanley, ML, Elion, GB, Colvin, OM, Modrich, PL, Keir, S, Adams, DJ, Bigner, DD, and Friedman, HS. "Therapeutic efficacy of vinorelbine against pediatric and adult central nervous system tumors." Cancer Chemother Pharmacol 42.6 (1998): 479-482.
PMID
9788574
Source
pubmed
Published In
Cancer Chemotherapy and Pharmacology
Volume
42
Issue
6
Publish Date
1998
Start Page
479
End Page
482

Erratum: Isolation of MutSβ from human cells and comparison of the mismatch repair specificities of MutSβ and MutSα (Journal of Biological Chemistry (1998) 273 (19895-19901))

Authors
Genschel, J; Littman, SJ; Drummond, JT; Modrich, P
MLA Citation
Genschel, J, Littman, SJ, Drummond, JT, and Modrich, P. "Erratum: Isolation of MutSβ from human cells and comparison of the mismatch repair specificities of MutSβ and MutSα (Journal of Biological Chemistry (1998) 273 (19895-19901))." Journal of Biological Chemistry 273.41 (1998): 27034--.
Source
scival
Published In
Journal of Biological Chemistry
Volume
273
Issue
41
Publish Date
1998
Start Page
27034-

Isolation of MutSβ from human cells and comparison of the mismatch repair specificities of MutSβ and MutSα

A human MSH2-human MSH3 (hMSH2·hMSH3) complex of approximately 1:1 stoichiometry (human MutSβ (hMutSβ)) has been demonstrated in several human tumor cell lines and purified to near homogeneity. In vitro, hMutSβ supports the efficient repair of insertion/deletion (I/D) heterologies of 2-8 nucleotides, is weakly active on a single-nucleotide I/D mispair, and is not detectably active on the eight base-base mismatches. Human MutSα (hMutSα), a heterodimer of hMSH2 and hMSH6, efficiently supports the repair of single- nucleotide I/D mismatches, base-base mispairs, and all substrates tested that were repaired by hMutSβ. Thus, the repair specificities of hMutSα and hMutSβ are redundant with respect to the repair of I/D heterologies of 2-8 nucleotides. The hMutSα level in repair-proficient HeLa cells (1.5 μg/mg nuclear extract) is approximately 10 times that of hMutSβ. In HCT-15 colorectal tumor cells, which do not contain hMSH6 and consequently lack hMutSα, the hMutSβ level is elevated severalfold relative to that in HeLa cells and is responsible for the repair of I/D mismatches that has been observed in this cell line. LoVo tumor cells, which are genetically deficient in hMSH2, lack both hMutSα and hMutSβ, and hMSH3 and hMSH6 levels are less than 4% of those found in repair-proficient cells. Coupled with previous findings (J. T. Drummond, J. Genschel, E. Wolf, and P. Modrich (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 10144-10149), these results suggest that hMSH2 partitions between available pools of hMSH3 and hMSH6 and indicate that hMSH2 positively modulates hMSH6 and hMSH3 levels, perhaps by stabilization of the polypeptides upon heterodimer formation.

Authors
Genschel, J; Littman, SJ; Drummond, JT; Modrich, P
MLA Citation
Genschel, J, Littman, SJ, Drummond, JT, and Modrich, P. "Isolation of MutSβ from human cells and comparison of the mismatch repair specificities of MutSβ and MutSα." Journal of Biological Chemistry 273.31 (1998): 19895-19901.
PMID
9677427
Source
scival
Published In
Journal of Biological Chemistry
Volume
273
Issue
31
Publish Date
1998
Start Page
19895
End Page
19901
DOI
10.1074/jbc.273.31.19895

Mismatch-, MutS-, MutL-, and helicase II-dependent unwinding from the single-strand break of an incised heteroduplex

Escherichia coli MutS, MutL, and DNA helicase II are sufficient to initiate mismatch-dependent unwinding of an incised heteroduplex (Yamaguchi, M., Dao, V., and Modrich, P. (1998) J. Biol. Chem., 273, 9197-9201). We have studied unwinding of 6.4-kilobase circular G-T heteroduplexes that contain a single-strand incision, 808 base pairs 5' to the mismatch or 1023 base pairs 3' to the mispair as viewed along the shorter path between the two DNA sites. Unwinding of both substrates in the presence of MutS, MutL, DNA helicase II, and single-stranded DNA binding protein was mismatch-dependent and initiated at the single-strand break. Although unwinding occurred in both directions from the strand break, it was biased toward the shorter path linking the strand break and the mispair. MutS and MutL are thus sufficient to coordinate mismatch recognition to the orientation-dependent activation of helicase II unwinding at a single-strand break located a kilobase mispair.

Authors
Dao, V; Modrich, P
MLA Citation
Dao, V, and Modrich, P. "Mismatch-, MutS-, MutL-, and helicase II-dependent unwinding from the single-strand break of an incised heteroduplex." Journal of Biological Chemistry 273.15 (1998): 9202-9207.
PMID
9535911
Source
scival
Published In
The Journal of biological chemistry
Volume
273
Issue
15
Publish Date
1998
Start Page
9202
End Page
9207
DOI
10.1074/jbc.273.15.9202

MutS and MutL activate DNA helicase II in a mismatch-dependent manner

MutS, MutL, and DNA helicase II are required for the mismatch-provoked excision step that occurs during Escherichia coli methyl-directed mismatch repair. In this study MutL is shown to enhance the unwinding activity of DNA helicase II more than 10-fold on a conventional helicase substrate in which a 35-residue oligonucleotide is annealed to a M13 circular single-stranded phage DNA under conditions where the two proteins are present at approximately molar stoichiometry with respect to the substrate. MutS- and MutL-dependent activation of DNA helicase II has also been demonstrated with a model substrate in which a 138-residue oligonucleotide was hybridized to a 138-nucleotide gap in an otherwise duplex 7,100-base pair circular DNA. Displacement of the oligonucleotide requires MutS, MutL, DNA helicase II, and ATP and is dependent on the presence of a mismatch within the hybrid region. Although DNA helicase II and Rep helicase share substantial sequence homology and features of mechanism, Rep helicase is inactive in this reaction.

Authors
Yamaguchi, M; Dao, V; Modrich, P
MLA Citation
Yamaguchi, M, Dao, V, and Modrich, P. "MutS and MutL activate DNA helicase II in a mismatch-dependent manner." Journal of Biological Chemistry 273.15 (1998): 9197-9201.
PMID
9535910
Source
scival
Published In
The Journal of biological chemistry
Volume
273
Issue
15
Publish Date
1998
Start Page
9197
End Page
9201
DOI
10.1074/jbc.273.15.9197

Increased transversions in a novel mutator colon cancer cell line

We describe a novel mutator phenotype in the Vaco411 colon cancer cell line which increases the spontaneous mutation rate 10-100-fold over background. This mutator results primarily in transversion base substitutions which are found infrequently in repair competent cells. Of the four possible types of transversions, only three were principally recovered. Spontaneous mutations recovered also included transitions and large deletions, but very few frameshifts were recovered. When compared to known mismatch repair defective colon cancer mutators, the distribution of mutations in Vaco411 is significantly different. Consistent with this difference, Vaco411 extracts are proficient in assays of mismatch repair. The Vaco411 mutator appears to be novel, and is not an obvious human homologue of any of the previously characterized bacterial or yeast transversion phenotypes. Several hypotheses by which this mutator may produce transversions are presented.

Authors
Eshleman, JR; Donover, PS; Littman, SJ; Swinler, SE; Li, G-M; Lutterbaugh, JD; Willson, JKV; Modrich, P; Sedwick, WD; Markowitz, SD; Veigl, ML
MLA Citation
Eshleman, JR, Donover, PS, Littman, SJ, Swinler, SE, Li, G-M, Lutterbaugh, JD, Willson, JKV, Modrich, P, Sedwick, WD, Markowitz, SD, and Veigl, ML. "Increased transversions in a novel mutator colon cancer cell line." Oncogene 16.9 (1998): 1125-1130.
PMID
9528854
Source
scival
Published In
Oncogene: Including Oncogene Reviews
Volume
16
Issue
9
Publish Date
1998
Start Page
1125
End Page
1130

A naturally occurring hPMS2 mutation can confer a dominant negative mutator phenotype

Defects in mismatch repair (MMR) genes result in a mutator phenotype by inducing microsatellite instability (MI), a characteristic of hereditary nonpolyposis colorectal cancers (HNPCC) and a subset of sporadic colon tumors. Present models describing the mechanism by which germ line mutations in MMR genes predispose kindreds to HNPCC suggest a 'two-hit' inactivation of both alleles of a particular MMR gene. Here we present experimental evidence that a nonsense mutation at codon 134 of the hPMS2 gene is sufficient to reduce MMR and induce MI in cells containing a wild-type hPMS2 allele. These results have significant implications for understanding the relationship between mutagenesis and carcinogenesis and the ability to generate mammalian cells with mutator phenotypes.

Authors
Nicolaides, NC; Littman, SJ; Modrich, P; Kinzler, KW; Vogelstein, B
MLA Citation
Nicolaides, NC, Littman, SJ, Modrich, P, Kinzler, KW, and Vogelstein, B. "A naturally occurring hPMS2 mutation can confer a dominant negative mutator phenotype." Molecular and Cellular Biology 18.3 (1998): 1635-1641.
PMID
9488480
Source
scival
Published In
Molecular and Cellular Biology
Volume
18
Issue
3
Publish Date
1998
Start Page
1635
End Page
1641

Methylator resistance mediated by mismatch repair deficiency in a glioblastoma multiforme xenograft.

A methylator-resistant human glioblastoma multiforme xenograft, D-245 MG (PR), in athymic nude mice was established by serially treating the parent xenograft D-245 MG with procarbazine. D-245 MG xenografts were sensitive to procarbazine, temozolomide, N-methyl-N-nitrosourea, 1,3-bis(2-chloroethyl)-1-nitrosourea, 9-aminocamptothecin, topotecan, CPT-11, cyclophosphamide, and busulfan. D-245 MG (PR) xenografts were resistant to procarbazine, temozolomide, N-methyl-N-nitrosourea, and busulfan, but they were sensitive to the other agents. Both D-245 MG and D-245 MG (PR) xenografts displayed no O6-alkylguanine-DNA alkyltransferase activity, and their levels of glutathione and glutathione-S-transferase were similar. D-245 MG xenografts expressed the human mismatch repair proteins hMSH2 and hMLH1, whereas D-245 MG (PR) expressed hMLH1 but not hMSH2.

Authors
Friedman, HS; Johnson, SP; Dong, Q; Schold, SC; Rasheed, BK; Bigner, SH; Ali-Osman, F; Dolan, E; Colvin, OM; Houghton, P; Germain, G; Drummond, JT; Keir, S; Marcelli, S; Bigner, DD; Modrich, P
MLA Citation
Friedman, HS, Johnson, SP, Dong, Q, Schold, SC, Rasheed, BK, Bigner, SH, Ali-Osman, F, Dolan, E, Colvin, OM, Houghton, P, Germain, G, Drummond, JT, Keir, S, Marcelli, S, Bigner, DD, and Modrich, P. "Methylator resistance mediated by mismatch repair deficiency in a glioblastoma multiforme xenograft." Cancer Res 57.14 (July 15, 1997): 2933-2936.
PMID
9230204
Source
pubmed
Published In
Cancer Research
Volume
57
Issue
14
Publish Date
1997
Start Page
2933
End Page
2936

Removal of polymerase-produced mutant sequences from PCR products.

Heteroduplex DNA lacking d(GATC) methylation is subject to mismatch-provoked double-strand cleavage at d(GATC) sites in a reaction dependent on MutH, MutL, MutS, and ATP. We have exploited this reaction to develop a method for removal of polymerase-produced mutant sequences that arise during sequence amplification by PCR. After denaturation and reannealing, the PCR product pool is subjected to MutH, MutL, and MutS mismatch repair proteins under double-strand cleavage conditions, followed by isolation of uncleaved product by size selection. Use of an Escherichia coli lac forward mutation assay has shown that this procedure reduces the incidence of polymerase-induced mutant sequences by an order of magnitude. Twenty mutants that originated from three independent PCR amplification reactions and survived MutHLS treatment all were found to contain an infrequently occurring A.T --> T.A transversion mutation at a unique position within the product. By contrast, the majority of mutations in untreated PCR products were transitions occurring throughout the amplified region, although frameshifts and transversions also were observed. The MutHLS method thus can be used to effectively remove the majority of mutant sequences produced by polymerase errors during PCR amplification.

Authors
Smith, J; Modrich, P
MLA Citation
Smith, J, and Modrich, P. "Removal of polymerase-produced mutant sequences from PCR products." Proc Natl Acad Sci U S A 94.13 (June 24, 1997): 6847-6850.
PMID
9192654
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
94
Issue
13
Publish Date
1997
Start Page
6847
End Page
6850

Strand-specific mismatch repair in mammalian cells

Authors
Modrich, P
MLA Citation
Modrich, P. "Strand-specific mismatch repair in mammalian cells." Journal of Biological Chemistry 272.40 (1997): 24727-24730.
PMID
9312062
Source
scival
Published In
The Journal of biological chemistry
Volume
272
Issue
40
Publish Date
1997
Start Page
24727
End Page
24730
DOI
10.1074/jbc.272.40.24727

DHFR/MSH3 amplification in methotrexate-resistant cells alters the hMutSα/hMutSβ ratio and reduces the efficiency of base-base mismatch repair

The level and fate of hMSH3 (human MutS homolog 3) were examined in the promyelocytic leukemia cell line HL-60 and its methotrexate-resistant derivative HL-60R, which is drug resistant by virtue of an amplification event that spans the dihydrofolate reductase (DHFR) and MSH3 genes. Nuclear extracts from HL-60 and HL-60R cells were subjected to an identical, rapid purification protocol that efficiently captures heterodimeric hMutSα (hMSH2·hMSH6) and hMutSβ (hMSH2·hMSH3). In HL-60 extracts the hMutSα to hMutSβ ratio is roughly 6:1, whereas in methotrexate-resistant HL-60R cells the ratio is less than 1:100, due to overproduction of hMSH3 and heterodimer formation of this protein with virtually all the nuclear hMSH2. This shift is associated with marked reduction in the efficiency of base-base mismatch and hypermutability at the hypoxanthine phosphoribosyltransferase (HPRT) locus. Purified hMutSα and hMutSβ display partial overlap in mismatch repair specificity: both participate in repair of a dinucleotide insertion-deletion heterology, but only hMutSα restores base-base mismatch repair to extracts of HL-60R cells or hMSH2-deficient LoVo colorectal tumor cells.

Authors
Drummond, JT; Genschel, J; Wolf, E; Modrich, P
MLA Citation
Drummond, JT, Genschel, J, Wolf, E, and Modrich, P. "DHFR/MSH3 amplification in methotrexate-resistant cells alters the hMutSα/hMutSβ ratio and reduces the efficiency of base-base mismatch repair." Proceedings of the National Academy of Sciences of the United States of America 94.19 (1997): 10144-10149.
PMID
9294177
Source
scival
Published In
Proceedings of the National Academy of Sciences of USA
Volume
94
Issue
19
Publish Date
1997
Start Page
10144
End Page
10149
DOI
10.1073/pnas.94.19.10144

MutS mediates heteroduplex loop formation by a translocation mechanism

Interaction of Escherichia coli MutS and MutL with heteroduplex DNA has been visualized by electron microscopy. In a reaction dependent on ATP hydrolysis, complexes between a MutS diner and a DNA heteroduplex are converted to protein-stabilized, α-shaped loop structures with the mismatch in most cases located within the DNA loop. Loop formation depends on ATP hydrolysis and loop size increases linearly with time at a rate of 370 base pairs/min in phosphate buffer and about 10,000 base pairs/min in the HEPES buffer used for repair assay. These observations suggest a translocation mechanism in which a MutS dimer bound to a mismatch subsequently leaves this site by ATP-dependent tracking or unidimensional movement that is in most cases bidirectional from the mispair. In view of the bidirectional capability of the methyl-directed pathway, this reaction may play a role in determination of heteroduplex orientation. The rate of MutS-mediated DNA loop growth is enhanced by MutL, and when both proteins are present, both are found at the base of α-loop structures, and both can remain associated with excision intermediates produced in later stages of the reaction.

Authors
Allen, DJ; Makhov, A; Grilley, M; Taylor, J; Thresher, R; Modrich, P; Griffith, JD
MLA Citation
Allen, DJ, Makhov, A, Grilley, M, Taylor, J, Thresher, R, Modrich, P, and Griffith, JD. "MutS mediates heteroduplex loop formation by a translocation mechanism." EMBO Journal 16.14 (1997): 4467-4476.
PMID
9250691
Source
scival
Published In
EMBO Journal
Volume
16
Issue
14
Publish Date
1997
Start Page
4467
End Page
4476
DOI
10.1093/emboj/16.14.4467

Mapping individual cosmid DNAs by direct AFM imaging

Individual cosmid clones have been restriction mapped by directly imaging, with the atomic force microscope (AFM), a mutant EcoRI endonuclease site-specifically bound to DNA. Images and data are presented that locate six restriction sites, predicted from gel electrophoresis, on a 35-kb cosmid isolated from mouse chromosome 7. Measured distances between endonuclease molecules bound to λ DNA, when compared to known values, demonstrate the accuracy of AFM mapping to better than 1%. These results may be extended to identify other important site-specific protein-DNA interactions, such as transcription factor and mismatch repair enzyme binding, difficult to resolve by current techniques.

Authors
Allison, DP; Kerper, PS; Doktycz, MJ; Thundat, T; Modrich, P; Larimer, FW; Johnson, DK; Hoyt, PR; Mucenski, ML; Warmack, RJ
MLA Citation
Allison, DP, Kerper, PS, Doktycz, MJ, Thundat, T, Modrich, P, Larimer, FW, Johnson, DK, Hoyt, PR, Mucenski, ML, and Warmack, RJ. "Mapping individual cosmid DNAs by direct AFM imaging." Genomics 41.3 (1997): 379-384.
PMID
9169135
Source
scival
Published In
Genomics
Volume
41
Issue
3
Publish Date
1997
Start Page
379
End Page
384
DOI
10.1006/geno.1997.4686

DNA polymerase δ is required for human mismatch repair in vitro

HeLa nuclear extract was resolved into a depleted fraction incapable of supporting mismatch repair in vitro, and repair activity was restored upon the addition of a purified fraction isolated from HeLa cells by in vitro complementation assay. The highly enriched complementing activity copurified with a DNA polymerase, and the most pure fraction contained DNA polymerase δ but was free of detectable DNA polymerases α and ε. Calf thymus DNA polymerase δ also fully restored mismatch repair to the depleted extract, indicating DNA polymerase δ is required for mismatch repair in human cells. However, due to the presence of DNA polymerases α and ε in the depleted extract, potential involvement of one or both of these activities in the reaction cannot be excluded.

Authors
Longley, MJ; Pierce, AJ; Modrich, P
MLA Citation
Longley, MJ, Pierce, AJ, and Modrich, P. "DNA polymerase δ is required for human mismatch repair in vitro." Journal of Biological Chemistry 272.16 (1997): 10917-10921.
PMID
9099749
Source
scival
Published In
The Journal of biological chemistry
Volume
272
Issue
16
Publish Date
1997
Start Page
10917
End Page
10921
DOI
10.1074/jbc.272.16.10917

Recognition and repair of compound DNA lesions (base damage and mismatch) by human mismatch repair and excision repair systems

Nucleotide excision repair and the long-patch mismatch repair systems correct abnormal DNA structures arising from DNA damage and replication errors, respectively. DNA synthesis past a damaged base (translesion replication) often causes misincorporation at the lesion site. In addition, mismatches are hot spots for DNA damage because of increased susceptibility of unpaired bases to chemical modification. We call such a DNA lesion, that is, a base damage superimposed on a mismatch, a compound lesion. To learn about the processing of compound lesions by human cells, synthetic compound lesions containing UV photoproducts or cisplatin 1,2-d(GpG) intrastrand cross-link and mismatch were tested for binding to the human mismatch recognition complex hMutSα and for excision by the human excision nuclease. No functional overlap between excision repair and mismatch repair was observed. The presence of a thymine dimer or a cisplatin diadduct in the context of a G-T mismatch reduced the affinity of hMutSα for the mismatch. In contrast, the damaged bases in these compound lesions were excised three- to fourfold faster than simple lesions by the human excision nuclease, regardless of the presence of hMutSα in the reaction. These results provide a new perspective on how excision repair, a cellular defense system for maintaining genomic integrity, can fix mutations under certain circumstances.

Authors
Mu, D; Tursun, M; Duckett, DR; Drummond, JT; Modrich, P; Sancar, A
MLA Citation
Mu, D, Tursun, M, Duckett, DR, Drummond, JT, Modrich, P, and Sancar, A. "Recognition and repair of compound DNA lesions (base damage and mismatch) by human mismatch repair and excision repair systems." Molecular and Cellular Biology 17.2 (1997): 760-769.
PMID
9001230
Source
scival
Published In
Molecular and Cellular Biology
Volume
17
Issue
2
Publish Date
1997
Start Page
760
End Page
769

MutSα MutSβ, MutLα and DNA polymerase ôin human mismatch repair

The human mismatch repair system corrects the eight hase-hase mismatches and insertion/deletion loops of 1-10 nucleotides. Using an in vitro assay, we have screened extracts from twenty-two cell lines derived from sporadic and hereditary RER (replication error prone) cancers of colorectal. endometrial, ovarian and lymphoid origin. Twenty-one of these were found to be deficient in mismatch repair, with the deficiency in each case atlrihutahle to molecular defects m MutSa (a heterodimer of MSH2 and MSH6i or MutLa (a heterodimer ot MLH1 and PMS2), suggesting that defects in either of these activities account for the vast majority of RER+ tumors In addition to MutSa and MutLa, recent work has implicated two additional activities in human mismatch repair: DNA polymerase ôand MutSβ (a heterodimer of MSH2 and MSH3). In contrast to MutSa.which is required for hase-hase mismatch repair and plays a role in correction of insertion/deletion heterologies, MutSβ contributes only to correction of the latter class of mispairs. The majority of nuclear MSH2 is eomplexed with MSH6 in the form of MutSa. with a minor fraction present in the MutSβ complex with MSH3. In addition to its ability to recognize mismatches, MutSa recognises the cytotoxic lesions produced by DNA methylating agents and cisplatin. Collaborative experiments with William Thilly (M.I.T.) and Robert Brown (CRC Beatson Laboratories) have demonstrated that cell lines deficient in MutSa or MutLa are resistant to the cytotoxic effects of these agents.

Authors
Drummond, J; Duckett, D; Genschel, J; Lonyley, M; Pierce, A; Litlman, S; Li, G-M; Modrich, P
MLA Citation
Drummond, J, Duckett, D, Genschel, J, Lonyley, M, Pierce, A, Litlman, S, Li, G-M, and Modrich, P. "MutSα MutSβ, MutLα and DNA polymerase ôin human mismatch repair." FASEB Journal 11.9 (1997): A1012-.
Source
scival
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
11
Issue
9
Publish Date
1997
Start Page
A1012

Methotrexate-inducfd amplification of the human DHHFR/MSH3 region alters the ratio of mutsd and mutsβ and reduces the efficacy of base-base mismatch repair

We examined the expression and fate of human MSH3 in the promyelocytic leukemia cell lines HI.-60 and HL-60R, where HL-60R is resistant to methotrexate by virtue of a DNA amplification event that includes the dihydrolblate reductase (DHFR) gene. This gene amplification directly impacts post-replication repair because human MSH3 is divergently transcribed from the same promoter region as the DHFR gene, and we find that MSH3 is also overexpressed. Nuclear extracts from HL-60 and HL-60R cells were subjected to an identical, rapid purification protocol that captures both heterodimeric hMutSa (MSH2/MSH6) and hMutSfi (MSH2/MSH3). In HL-60 cells the hMutSa to hMutS ratio is roughly 6:1, while in the methotrexate-resistam HL-60R cells, the ratio reverses to <1:100, where MSH2 is now complexed primarily with MSH3. Purified hMutSa and hMutSβ display partial overlap in mismatch repair specificity in complementation assays, where both participate in the repair of a dinucleotide insertion mismatch but only hMutSoc participates in base-base repair. We propose that treatment of tumor cells with methotrexate is likely to produce an unintended alteration in the mismatch repair profile of these cells in vivo, potentially generating hypermutable derivatives that may become more aggressive or resistant to other drugs by virtue of this repair detect. This work was supported by the Howard Hughes Medical Institute.

Authors
Drummond, J; Modrich, P
MLA Citation
Drummond, J, and Modrich, P. "Methotrexate-inducfd amplification of the human DHHFR/MSH3 region alters the ratio of mutsd and mutsβ and reduces the efficacy of base-base mismatch repair." FASEB Journal 11.9 (1997): A1192-.
Source
scival
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
11
Issue
9
Publish Date
1997
Start Page
A1192

Cisplatin and adriamycin resistance are associated with MutLalpha and mismatch repair deficiency in an ovarian tumor cell line.

In contrast to parental A2780 ovarian tumor cells, extracts of one doxorubicin-resistant and two independent cis-diamminedichloroplatinum(II)-resistant derivatives are defective in strand-specific mismatch repair. The repair defect of the three hypermutable, drug-resistant cell lines is only evident when the strand break that directs the reaction is located 3' to the mismatch, and in each case repair is restored to extracts by addition of purified MutLalpha heterodimer. As judged by immunological assay, drug resistance is associated with the virtual absence of the MutLalpha MLH1 subunit and greatly reduced levels of the PMS2 subunit. These findings implicate a functional mismatch repair system in the cytotoxic effects of these antitumor drugs and may have ramifications for their clinical application.

Authors
Drummond, JT; Anthoney, A; Brown, R; Modrich, P
MLA Citation
Drummond, JT, Anthoney, A, Brown, R, and Modrich, P. "Cisplatin and adriamycin resistance are associated with MutLalpha and mismatch repair deficiency in an ovarian tumor cell line." J Biol Chem 271.33 (August 16, 1996): 19645-19648.
PMID
8702663
Source
pubmed
Published In
The Journal of biological chemistry
Volume
271
Issue
33
Publish Date
1996
Start Page
19645
End Page
19648

Mutation detection with MutH, MutL, and MutS mismatch repair proteins.

Escherichia coli methyl-directed mismatch repair is initiated by MutS-, MutL-, and ATP-dependent activation of MutH endonuclease, which cleaves at d(GATC) sites in the vicinity of a mismatch. This reaction provides an efficient method for detection of mismatches in heteroduplexes produced by hybridization of genetically distinct sequences after PCR amplification. Multiple examples of transition and transversion mutations, as well as one, two, and three nucleotide insertion/deletion mutants, have been detected in PCR heteroduplexes ranging in size from 400 bp to 2.5 kb. Background cleavage of homoduplexes is largely due to polymerase errors that occur during amplification, and the MutHLS reaction provides an estimate of the incidence of mutant sequences that arise during PCR.

Authors
Smith, J; Modrich, P
MLA Citation
Smith, J, and Modrich, P. "Mutation detection with MutH, MutL, and MutS mismatch repair proteins." Proc Natl Acad Sci U S A 93.9 (April 30, 1996): 4374-4379.
PMID
8633074
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
93
Issue
9
Publish Date
1996
Start Page
4374
End Page
4379

Direct atomic force microscope imaging of EcoRI endonuclease site specifically bound to plasmid DNA molecules

Direct imaging with the atomic force microscope has been used to identify specific nucleotide sequences in plasmid DNA molecules. This was accomplished using EcoRI(Gln-111), a mutant of the restriction enzyme that has a 1000-fold greater binding affinity than the wild-type enzyme but with cleavage rate constants reduced by a factor of 104. ScaI-linearized plasmids with single (pBS+) and double (pGEM-luc and pSV-β-galactosidase) EcoRI recognition sites were imaged, and the bound enzyme was localized to a 50- to 100-nt resolution. The high affinity for the EcoRI binding site exhibited by this mutant endonuclease, coupled with an observed low level of nonspecific binding, should prove valuable for physically mapping large DNA clones by direct atomic force microscope imaging.

Authors
Allison, DP; Kerper, PS; Doktycz, MJ; Spain, JA; Modrich, P; Larimer, FW; Thundat, T; Warmack, RJ
MLA Citation
Allison, DP, Kerper, PS, Doktycz, MJ, Spain, JA, Modrich, P, Larimer, FW, Thundat, T, and Warmack, RJ. "Direct atomic force microscope imaging of EcoRI endonuclease site specifically bound to plasmid DNA molecules." Proceedings of the National Academy of Sciences of the United States of America 93.17 (1996): 8826-8829.
PMID
8799111
Source
scival
Published In
Proceedings of the National Academy of Sciences of USA
Volume
93
Issue
17
Publish Date
1996
Start Page
8826
End Page
8829
DOI
10.1073/pnas.93.17.8826

Humnan MutSα recognizes damaged DNA base pairs containing O6-methylguanine, O4-methylthymine, or the cisplatin-d(GpG) adduct

Bacterial and mammalian mismatch repair systems have been implicated in the cellular response to certain types of DNA damage, and genetic defects in this pathway are known to confer resistance to the cytotoxic effects of DNA-methylating agents. Such observations suggest that in addition to their ability to recognize DNA base-pairing errors, members of the MutS family may also respond to genetic lesions produced by DNA damage. We show that the human mismatch recognition activity MutSα recognizes several types of DNA lesion including the 1,2-intrastrand d(GpG) crosslink produced by cis-diamminedichloroplatinum(II), as well as base pairs between O6-methylguanine and thymine or cytosine, or between O4-methylthymine and adenine. However, the protein fails to recognize 1,3-intrastrand adduct produced by transdiamminedichloroplatinum(II) at a d (GpTpG) sequence. These observations imply direct involvement of the mismatch repair system in the cytotoxic effects of DNA-methylating agents and suggest that recognition of 1,2-intrastrand cis-diamminedichloroplatinum(II) adducts by MutSα may be involved in the cytotoxic action of this chemotherapeutic agent.

Authors
Duckett, DR; Drummond, JT; Murchie, AIH; Reardon, JT; Sancar, A; Lilley, DMJ; Modrich, P
MLA Citation
Duckett, DR, Drummond, JT, Murchie, AIH, Reardon, JT, Sancar, A, Lilley, DMJ, and Modrich, P. "Humnan MutSα recognizes damaged DNA base pairs containing O6-methylguanine, O4-methylthymine, or the cisplatin-d(GpG) adduct." Proceedings of the National Academy of Sciences of the United States of America 93.13 (1996): 6443-6447.
PMID
8692834
Source
scival
Published In
Proceedings of the National Academy of Sciences of the United States of America
Volume
93
Issue
13
Publish Date
1996
Start Page
6443
End Page
6447

Diverse hypermutability of multiple expressed sequence motifs present in a cancer with microsatellite instability

Colon cancer and an increasing number of other cancers have been found to exhibit instability of DNA microsatellite sequences. Such tumors have been designated as replication errors (RER) tumors. However, as microsatellites are only rarely found within coding regions of the genome, instability of these sequences cannot directly contribute to carcinogenesis. Recently, we have shown RER colon cancers also demonstrate a marked 100-fold increase in mutation rates measured within an expressed gene, hprt, suggesting the mutator phenotype in these tumors extends beyond microsatellite sequences. To determine whether the RER phenotype indeed destabilizes non-repetitive DNA sequences we have sequenced hprt gene mutations recovered from the RER colon cancer cell line RKO. Greater than 10% of hprt mutants proved to be a single 3 bp deletion located in a nonrepetitive ATTAT sequence motif. Additionally, 1-4 bp deletions or insertions were found to be randomly located throughout the hprt gene. Lastly, one third of hprt mutations proved to be transitions or transversions. The microsatellite instability demonstrated in RKO is thus a global mutator phenotype which destabilizes DNA sequences both inside and outside of repetitive sequence elements and which augments base substitutions as well as frameshifts. These findings extend the characteristics of mutations associated with RER tumors and suggest additional mechanisms by which mutator phenotypes may alter target oncogenes and tumor suppressor genes.

Authors
Eshleman, JR; Markowitz, SD; Donover, PS; Lang, EZ; Lutterbaugh, JD; Li, G-M; Longley, M; Modrich, P; Veigl, ML; Sedwick, WD
MLA Citation
Eshleman, JR, Markowitz, SD, Donover, PS, Lang, EZ, Lutterbaugh, JD, Li, G-M, Longley, M, Modrich, P, Veigl, ML, and Sedwick, WD. "Diverse hypermutability of multiple expressed sequence motifs present in a cancer with microsatellite instability." Oncogene 12.7 (1996): 1425-1432.
PMID
8622858
Source
scival
Published In
Oncogene: Including Oncogene Reviews
Volume
12
Issue
7
Publish Date
1996
Start Page
1425
End Page
1432

Hmutlα and hmutsα in human mismatch repair

Strand-specific mismatch correction in human cells occurs by a mechanism similar to that of the E. coli reaction, and like the bacterial pathway, the human system functions in mutation avoidance. Using an in vitro assay, we have identified mismatch repair defects in a number of hypermutable human cell lines, including a set of lines derived from RER+ tumors. More than 90% of the genetically unstable tumor cell lines tested to date are deficient in the reaction, suggesting that mismatch repair defects are associated with the vast majority of RER+ tumors. These hypermutable cell lines define several in vitro complementation groups, and activities that complement two of these have been isolated in pure form. hMutLα is a 1:1 complex of the MutL homologs MLH1 and PMS2, while tiMutSα is also a heterodimer of the MutS homologs MSH2 and p160 (GTBP). The hMutSα heterodimer specifically recognizes base-base mismatches, small insertion/deletion mispairs, and several types of chemical lesions.

Authors
Drummond, J; Li, G-M; Duckett, D; Modrich, P
MLA Citation
Drummond, J, Li, G-M, Duckett, D, and Modrich, P. "Hmutlα and hmutsα in human mismatch repair." FASEB Journal 10.6 (1996): A1000-.
Source
scival
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
10
Issue
6
Publish Date
1996
Start Page
A1000

MSH6, a Saccharomyces cerevisiae protein that binds to mismatches as a heterodimer with MSH2

The process of post-replicative DNA-mismatch repair seems to be highly evolutionary conserved. In Escherichia coli, DNA mismatches are recognized by the MutS protein [1,2]. Homologues of the E. coli mutS and mutL mismatch-repair genes have been identified in other prokaryotes, as well as in yeast and mammals (see [3] for review). Recombinant Saccharomyces cerevisiae MSH2 (MSH for MutS homologue) [4] and human hMSH2 proteins [5,6] have been shown to bind to mismatch-containing DNA in vitro. However, the physiological role of hMSH2 is unclear, as shown by the recent finding that the mismatch-binding factor hMutSα isolated from extracts of human cells is a heterodimer of hMSH2 and another member of the MSH family, GTBP [7,8]. It has been reported that S. cerevisiae possesses a mismatch-binding activity, which most probably contains MSH2 [9]. We show here that, as in human cells, the S. cerevisiae binding factor is composed of MSH2 and a new functional MutS homologue, MSH6, identified by its homology to GTBP. © Current Biology Ltd. ISSN 0960-9822.

Authors
Iaccarino, I; Palombo, F; Drummond, J; Totty, NF; Hsuan, JJ; Modrich, P; Jiricny, J
MLA Citation
Iaccarino, I, Palombo, F, Drummond, J, Totty, NF, Hsuan, JJ, Modrich, P, and Jiricny, J. "MSH6, a Saccharomyces cerevisiae protein that binds to mismatches as a heterodimer with MSH2." Current Biology 6.4 (1996): 484-486.
PMID
8723353
Source
scival
Published In
Current Biology
Volume
6
Issue
4
Publish Date
1996
Start Page
484
End Page
486

Mismatch repair in replication fidelity, genetic recombination, and cancer biology

Mismatch repair stabilizes the cellular genome by correcting DNA replication errors and by blocking recombination events between divergent DNA sequences. The reaction responsible for strand-specific correction of mispaired bases has been highly conserved during evolution, and homologs of bacterial MutS and MutL, which play key roles in mismatch recognition and initiation of repair, have been identified in yeast and mammalian cells. Inactivation of genes encoding these activities results in a large increase in spontaneous mutability, and in the case of mice and men, predisposition to tumor development.

Authors
Modrich, P; Lahue, R
MLA Citation
Modrich, P, and Lahue, R. "Mismatch repair in replication fidelity, genetic recombination, and cancer biology." Annual Review of Biochemistry 65 (1996): 101-133.
PMID
8811176
Source
scival
Published In
Annual Review of Biochemistry
Volume
65
Publish Date
1996
Start Page
101
End Page
133

A structural basis for a phosphoramide mustard-induced DNA interstrand cross-link at 5'-d(GAC).

Phosphoramide mustard-induced DNA interstrand cross-links were studied both in vitro and by computer simulation. The local determinants for the formation of phosphoramide mustard-induced DNA interstrand cross-links were defined by using different pairs of synthetic oligonucleotide duplexes, each of which contained a single potentially cross-linkable site. Phosphoramide mustard was found to cross-link dG to dG at a 5'-d(GAC)-3'. The structural basis for the formation of this 1,3 cross-link was studied by molecular dynamics and quantum chemistry. Molecular dynamics indicated that the geometrical proximity of the binding sites also favored a 1,3 dG-to-dG linkage over a 1,2 dG-to-dG linkage in a 5'-d(GCC)-3' sequence. While the enthalpies of 1,2 and 1,3 mustard cross-linked DNA were found to be very close, a 1,3 structure was more flexible and may therefore be in a considerably higher entropic state.

Authors
Dong, Q; Barsky, D; Colvin, ME; Melius, CF; Ludeman, SM; Moravek, JF; Colvin, OM; Bigner, DD; Modrich, P; Friedman, HS
MLA Citation
Dong, Q, Barsky, D, Colvin, ME, Melius, CF, Ludeman, SM, Moravek, JF, Colvin, OM, Bigner, DD, Modrich, P, and Friedman, HS. "A structural basis for a phosphoramide mustard-induced DNA interstrand cross-link at 5'-d(GAC)." Proc Natl Acad Sci U S A 92.26 (December 19, 1995): 12170-12174.
PMID
8618865
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
92
Issue
26
Publish Date
1995
Start Page
12170
End Page
12174

Restoration of mismatch repair to nuclear extracts of H6 colorectal tumor cells by a heterodimer of human MutL homologs

Hypermutable H6 colorectal tumor cells are defective in strand-specific mismatch repair and bear defects in both alleles of the hMLHI gene. We have purified to near homogeneity an activity from HeLa cells that complements H6 nuclear extracts to restore repair proficiency on a set of heteroduplex DNAs representing the eight base-base mismatches as well as a number of slipped- strand, insertion/deletion mispairs. This activity behaves as a single species during fractionation and copurifies with proteins of 85 and 110 kDa. Microsequence analysis demonstrated both of these proteins to be homologs of bacterial MutL, with the former corresponding to the hMLHI product and the latter to the product of hPMS2 or a closely related gene. The 1:1 molar stoichiometry of the two polypeptides and their hydrodynamic behavior indicate formation of a heterodimer, which we have designated hMutLα. These observations indicate that interactions between members of the family of human MutL homologs may be restricted.

Authors
Li, G-M; Modrich, P
MLA Citation
Li, G-M, and Modrich, P. "Restoration of mismatch repair to nuclear extracts of H6 colorectal tumor cells by a heterodimer of human MutL homologs." Proceedings of the National Academy of Sciences of the United States of America 92.6 (1995): 1950-1954.
PMID
7892206
Source
scival
Published In
Proceedings of the National Academy of Sciences of USA
Volume
92
Issue
6
Publish Date
1995
Start Page
1950
End Page
1954
DOI
10.1073/pnas.92.6.1950

A human protein with antimutator activity.

Authors
Modrich, P
MLA Citation
Modrich, P. "A human protein with antimutator activity." Japanese journal of cancer research : Gann 86.2 (1995): inside front cover-.
PMID
7730133
Source
scival
Published In
Japanese journal of cancer research : Gann
Volume
86
Issue
2
Publish Date
1995
Start Page
inside front cover

Human protein with antimutator activity

Authors
Modrich, P
MLA Citation
Modrich, P. "Human protein with antimutator activity." Japanese Journal of Cancer Research 86.12 (1995): 1226-1227.
Source
scival
Published In
Japanese Journal of Cancer Research
Volume
86
Issue
12
Publish Date
1995
Start Page
1226
End Page
1227

Mismatch repair, genetic stability and tumour avoidance.

Escherichia coli methyl-directed mismatch repair eliminates premutagenic lesions that arise via DNA biosynthetic errors; components of the repair system also block ectopic recombination between diverged DNA sequences. A mismatch-dependent, methyl-directed excision reaction that accounts for function of the system in replication fidelity has been reconstituted in a purified system dependent on ten activities. The reaction displays a broad specificity for mismatched base pairs and is characterized by an unusual bidirectional excision capability. Human cell nuclear extracts support strand-specific mismatch correction in a reaction that is similar to bacterial repair, with respect to both mismatch specificity and unusual features of mechanism. Like the bacterial system, the human pathway also functions in mutation avoidance because several classes of mutator human cells are deficient in the reaction. These include an alkylation-tolerance cell line that is resistant to the cytotoxic action of N-methyl-N'-nitro-nitrosoguanidine, as well as hypermutable RER+ tumour cells such as those associated with hereditary non-polyposis colon cancer. In vitro experiments indicate that the human repair reaction is dependent on at least six activities, excluding DNA ligase, and that distinct defects in the system can lead to the RER+ phenotype.

Authors
Modrich, P
MLA Citation
Modrich, P. "Mismatch repair, genetic stability and tumour avoidance." Philosophical transactions of the Royal Society of London. Series B: Biological sciences 347.1319 (1995): 89-95.
PMID
7746860
Source
scival
Published In
Philosophical transactions of the Royal Society of London. Series B: Biological sciences
Volume
347
Issue
1319
Publish Date
1995
Start Page
89
End Page
95

Mapping site-specific endonuclease binding to DNA by direct imaging with atomic force microscopy (AFM)

Physical mapping of DNA can be accomplished by direct AFM imaging of site specific proteins bound to DNA molecules. Using Gln-111, a mutant of EcoRI endonuclease with a specific affinity for EcoRI sites 1000 times greater than wild type enzyme but with cleavage rate constants reduced by a factor of 10 4, we demonstrate site-specific mapping by direct AFM imaging. Images are presented showing specific-site binding of Gln-111 to plasmids having either one (pBS +) or two (pMP 32) EcoRI sites. Identification of the Gln-111/DNA complex is greatly enhanced by biotinylation of the complex followed by reaction with streptavidin gold prior to imaging. Image enhancement coupled with improvements in our preparation techniques for imaging large DNA molecules, such as lambda DNA (47 kb), has the potential to contribute to direct AFM restriction mapping of cosmid-sized genomic DNAs.

Authors
Allison, DP; Thundat, TG; Modrich, P; Isfort, RJ; Doktycz, MJ; Kerper, PS; Warmack, RJ
MLA Citation
Allison, DP, Thundat, TG, Modrich, P, Isfort, RJ, Doktycz, MJ, Kerper, PS, and Warmack, RJ. "Mapping site-specific endonuclease binding to DNA by direct imaging with atomic force microscopy (AFM)." Proceedings of SPIE - The International Society for Optical Engineering 2386 (1995): 24-29.
Source
scival
Published In
Proceedings of SPIE - The International Society for Optical Engineering
Volume
2386
Publish Date
1995
Start Page
24
End Page
29

Isolation of an hMSH2-p160 heterodimer that restores DNA mismatch repair to tumor cells

A mismatch-binding heterodimer of hMSH2 and a 160-kilodalton polypeptide has been isolated from HeLa cells by virtue of its ability to restore mismatch repair to nuclear extracts of hMSH2-deficient LoVo colorectal tumor cells. This heterodimer, designated hMutSα; also restores mismatch repair to extracts of alkylation-tolerant MT1 lymphoblastoid cells and HCT-15 colorectal tumor cells, which are selectively defective in the repair of base-base and single-nucleotide insertion-deletion mismatches. Because HCT-15 cells appear to be free of hMSH2 mutations, this selective repair defect is likely a result of a deficiency of the hMutSα 160-kilodalton subunit, and mutations in the corresponding gene may confer hypermutability and cancer predisposition.

Authors
Drummond, JT; Li, G-M; Longley, MJ; Modrich, P
MLA Citation
Drummond, JT, Li, G-M, Longley, MJ, and Modrich, P. "Isolation of an hMSH2-p160 heterodimer that restores DNA mismatch repair to tumor cells." Science 268.5219 (1995): 1909-1912.
PMID
7604264
Source
scival
Published In
Science
Volume
268
Issue
5219
Publish Date
1995
Start Page
1909
End Page
1912

Mismatch repair deficiency in phenotypically normal human cells

Tumor cells in patients with hereditary nonpolyposis colorectal cancer (HNPCC) are characterized by a genetic hypermutability caused by defects in DNA mismatch repair. A subset of HNPCC patients was found to have widespread mutations not only in their tumors, but also in their non-neoplastic cells. Although these patients had numerous mutations in all tissues examined, they had very few tumors. The hypermutability was associated with a profound defect in mismatch repair at the biochemical level. These results have implications for the relation between mutagenesis and carcinogenesis, and they suggest that mismatch repair deficiency is compatible with normal human development.

Authors
Parsons, R; Li, G-M; Longley, M; Modrich, P; Liu, B; Berk, T; Hamilton, SR; Kinzler, KW; Vogelstein, B
MLA Citation
Parsons, R, Li, G-M, Longley, M, Modrich, P, Liu, B, Berk, T, Hamilton, SR, Kinzler, KW, and Vogelstein, B. "Mismatch repair deficiency in phenotypically normal human cells." Science 268.5211 (1995): 738-740.
PMID
7632227
Source
scival
Published In
Science
Volume
268
Issue
5211
Publish Date
1995
Start Page
738
End Page
740

Misincorporation and mispaired primer extension by human immunodeficiency virus reverse transcriptase

Pre-steady-state methods were used to study the fidelity of human immunodeficiency virus reverse transcriptase. Fidelity of DNA-directed DNA synthesis can be attributed to a 1-2 order of magnitude reduction in affinity for noncomplementary dNTPs, and a 1-4 order of magnitude reduction in the rate of the conformational change that limits the rate of nucleotide addition. Affinities of reverse transcriptase for paired or mispaired primer termini are similar. Discrimination against a mispaired primer is due to reduction in affinity for the next dNTP and reduction in rate of extension. Extension of mispaired termini proceeds 20-700-fold faster than the rate of dissociation of reverse transcriptase from the primer-template and is 2-3 orders of magnitude more frequent than nucleotide misincorporation. The rate- limiting step for extension of a mispaired terminus occurs at the conformational change or chemical step, depending on the nature of the mispair. Presence of a mismatch at the 3' penultimate position reduces pyrophosphorolysis of the primer by a factor of 103, indicating that mispairs 5' to the site of chemistry can also affect catalysis.

Authors
Zinnen, S; Hsieh, J-C; Modrich, P
MLA Citation
Zinnen, S, Hsieh, J-C, and Modrich, P. "Misincorporation and mispaired primer extension by human immunodeficiency virus reverse transcriptase." Journal of Biological Chemistry 269.39 (1994): 24195-24202.
PMID
7523369
Source
scival
Published In
Journal of Biological Chemistry
Volume
269
Issue
39
Publish Date
1994
Start Page
24195
End Page
24202

Mismatch repair, genetic stability, and cancer

Authors
Modrich, P
MLA Citation
Modrich, P. "Mismatch repair, genetic stability, and cancer." Science 266.5193 (1994): 1959-1960.
PMID
7801122
Source
scival
Published In
Science
Volume
266
Issue
5193
Publish Date
1994
Start Page
1959
End Page
1960

Mismatch repair proteins MutS and MutL inhibit RecA-catalyzed strand transfer between diverged DNAs

Bacterial mutS and mutL mutations confer large increases in recombination between sequences that are divergent by several percent at the nucleotide level, an effect attributed to a role for products of these genes in control of recombination fidelity. Since MutS and MutL are proteins involved in the earliest steps of mismatch repair, including mismatch recognition by MutS, we have tested the possibility that they may affect strand exchange in response to occurrence of mispairs within the recombination heteroduplex. We show that MutS abolishes RecA-catalyzed strand transfer between fd and M13 bacteriophage DNAs, which vary by 3% at the nucleotide level, but is without effect on M13-M13 or fd-fd exchange. Although MutL alone has no effect on M13-fd heteroduplex formation, the protein dramatically enhances the inhibition of strand transfer mediated by MutS. Analysis of strand-transfer intermediates that accumulate in the presence of MutS and MutL indicates that the proteins block branch migration, presumably in response to occurrence of mispairs within newly formed heteroduplex.

Authors
Jr, LW; Clark, S; Radman, M; Modrich, P
MLA Citation
Jr, LW, Clark, S, Radman, M, and Modrich, P. "Mismatch repair proteins MutS and MutL inhibit RecA-catalyzed strand transfer between diverged DNAs." Proceedings of the National Academy of Sciences of the United States of America 91.8 (1994): 3238-3241.
PMID
8159731
Source
scival
Published In
Proceedings of the National Academy of Sciences of the United States of America
Volume
91
Issue
8
Publish Date
1994
Start Page
3238
End Page
3241

Genomic mismatch scanning: a new approach to genetic linkage mapping.

Genomic mismatch scanning (GMS) is a new method of genetic linkage analysis that does not require conventional polymorphic markers or gel electrophoresis. GMS is ideally suited to affected-relative-pair mapping. DNA fragments from all regions of identity-by-descent between two relatives are isolated based on their ability to form extensive mismatch-free hybrid molecules. The genomic origin of this selected pool of DNA fragments is then mapped in a single hybridization step. Here we demonstrate the practicality of GMS in a model organism, Saccharomyces cerevisiae. GMS is likely to be applicable to other organisms, including humans, and may be of particular value in mapping complex genetic traits.

Authors
Nelson, SF; McCusker, JH; Sander, MA; Kee, Y; Modrich, P; Brown, PO
MLA Citation
Nelson, SF, McCusker, JH, Sander, MA, Kee, Y, Modrich, P, and Brown, PO. "Genomic mismatch scanning: a new approach to genetic linkage mapping." Nat Genet 4.1 (May 1993): 11-18.
PMID
8513319
Source
pubmed
Published In
Nature Genetics
Volume
4
Issue
1
Publish Date
1993
Start Page
11
End Page
18
DOI
10.1038/ng0593-11

Mismatch repair and genetic stability in human cells.

Authors
Fang, WH; Li, GM; Longley, M; Holmes, J; Thilly, W; Modrich, P
MLA Citation
Fang, WH, Li, GM, Longley, M, Holmes, J, Thilly, W, and Modrich, P. "Mismatch repair and genetic stability in human cells." Cold Spring Harb Symp Quant Biol 58 (1993): 597-603.
PMID
7956074
Source
pubmed
Published In
Cold Spring Harbor Laboratory: Symposia on Quantitative Biology
Volume
58
Publish Date
1993
Start Page
597
End Page
603

Kinetic mechanism of the DNA-dependent DNA polymerase activity of human immunodeficiency virus reverse transcriptase

The kinetic pathway of DNA-dependent DNA polymerase activity of human immunodeficiency virus reverse transcriptase (HIV RT) as determined by pre-steady-state methods using a defined primer/template is as follows, equation presented where E is RT, Dn,n+1 is primer/template, dNTP is deoxyribonucleoside triphosphate, and PPi is pyrophosphate. The rate-determining step for enzyme turnover in single nucleotide addition is the dissociation of enzyme from DNA (k6 = 0.11 S-1). The observation of an E′·DNA·dNTP intermediate by pulse-chase analysis and the absence of a phosphorothioate elemental effect identified the rate-limiting step for nucleotide addition as a conformational change of the E·DNA·dNTP complex (K3 = 83 s-1) prior to the chemical step. Biphasic kinetics of single-turnover pyrophosphorolysis suggested that this conformational change (k-3 = 0.3 s-1 ) is also rate-limiting for the reverse reaction. The equilibrium constant for the chemical step (K4) is 3.8, in slight favor of the forward reaction. The large equilibrium constant (K3 = 280) for the conformational change effectively renders nucleotide addition kinetically irreversible. The dissociation constant for primer/template is 26 nM, and the association rate of enzyme and DNA (K1) is 2.3 × 106 M-1 s-1. Equilibrium dissociation constants for dTTP and PPi are 18 μM and 7.2 mM, respectively. Mg2+ enhances productive interaction of RT with DNA as judged by a 50% increase in burst amplitude in the single nucleotide addition reaction and by an 8-fold decrease in KD for the RT·DNA complex as determined by gel mobility shift assay. Secondary interactions of the RT·DNA complex with free DNA were observed in the absence of Mg2+.

Authors
Hsieh, J-C; Zinnen, S; Modrich, P
MLA Citation
Hsieh, J-C, Zinnen, S, and Modrich, P. "Kinetic mechanism of the DNA-dependent DNA polymerase activity of human immunodeficiency virus reverse transcriptase." Journal of Biological Chemistry 268.33 (1993): 24607-24613.
PMID
7693703
Source
scival
Published In
Journal of Biological Chemistry
Volume
268
Issue
33
Publish Date
1993
Start Page
24607
End Page
24613

Methyl-directed mismatch repair is bidirectional

Methyl-directed mismatch repair is initiated by the mismatch-provoked, MutHLS-dependent cleavage of the unmodified strand at a hemimethylated d(GATC) sequence. This reaction is independent of the polarity of the unmodified strand and can occur either 3′ or 5′ to the mismatch on the unmethylated strand (Au, K. G., Welsh, K., and Modrich, P. (1992) J. Biol. Chem. 267, 12142-12148). The overall repair reaction also occurs without regard to polarity of the unmethylated strand. Both hemimethylated configurations of a linear heteroduplex containing a single d(GATC) sequence are subject to methyl-directed correction in Escherichia coli extracts and in a purified repair system. Repair of both heteroduplex orientations requires MutH, MutL, MutS, DNA helicase II, SSB, and DNA polymerase III holoenzyme, but the two substrates differ with respect to exonuclease requirements for correction. When the unmethylated d(GATC) sequence that directs repair is located 5′ to the mismatch on the unmodified strand, mismatch correction requires the 5′ → 3′ hydrolytic activity of exonuclease VII or RecJ exonuclease. Repair directed by an unmodified d(GATC) sequence situated 3′ to the mismatch depends on the 3′ → 5′ activity of exonuclease I. Specific requirements for these activities are evident with circular heteroduplexes containing a single asymmetrically placed d(GATC) sequence, with the requirement for a 5′ → 3′ or 3′ → 5′ hydrolytic activity being determined by the orientation of the unmethylated strand along the shorter path joining the two sites in the DNA circle. This observation suggests that the methyl-directed repair system utilizes the proximal d(GATC) sequence to direct correction. To our knowledge, these experiments represent the first instance in which exonuclease I, exonuclease VII, and RecJ have been implicated in a particular DNA metabolic pathway.

Authors
Cooper, DL; Lahue, RS; Modrich, P
MLA Citation
Cooper, DL, Lahue, RS, and Modrich, P. "Methyl-directed mismatch repair is bidirectional." Journal of Biological Chemistry 268.16 (1993): 11823-11829.
PMID
8389365
Source
scival
Published In
Journal of Biological Chemistry
Volume
268
Issue
16
Publish Date
1993
Start Page
11823
End Page
11829

An alkylation-tolerant, mutator human cell line is deficient in strand-specific mismatch repair

The human lymphoblastoid MTl B-cell line was previously isolated as one of a series of mutant cells able to survive the cytotoxic effects of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). MT1 cells nevertheless remain sensitive to mutagenesis by MNNG and display a mutator phenotype. These phenotypes have been attributed to a single genetic alteration postulated to confer a defect in strand-specific mismatch repair, a proposal that attributes the cytotoxic effect of DNA alkylation in wild-type cells to futile attempts to correct mispairs that arise during replication of alkylated template strands. Our results support this view. MNNG-induced mutations in the HPRT gene of MT1 cells are almost exclusively G·C → A·T transitions, while spontaneous mutations observed in this mutator cell line are single-nucleotide insertions, transversions, and A·T → G·C transitions. In vitro assay has demonstrated that the MT1 line is in fact deficient in strand-specific correction of all eight base-base mispairs. This defect, which is manifest at or prior to the excision stage of the reaction, is due to simple deficiency of a required activity because MT1 nuclear extracts can be complemented by a partially purified HeLa fraction to restore in vitro repair. These findings substantiate the idea that strand-specific mismatch repair contributes to alkylation-induced cytotoxicity and imply that this process serves as a barrier to spontaneous transition, transversion, and insertion/deletion mutations in mammalian cells.

Authors
Kat, A; Thilly, WG; Fang, W-H; Longley, MJ; Li, G-M; Modrich, P
MLA Citation
Kat, A, Thilly, WG, Fang, W-H, Longley, MJ, Li, G-M, and Modrich, P. "An alkylation-tolerant, mutator human cell line is deficient in strand-specific mismatch repair." Proceedings of the National Academy of Sciences of the United States of America 90.14 (1993): 6424-6428.
PMID
8341649
Source
scival
Published In
Proceedings of the National Academy of Sciences of the United States of America
Volume
90
Issue
14
Publish Date
1993
Start Page
6424
End Page
6428

Human strand-specific mismatch repair occurs by a bidirectional mechanism similar to that of the bacterial reaction

Nuclear extracts prepared from a HeLa cell line have been previously shown to support strand-specific repair of heteroduplex DNAs containing a site-specific, strand-specific incision (Holmes, J. J., Clark, S., and Modrich, P. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 5837-5841; Thomas, D. C., Roberts, J. D., and Kunkel, T. A. (1991) J. Biol. Chem. 266, 3744-3751). Further analysis of the substrate specificity of the reaction has shown that in addition to G-T, A-C, G-G, and C-C, nuclear extracts also recognize and correct in a strand-specific manner A-A, A-G, T-T, and C-T mismatches, with repair in each case being inhibited by aphidicolin. The rate of repair of a circular G-T heteroduplex was found to decrease monotonically with increasing separation between the mismatch and the strand break that targets repair, as viewed along the shorter path joining the two sites in the circular substrate. This decrease is independent of the polarity of the incised strand, suggesting that the human pathway of mismatch correction may possess a bidirectional excision capability similar to that of the Escherichia coli methyl-directed system. This possibility was confirmed by analysis of excision tracts associated with the reaction. Inhibition of DNA synthesis by aphidicolin or by omission of exogenous dNTPs leads to the mismatch-provoked formation of a single-strand gap that spans the shorter path between the strand break and the mismatch, irrespective of the polarity of the incised strand. Formation of these gaps, which extend from the site of the strand break to terminate at a number of discrete sites in the region 90 to 170 nucleotides beyond the mismatch, is therefore independent of the relative orientation of the two sites. Based on similar mismatch specificities and common features of mechanism, we have concluded that the human strandspecific mismatch repair system is functionally homologous to the bacterial methyl-directed pathway.

Authors
Fang, W-H; Modrich, P
MLA Citation
Fang, W-H, and Modrich, P. "Human strand-specific mismatch repair occurs by a bidirectional mechanism similar to that of the bacterial reaction." Journal of Biological Chemistry 268.16 (1993): 11838-11844.
PMID
8505312
Source
scival
Published In
Journal of Biological Chemistry
Volume
268
Issue
16
Publish Date
1993
Start Page
11838
End Page
11844

Bidirectional excision in methyl-directed mismatch repair

Using electron microscopy and indirect end-labeling methods, we have examined excision tracts produced by the Escherichia coli methyl-directed mismatch repair system on a closed circular G-T heteroduplex that contains a single d(GATC) site. Despite differing polarities of the unmodified strand in the two hemimethylated derivatives of the heteroduplex, that portion of the unmethylated strand spanning the shorter path between the d(GATC) site and mismatch is targeted for excision in both cases. Mismatch-provoked excision occurring on both hemimethylated DNAs requires DNA helicase II, but exonuclease requirements for the reaction depend on heteroduplex orientation. When the d(GATC) sequence on the unmodified strand resides 3′ to the mismatch as viewed along the shorter path, excision requires exonuclease I. Excision occurring on the alternate hemimethylated heteroduplex depends on the 5′ → 3′' hydrolytic activity of exonuclease VII. Coupled with the previous demonstration that repair initiates via the mismatch-provoked, MutHLS-dependent incision of the unmethylated strand at a d(GATC) sequence (Au, K. G., Welsh, K., and Modrich, P. (1992) J. Biol. Chem. 267, 12142-12148), these findings indicate an excision mechanism in which helicase II displacement renders the incised strand sensitive to the appropriate single-strand exonuclease. Our data imply that hydrolysis commences at the d(GATC) site, proceeds to a point beyond the mismatch, and terminates at a number of discrete sites within a 100-nucleotide region just beyond this site. The extent of excision is therefore controlled by one or more components of the repair system.

Authors
Grilley, M; Griffith, J; Modrich, P
MLA Citation
Grilley, M, Griffith, J, and Modrich, P. "Bidirectional excision in methyl-directed mismatch repair." Journal of Biological Chemistry 268.16 (1993): 11830-11837.
PMID
8505311
Source
scival
Published In
Journal of Biological Chemistry
Volume
268
Issue
16
Publish Date
1993
Start Page
11830
End Page
11837

The DNA helicase activities of Rad3 protein of Saccharomyces cerevisiae and helicase II of Escherichia coli are differentially inhibited by covalent and noncovalent DNA modifications

Rad3 protein of Saccharomyces cerevisiae is a DNA-dependent ATPase that acts as a DNA helicase on partially duplex substrates. Rad3 protein is required for damage-specific incision of DNA during the nucleotide excision repair (NER) pathway in yeast. Helicase II of Escherichia coli is also a DNA helicase, but it is involved in postincision events in NER. Previous investigations have demonstrated that the DNA helicase activities of Rad3 protein and helicase II are both inhibited by DNA damage. In the present study we have compared the response of yeast Rad3 protein and E. coli helicase II to a broad spectrum of DNA modifications. The Rad3 helicase activity is considerably more sensitive to ultraviolet radiation damage and cisplatin adducts in DNA than to drugs that interact noncovalently with duplex DNA. Conversely, E. coli helicase II is highly sensitive to noncovalent DNA modifications but less sensitive than Rad3 protein to ultraviolet radiation damage or cisplatin adducts. We also show that Rad3 protein and helicase II differ in their ability to form stable protein-DNA complexes at sites of DNA damage. Hence, DNA helicases that catalyze distinct steps in NER respond differently to chemical and conformational states of the DNA substrate. The observation that Rad3 protein is particularly sensitive to covalent but not noncovalent alterations in DNA structure is consistent with the hypothesis that this enzyme may have adopted a highly specialized role in damage-specific recognition during NER.

Authors
Naegeli, H; Modrich, P; Friedberg, EC
MLA Citation
Naegeli, H, Modrich, P, and Friedberg, EC. "The DNA helicase activities of Rad3 protein of Saccharomyces cerevisiae and helicase II of Escherichia coli are differentially inhibited by covalent and noncovalent DNA modifications." Journal of Biological Chemistry 268.14 (1993): 10386-10392.
PMID
8387518
Source
scival
Published In
Journal of Biological Chemistry
Volume
268
Issue
14
Publish Date
1993
Start Page
10386
End Page
10392

Hypermutability and mismatch repair deficiency in RER+ tumor cells

A subset of sporadic colorectal tumors and most tumors developing in hereditary nonpolyposis colorectal cancer patients display frequent alterations in microsatellite sequences. Such tumors have been thought to manifest replication errors (RER+), but the basis for the alterations has remained conjectural. We demonstrate that the mutation rate of (CA)n repeats in RER+ tumor cells is at least 100-fold that in RER- tumor cells and show by in vitro assay that increased mutability of RER+ cells is associated with a profound defect in strand-specific mismatch repair. This deficiency was observed with microsatellite heteroduplexes as well as with heteroduplexes containing single base-base mismatches and affected an early step in the repair pathway. Thus, a true mutator phenotype exists in a subset of tumor cells, the responsible defect is likely to cause transitions and transversions in addition to microsatellite alterations, and a biochemical basis for this phenotype has been identified.

Authors
Parsons, R; Li, G-M; Longley, MJ; Fang, W-H; Papadopoulos, N; Jen, J; Chapelle, ADL; Kinzler, KW; Vogelstein, B; Modricht, P
MLA Citation
Parsons, R, Li, G-M, Longley, MJ, Fang, W-H, Papadopoulos, N, Jen, J, Chapelle, ADL, Kinzler, KW, Vogelstein, B, and Modricht, P. "Hypermutability and mismatch repair deficiency in RER+ tumor cells." Cell 75.6 (1993): 1227-1236.
PMID
8261516
Source
scival
Published In
Cell
Volume
75
Issue
6
Publish Date
1993
Start Page
1227
End Page
1236

Initiation of methyl-directed mismatch repair

Escherichia coli MutH possesses an extremely weak d(GATC) endonuclease that responds to the state of methylation of the sequence (Welsh, K. M., Lu, A.-L., Clark, S., and Modrich, P. (1987) J. Biol. Chem. 262, 15624-15629). MutH endonuclease is activated in a reaction that requires MutS, MutL, ATP, and Mg2+ and depends upon the presence of a mismatch within the DNA. The degree of activation correlates with the efficiency with which a particular mismatch is subject to methyl-directed repair (G-T > G-G > A-C > C-C), and activated MutH responds to the state of DNA adenine methylation. Incision of an unmethylated strand occurs immediately 5′ to a d(GATC) sequence, leaving 5′ phosphate and 3′ hydroxy termini (p N ↓ p G p A p-TpC). Unmethylated d(GATC) sites are subject to double strand cleavage by activated MutH, an effect that may account for the killing of dam- mutants by 2-aminopurine. The mechanism of activation apparently requires ATP hydrolysis since adenosine-5′-O-(3-thiotriphosphate) not only fails to support the reaction but also inhibits activation promoted by ATP. The process has no obligate polarity as d(GATC) site incision by the activated nuclease can occur either 3′ or 5′ to the mismatch on an unmethylated strand. However, activation is sensitive to DNA topology. Circular heteroduplexes are better substrates than linear molecules, and activity of DNAs of the latter class depends on placement of the mismatch and d(GATC) site within the molecule. MutH activation is supported by a 6-kilobase linear heteroduplex in which the mismatch and d(GATC) site are centrally located and separated by 1 kilobase, but a related molecule, in which the two sites are located near opposite ends of the DNA, is essentially inactive as substrate. We conclude that MutH activation represents the initiation stage of methyl-directed repair and suggest that interaction of a mismatch and a d(GATC) site is provoked by MutS binding to a mispair, with subsequent ATP-dependent translocation of one or more Mut proteins along the helix leading to cleavage at a d(GATC) sequence on either side of the mismatch.

Authors
Au, KG; Welsh, K; Modrich, P
MLA Citation
Au, KG, Welsh, K, and Modrich, P. "Initiation of methyl-directed mismatch repair." Journal of Biological Chemistry 267.17 (1992): 12142-12148.
PMID
1601880
Source
scival
Published In
The Journal of biological chemistry
Volume
267
Issue
17
Publish Date
1992
Start Page
12142
End Page
12148

Mechanisms and biological effects of mismatch repair

Authors
Modrich, P
MLA Citation
Modrich, P. "Mechanisms and biological effects of mismatch repair." Annual Review of Genetics 25 (1991): 229-253.
PMID
1812808
Source
scival
Published In
Annual Review of Genetics
Volume
25
Publish Date
1991
Start Page
229
End Page
253

Mechanisms of DNA-mismatch correction.

Authors
Grilley, M; Holmes, J; Yashar, B; Modrich, P
MLA Citation
Grilley, M, Holmes, J, Yashar, B, and Modrich, P. "Mechanisms of DNA-mismatch correction." Mutat Res 236.2-3 (September 1990): 253-267. (Review)
PMID
2144613
Source
pubmed
Published In
Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis
Volume
236
Issue
2-3
Publish Date
1990
Start Page
253
End Page
267

Strand-specific mismatch correction in nuclear extracts of human and Drosophila melanogaster cell lines.

Nuclear extracts derived from HeLa and Drosophila melanogaster KC cell lines have been found to correct single base-base mispairs within open circular DNA heteroduplexes containing a strand-specific, site-specific incision located 808 base pairs from the mismatch. Correction in both extract systems is strand specific, being highly biased to the incised DNA strand. Different mispairs within a homologous set of heteroduplexes were processed with different efficiencies (G.T greater than G.G approximately equal to A.C greater than C.C), and correction was accompanied by mismatch-dependent DNA synthesis localized to the region spanning the mispair and the strand break, thus demonstrating that mismatch recognition is associated with the repair reaction. Correction of each of these heteroduplexes was abolished by aphidicolin but was relatively insensitive to the presence of high concentrations of ddTTP, indicating probable involvement of alpha and/or delta class DNA polymerase(s). These findings suggest that higher eukaryotic cells possess a general, strand-specific mismatch repair system analogous to the Escherichia coli mutHLS and the Streptococcus pneumoniae hexAB pathways, systems that contribute in a major way to the genetic stability of these bacterial species.

Authors
Holmes, J; Clark, S; Modrich, P
MLA Citation
Holmes, J, Clark, S, and Modrich, P. "Strand-specific mismatch correction in nuclear extracts of human and Drosophila melanogaster cell lines." Proc Natl Acad Sci U S A 87.15 (August 1990): 5837-5841.
PMID
2116007
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
87
Issue
15
Publish Date
1990
Start Page
5837
End Page
5841

Methyl-directed DNA mismatch correction

Authors
Modrich, P
MLA Citation
Modrich, P. "Methyl-directed DNA mismatch correction." Journal of Biological Chemistry 264.12 (1989): 6597-6600.
PMID
2651430
Source
scival
Published In
Journal of Biological Chemistry
Volume
264
Issue
12
Publish Date
1989
Start Page
6597
End Page
6600

Gap formation is associated with methyl-directed mismatch correction under conditions of restricted DNA synthesis.

A covalently closed, circular heteroduplex containing a G-T mismatch and a single hemimethylated d(GATC) site is subject to efficient methyl-directed mismatch correction in Escherichia coli extracts when repair DNA synthesis is severely restricted by limiting the concentration of exogenously supplied deoxyribonucleoside-5'-triphosphates or by supplementing reactions with chain-terminating 2',3'-dideoxynucleoside triphosphates. However, repair under these conditions results in formation of a single-strand gap in the region of the molecule containing the mismatch and the d(GATC) site. These findings indicate that repair DNA synthesis required for methyl-directed correction can initiate in the vicinity of the mispair, and they are most consistent with a repair reaction involving 3'----5' excision (or strand displacement) from the d(GATC) site followed by 5'----3' repair DNA synthesis initiating in the vicinity of the mismatch.

Authors
Su, SS; Grilley, M; Thresher, R; Griffith, J; Modrich, P
MLA Citation
Su, SS, Grilley, M, Thresher, R, Griffith, J, and Modrich, P. "Gap formation is associated with methyl-directed mismatch correction under conditions of restricted DNA synthesis." Genome / National Research Council Canada = Genome / Conseil national de recherches Canada 31.1 (1989): 104-111.
PMID
2687086
Source
scival
Published In
Genome / National Research Council Canada = Genome / Conseil national de recherches Canada
Volume
31
Issue
1
Publish Date
1989
Start Page
104
End Page
111

Isolation and characterization of the Escherichia coli mutL gene product

The Escherichia coli mutL gene product has been purified to near homogeneity from an overproducing clone. The mutL locus encodes a polypeptide of 70,000 daltons as determined by denaturing gel electrophoresis. The native molecular weight of MutL protein as calculated from the sedimentation coefficient of 5.5 S and Stokes radius of 61 Å is 139,000 daltons, indicating that MutL exists as a dimer in solution. In addition to its ability to complement methyl-directed DNA mismatch repair in mutL-deficient cell-free extracts, DNase I protection experiments demonstrate that the purified MutL protein interacts with the MutS-heteroduplex DNA complex in the presence of ATP.

Authors
Grilley, M; Welsh, KM; Su, S-S; Modrich, P
MLA Citation
Grilley, M, Welsh, KM, Su, S-S, and Modrich, P. "Isolation and characterization of the Escherichia coli mutL gene product." Journal of Biological Chemistry 264.2 (1989): 1000-1004.
PMID
2536011
Source
scival
Published In
Journal of Biological Chemistry
Volume
264
Issue
2
Publish Date
1989
Start Page
1000
End Page
1004

DNA mismatch correction in a defined system

DNA mismatch correction is a strand-specific process involving recognition of noncomplementary Watson-Crick nucleotide pairs and participation of widely separated DNA sites. The Escherichia coli methyl-directed reaction has been reconstituted in a purified system consisting of MutH, MutL, and MutS proteins, DNA helicase II, single-strand DNA binding protein, DNA polymerase III holoenzyme, exonuclease I, DNA ligase, along with ATP (adenosine triphosphate), and the four deoxynucleoside triphosphates. This set of proteins can process seven of the eight base-base mismatches in a strand-specific reaction that is directed by the state of methylation of a single d(GATC) sequence located I kilobase from the mispair.

Authors
Lahue, RS; Modrich, P
MLA Citation
Lahue, RS, and Modrich, P. "DNA mismatch correction in a defined system." Science 245.4914 (1989): 160-164.
PMID
2665076
Source
scival
Published In
Science
Volume
245
Issue
4914
Publish Date
1989
Start Page
160
End Page
164

Glu-111 is required for activation of the DNA cleavage center of EcoRI endonuclease

Gap repair in the presence of 2'-deoxycytosine 5'-O-(1-thiotriphosphate) has been utilized to mutagenize the amino-terminal one-half of the structural gene for EcoRI endonuclease. This approach has led to identification of over 200 mutants defective in endonuclease function. One mutant protein, which binds to the EcoRI sequence but displays greatly reduced cleavage activity, is the consequence of a Glu to Gly change at position 111. This protein has been purified to homogeneity and characterized in detail. Subunit interactions governing the tetramer to dimer transition of the mutant endonuclease are near normal as are parameters governing its interaction with specific and nonspecific DNA sequences. However, the rate constants for first and second strand cleavage steps are reduced by 60,000- and 30,000-fold, respectively, as a consequence of the Glu→Gly change. The defect in chemical cleavage steps can be partially overcome by elevating the pH of the reaction buffer from 7.6 to 8.5, conditions which enhance the rate of EcoRI* strand cleavage by wild type enzyme to a similar degree. We suggest that the Glu-111 mutation affects an interface between recognition and cleavage functions of the enzyme, an idea consistent with the suggestion that the cleavage center of the endonuclease is subject to activation upon specific recognition of the EcoRI sequence.

Authors
King, K; Benkovic, SJ; Modrich, P
MLA Citation
King, K, Benkovic, SJ, and Modrich, P. "Glu-111 is required for activation of the DNA cleavage center of EcoRI endonuclease." Journal of Biological Chemistry 264.20 (1989): 11807-11815.
PMID
2745417
Source
scival
Published In
Journal of Biological Chemistry
Volume
264
Issue
20
Publish Date
1989
Start Page
11807
End Page
11815

The negative charge of Glu-111 is required to activate the cleavage center of EcoRI endonuclease

King et al. (King, K., Benkovic, S.J., and Modrich, P. (1989) 264, 11807-11815) have shown that Glu-111 is required for DNA cleavage by EcoRI endonuclease and have suggested that this residue is required for activation of the cleavage center upon specific recognition. We have substituted Gln or Asp for Glu-111 by oligonucleotide-directed mutagenesis. First and second strand cleavage rate constants are reduced by a factor of more than 104 by the Gln-111 substitution. However, these rate constants are enhanced 9-fold when pH is increased from 7.6 to 8.5, which enhances strand cleavage at EcoRI* sites by wild type endonuclease to a similar degree. The specific affinity of Gln-111 endonuclease for EcoRI sites is 1000 times greater than that of wild type enzyme reflecting a decrease in the rate constant governing specific complex dissociation. In contrast to Gln-111 endonuclease, the equilibrium specific affinity of Asp-111 endonuclease for the EcoRI sequence is similar to that of wild type enzyme, and first and second strand cleavage rate constants are reduced only 100-fold relative to wild type enzyme. These results suggest that a negative charge on residue 111 is required for strand cleavage and are consistent with participation of Glu-111 in activation of the DNA cleavage center, with energy associated with specific sequence recognition driving this process.

Authors
Wright, DJ; King, K; Modrich, P
MLA Citation
Wright, DJ, King, K, and Modrich, P. "The negative charge of Glu-111 is required to activate the cleavage center of EcoRI endonuclease." Journal of Biological Chemistry 264.20 (1989): 11816-11821.
PMID
2745418
Source
scival
Published In
Journal of Biological Chemistry
Volume
264
Issue
20
Publish Date
1989
Start Page
11816
End Page
11821

Escherichia coli mutY gene encodes an adenine glycosylase active on G-A mispairs

Mutations in the mutY gene of Escherichia coli confer hypermutability reflecting G·C to T·A transversion mutations and result in a deficiency in methyl-independent G-A to G·C mismatch correction. In the present work, the mutY product has been purified to near homogeneity by virtue of its ability to restore G-A to G·C mismatch correction to cell-free extracts of a mutS mutY strain. The 36-Kda protein renders the strand containing the mispaired adenine labile to base-catalyzed cleavage and sensitive to cleavage by several apurinic/apyrimidinic-site endonucleases, with the sites of strand scission by both agents corresponding to the location of the mismatch. These findings indicate that MutY is a DNA glycosylase that hydrolyzes the glycosyl bond linking the mispaired adenine to deoxyribose. MutY, a 5'-apurinic/apyrimidinic-site endonuclease, DNA polymerase I, and DNA ligase are sufficient to reconstitute MutY-dependent G-A to G·C repair in vitro.

Authors
Au, KG; Clark, S; Miller, JH; Modrich, P
MLA Citation
Au, KG, Clark, S, Miller, JH, and Modrich, P. "Escherichia coli mutY gene encodes an adenine glycosylase active on G-A mispairs." Proceedings of the National Academy of Sciences of the United States of America 86.22 (1989): 8877-8881.
PMID
2682664
Source
scival
Published In
Proceedings of the National Academy of Sciences of USA
Volume
86
Issue
22
Publish Date
1989
Start Page
8877
End Page
8881
DOI
10.1073/pnas.86.22.8877

Mismatch-containing oligonucleotide duplexes bound by the E.coli mutS-encoded protein

The binding of the mutS gene product, a protein involved in at least two E. coli mismatch correction pathways, to a series of synthetic DNA duplexes containing mismatches or mismatch analogues of the purine/pyrimidine type was studied in order to establish whether a correlation exists between the recognition of these mispairs and the efficiency of their correction in vivo. Experiments using nitrocellulose filter binding or band-shift assays revealed that duplexes containing a G/T mismatch or its analogues I/T and DI/T were bound by the protein with affinities correlating to the efficiency of their repair in vivo. In contrast, the A/C mismatch, contained within the same sequence, was bound only poorly, despite being efficiently corrected in vivo. The analogues of the A/C mispair, uncorrected in vivo, were not detectably bound under the conditions of these assays. © 1988 IRL Press Limited, Oxford, England.

Authors
Jiricny, J; Su, S-S; Wood, SG; Modrich, P
MLA Citation
Jiricny, J, Su, S-S, Wood, SG, and Modrich, P. "Mismatch-containing oligonucleotide duplexes bound by the E.coli mutS-encoded protein." Nucleic Acids Research 16.16 (1988): 7843-7853.
PMID
3047673
Source
scival
Published In
Nucleic Acids Research
Volume
16
Issue
16
Publish Date
1988
Start Page
7843
End Page
7853
DOI
10.1093/nar/16.16.7843

Escherichia coli mutY gene product is required for specific A-G→C·G mismatch correction

A-G mispairs are subject to correction by two distinct pathways in cell-free extracts of Escherichia coli [Su, S.-S., Lahue, R.S., Au, K.G. & Modrich, P. (1988) J. Biol. Chem. 263, 6829-6835; Lu, A.-L. & Chang, D.Y. (1988) Genetics 118, 593-600]. One is the mutHLS-dependent, methyl-directed pathway that recognizes a variety of mismatches and repairs the unmethylated strand of DNA heteroduplexes that are hemimethylated at d(GATC) sequences. The other pathway appears to be specific for A-G mispairs, yields C·G base pairs exclusively, and is independent of the presence of d(GATC) sites. Analyses of cell-free extracts prepared from E. coli mutY strains and isogenic parents have demonstrated that the mutY gene product is involved in the methyl-independent pathway, which converts A-G mispairs to C·G pairs. The specificity of this activity is consistent with the mutator phenotype associated with the mutY locus, which generates G·C→T·A transversions [Nghiem, Y., Cabrera, M., Cupples, C.G. & Miller, J.H. (1988) Proc. Natl. Acad. Sci. USA 85, 2709-2713]. We propose that the mutY product functions at a late stage of a pathway that excludes A-G mispairs during chromosome replication and that involves the function of the mutT gene product. This model suggests that the mutT function acts at an early stage of this pathway to exclude A-G mismatches where the adenine resides on the template DNA strand. A-G mispairs that persist after passage of the replication fork would contain guanine on the template strand and thus be processed to C·G base pairs by the mutY-dependent repair system.

Authors
Au, KG; Cabrera, M; Miller, JH; Modrich, P
MLA Citation
Au, KG, Cabrera, M, Miller, JH, and Modrich, P. "Escherichia coli mutY gene product is required for specific A-G→C·G mismatch correction." Proceedings of the National Academy of Sciences of the United States of America 85.23 (1988): 9163-9166.
PMID
3057502
Source
scival
Published In
Proceedings of the National Academy of Sciences of the United States of America
Volume
85
Issue
23
Publish Date
1988
Start Page
9163
End Page
9166

Mispair specificity of methyl-directed DNA mismatch correction in vitro.

To evaluate the substrate specificity of methyl-directed mismatch repair in Escherichia coli extracts, we have constructed a set of DNA heteroduplexes, each of which contains one of the eight possible single base pair mismatches and a single hemimethylated d(GATC) site. Although all eight mismatches were located at the same position within heteroduplex molecules and were embedded within the same sequence environment, they were not corrected with equal efficiencies in vitro. G-T was corrected most efficiently, with A-C, C-T, A-A, T-T, and G-G being repaired at rates 40-80% of that of the G-T mispair. Correction of each of these six mispairs occurred in a methyl-directed manner in a reaction requiring mutH, mutL, and mutS gene products. C-C and A-G mismatches showed different behavior. C-C was an extremely poor substrate for correction while repair of A-G was anomalous. Although A-G was corrected to A-T by the mutHLS-dependent, methyl-directed pathway, repair of A-G to C-G occurred largely by a pathway that is independent of the methylation state of the heteroduplex and which does not require mutH, mutL, or mutS gene products. Similar results were obtained with a second A-G mismatch in a different sequence environment suggesting that a novel pathway may exist for processing A-G mispairs to C-G base pairs. As judged by DNase I footprint analysis, MutS protein is capable of recognizing each of the eight possible base-base mismatches. Use of this method to estimate the apparent affinity of MutS protein for each of the mispairs revealed a rough correlation between MutS affinity and efficiency of correction by the methyl-directed pathway. However, the A-C mismatch was an exception in this respect indicating that interactions other than mismatch recognition may contribute to the efficiency of repair.

Authors
Su, SS; Lahue, RS; Au, KG; Modrich, P
MLA Citation
Su, SS, Lahue, RS, Au, KG, and Modrich, P. "Mispair specificity of methyl-directed DNA mismatch correction in vitro." Journal of Biological Chemistry 263.14 (1988): 6829-6835.
PMID
2834393
Source
scival
Published In
Journal of Biological Chemistry
Volume
263
Issue
14
Publish Date
1988
Start Page
6829
End Page
6835

Methyl-directed DNA mismatch repair in Escherichia coli

Some of the molecular aspects of methyl-directed mismatch repair in E. coli have been characterized. These include: mismatch recognition by mutS protein in which different mispairs are bound with different affinities; the direct involvement of d(GATC) sites; and strand scission by mutH protein at d(GATC) sequences with strand selection based on methylation of the DNA at those sites. In addition, communication over a distance between a mismatch and d(GATC) sites has been implicated. Analysis of mismatch correlation in a defined system (Lahue et al., unpublished) should provide a direct means to further molecular aspects of this process. © 1988.

Authors
Lahue, RS; Modrich, P
MLA Citation
Lahue, RS, and Modrich, P. "Methyl-directed DNA mismatch repair in Escherichia coli." Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis 198.1 (1988): 37-43.
PMID
3280983
Source
scival
Published In
Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis
Volume
198
Issue
1
Publish Date
1988
Start Page
37
End Page
43

Investigation of the complexes of EcoRI endonuclease with decanucleotides containing canonical and modified recognition sequences using fluorescence and optical detection of magnetic resonance spectroscopy

The binding of EcoRI endonuclease to the oligonucleotides d(GCGAATTCGC) and d(GCGAA) (5BrdU) (5BrdU) d(CGC) has been investigated to determine whether stacking interactions occur between tryptophan residues and the DNA bases. Fluorescence binding isotherms show that the decamer containing the canonical and that containing the modified recognition sequence bind with comparable affinity. Optically detected magnetic resonance spectra show limited perturbations of the Trp zero-field splitting parameters, which are assigned to electrical field effects. No evidence for Trp stacking interactions has been found. © 1988.

Authors
Jhon, N-I; Casas-Finet, JR; Maki, AH; Modrich, P
MLA Citation
Jhon, N-I, Casas-Finet, JR, Maki, AH, and Modrich, P. "Investigation of the complexes of EcoRI endonuclease with decanucleotides containing canonical and modified recognition sequences using fluorescence and optical detection of magnetic resonance spectroscopy." BBA - Gene Structure and Expression 949.2 (1988): 189-194.
PMID
2829965
Source
scival
Published In
BBA - Gene Structure and Expression
Volume
949
Issue
2
Publish Date
1988
Start Page
189
End Page
194

Isolation and characterization of the Escherichia coli mutH gene product.

The Escherichia coli mutH gene product has been isolated in near homogeneous form using an in vitro complementation assay for DNA mismatch correction (Lu, A.-L., Clark, S., and Modrich, P. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4639-4643) which is dependent on mutH function. The protein has a subunit Mr of 25,000, and purified preparations contain a Mg2+-dependent endonuclease activity which cleaves 5' to the dG of d(GATC) sequences to generate 5'-phosphoryl and 3'-hydroxyl termini. Symmetrically methylated d(GATC) sites are resistant to the endonuclease, hemimethylated sequences are cleaved on the unmethylated strand, and unmethylated d(GATC) sites are usually subject to scission on only one DNA strand. Although this endonuclease activity is extremely weak (less than 1 scission/h/mutH monomer equivalent) and cleavage at a d(GATC) site does not depend on the presence of a mismatched base pair within the DNA substrate, the activity does not appear to be a contaminant of mutH preparations. d(GATC) endonuclease activity and mutH complementing activity co-purify through multiple column steps without change in relative specific activities, and both activities co-electrophorese under native conditions. These findings suggest that the mutH product functions at the strand discrimination stage of mismatch correction and that this stage of the reaction involves scission of the unmethylated DNA strand.

Authors
Welsh, KM; Lu, AL; Clark, S; Modrich, P
MLA Citation
Welsh, KM, Lu, AL, Clark, S, and Modrich, P. "Isolation and characterization of the Escherichia coli mutH gene product." Journal of Biological Chemistry 262.32 (1987): 15624-15629.
PMID
2824465
Source
scival
Published In
Journal of Biological Chemistry
Volume
262
Issue
32
Publish Date
1987
Start Page
15624
End Page
15629

Mechanism of specific site location and DNA cleavage by EcoR I endonuclease.

Authors
Terry, BJ; Jack, WE; Modrich, P
MLA Citation
Terry, BJ, Jack, WE, and Modrich, P. "Mechanism of specific site location and DNA cleavage by EcoR I endonuclease." Gene amplification and analysis 5 (1987): 103-118.
PMID
3333364
Source
scival
Published In
Gene amplification and analysis
Volume
5
Publish Date
1987
Start Page
103
End Page
118

Requierement for d(GATC) sequences in Escherichia coli mutHLS mismatch correction

The involvement of d(GATC) sequences in Escherichia coli DNA mismatch correction was ascertained by analyzing in vitro repair efficiencies of a series of related, covalently closed circular DNA heteroduplexes that contained from zero to four d(GATC) sites. A heteroduplex with four d(GATC) sites was repaired with high efficiency by extracts of E. coli, whereas no significant correction occurred on a closely related molecule lacking such sequences. Heteroduplexes containing one or two d(GATC) sites were corrected at rates between 10% and 93% of that observed for the four-site molecule, but repair efficiency did not correlate in a simple way with the number of sites present. The methylation state at a single d(GATC) sequence was sufficient to direct strandedness of repair, and correction of heteroduplexes containing one or more d(GATC) sites required functional mutH, mutL, and mutS gene products. In addition, DNA repair synthesis dependent on mutH and mutS also required the presence of at least one d(GATC) site. Although mismatch correction was not observed on a covalently closed circular heteroduplex lacking a d(GATC) sequence, such molecules were subject to strand-specific repair if they contained a strand-specific single-strand break. However, this correction reaction did not require mutH, mutL, mutS, or uvrD gene products. Consequently, we have concluded that d(GATC) sequences are directly involved in mismatch correction mediated by the mutHLS system.

Authors
Lahue, RS; Su, S-S; Modrich, P
MLA Citation
Lahue, RS, Su, S-S, and Modrich, P. "Requierement for d(GATC) sequences in Escherichia coli mutHLS mismatch correction." Proceedings of the National Academy of Sciences of the United States of America 84.6 (1987): 1482-1486.
PMID
3550791
Source
scival
Published In
Proceedings of the National Academy of Sciences of the United States of America
Volume
84
Issue
6
Publish Date
1987
Start Page
1482
End Page
1486

DNA mismatch correction.

Authors
Modrich, P
MLA Citation
Modrich, P. "DNA mismatch correction." Annual Review of Biochemistry 56 (1987): 435-466.
PMID
3304141
Source
scival
Published In
Annual Review of Biochemistry
Volume
56
Publish Date
1987
Start Page
435
End Page
466

Nucleotide sequence of a cDNA for a member of the human 90-kDa heat-shock protein family

This paper describes the isolation and sequence of a human cDNA homologous to a class of proteins commonly referred to as 90-kDa heat-shock proteins. The complete nucleotide sequence of 2563 bp and the deduced amino acid sequence are presented. A single long open reading frame encodes a protein of 83 303 Da, the amino acid composition of which correlates well with that determined for the human 90-kDa heat-shock or 'stress' protein [Welch W.J. and Feramisco J.R., J. Biol. Chem. 257 (1982) 14949-14959]. Moreover, sequence analysis of this gene reveals extensive homology with the Drosophila 83-kDa and yeast 90-kDa heat-shock proteins. A comparison of the translated product of the human cDNA to the published yeast 90-kDa heat-shock protein reveals more than 60% homology at both the nucleotide and amino acid levels. Several regions of 50 aa or more show greater than 90% identity. This cDNA also hybridizes with an RNA species which increases upon heat shock of HeLa cells. © 1987.

Authors
Rebbe, NF; Ware, J; Bertina, RM; Modrich, P; Stafford, DW
MLA Citation
Rebbe, NF, Ware, J, Bertina, RM, Modrich, P, and Stafford, DW. "Nucleotide sequence of a cDNA for a member of the human 90-kDa heat-shock protein family." Gene 53.2-3 (1987): 235-245.
PMID
3301534
Source
scival
Published In
Gene
Volume
53
Issue
2-3
Publish Date
1987
Start Page
235
End Page
245

Mismatch correction.

Authors
Modrich, P
MLA Citation
Modrich, P. "Mismatch correction." Basic life sciences 38 (1986): 303-310.
PMID
2943263
Source
scival
Published In
Basic life sciences
Volume
38
Publish Date
1986
Start Page
303
End Page
310

Escherichia coli mutS-encoded protein binds to mismatched DNA base pairs

The Escherichia coli mutS gene product is involved in mismatch correction in this organism. We have purified a biologically active form of the 97,000 M(r) protein to near homogeneity from an overproducing strain. Enzymatic and chemical protection ('footprinting') experiments have demonstrated that mutS-encoded protein specifically binds to DNA regions containing a single base-pair mismatch. The protein displayed variable affinity for the limited set of mismatches tested (G-T > G-A ~ A-C > T-C).

Authors
Su, S-S; Modrich, P
MLA Citation
Su, S-S, and Modrich, P. "Escherichia coli mutS-encoded protein binds to mismatched DNA base pairs." Proceedings of the National Academy of Sciences of the United States of America 83.14 (1986): 5057-5061.
PMID
3014530
Source
scival
Published In
Proceedings of the National Academy of Sciences of the United States of America
Volume
83
Issue
14
Publish Date
1986
Start Page
5057
End Page
5061

Facilitated diffusion during catalysis by EcoRI endonuclease. Nonspecific interactions in EcoRI catalysis

Authors
Terry, BJ; Jack, WE; Modrich, P
MLA Citation
Terry, BJ, Jack, WE, and Modrich, P. "Facilitated diffusion during catalysis by EcoRI endonuclease. Nonspecific interactions in EcoRI catalysis." Journal of Biological Chemistry 260.24 (1985): 13130-13137.
PMID
2997157
Source
scival
Published In
Journal of Biological Chemistry
Volume
260
Issue
24
Publish Date
1985
Start Page
13130
End Page
13137

Extent of equilibrium perturbation of the DNA helix upon enzymatic methylation of adenine residues

The extent of equilibrium perturbation of the DNA helix associated with enzymatic methylation of dA residues has been determined by the agarose gel electrophoresis band-shift method. Utilization of EcoRI methylase under conditions of reduced specificity together with Escherichia coli dam methylase permitted modification of up to 300 dA residues/plasmid pBR322 dimer. A conformational change associated with methylation was observed, with the magnitude of the transition being linear with extent of modification of relaxed DNA circles. The conformational change corresponds to a unwinding of the DNA helix by 0.5°/methyl group transferred to relaxed molecules. The magnitude of the effect was independent of temperature from 5-37° C indicating that it is not the consequence of a thermal transition within this range.

Authors
Cheng, S-C; Herman, G; Modrich, P
MLA Citation
Cheng, S-C, Herman, G, and Modrich, P. "Extent of equilibrium perturbation of the DNA helix upon enzymatic methylation of adenine residues." Journal of Biological Chemistry 260.1 (1985): 191-194.
PMID
3880739
Source
scival
Published In
Journal of Biological Chemistry
Volume
260
Issue
1
Publish Date
1985
Start Page
191
End Page
194

In vitro maturation of circular bacteriophage P2 DNA. Purification of ter components and characterization of the reaction

Authors
Bowden, DW; Modrich, P
MLA Citation
Bowden, DW, and Modrich, P. "In vitro maturation of circular bacteriophage P2 DNA. Purification of ter components and characterization of the reaction." Journal of Biological Chemistry 260.11 (1985): 6999-7007.
PMID
2987239
Source
scival
Published In
Journal of Biological Chemistry
Volume
260
Issue
11
Publish Date
1985
Start Page
6999
End Page
7007

'interactive' recognition in EcoRl restriction enzyme-DNA complex

A solution study or interaction between DNA and EcoRI restriction enzyme snows that there is a definite distortion of DNA in the specific recognition complexes but no Measurable DNA distortion in the non-specific interaction. © 1984 IRL Press Ltd.

Authors
Kim, R; Modrich, P; Kim, S-H
MLA Citation
Kim, R, Modrich, P, and Kim, S-H. "'interactive' recognition in EcoRl restriction enzyme-DNA complex." Nucleic Acids Research 12.19 (1984): 7285-7292.
PMID
6093038
Source
scival
Published In
Nucleic Acids Research
Volume
12
Issue
19
Publish Date
1984
Start Page
7285
End Page
7292
DOI
10.1093/nar/12.19.7285

Prediction of secondary structure for ecoRI endonuclease

The circular dichroism of EcoRI restriction endonuclease was measured to 178 nm and analyzed for secondary structure. The results (33% α-helix, 25% β-sheet, 17% turns, and 25% other structures) compare well with our joint prediction from sequence data.

Authors
Manavalan, P; Jr, WCJ; Modrich, P
MLA Citation
Manavalan, P, Jr, WCJ, and Modrich, P. "Prediction of secondary structure for ecoRI endonuclease." Journal of Biological Chemistry 259.19 (1984): 11666-11667.
PMID
6090442
Source
scival
Published In
Journal of Biological Chemistry
Volume
259
Issue
19
Publish Date
1984
Start Page
11666
End Page
11667

Partial NH2- and COOH-terminal sequence and cyanogen bromide peptide analysis of Escherichia coli sn-glycerol-3-phosphate acyltransferase.

The sn-glycerol-3-phosphate acyltransferase from Escherichia coli, an integral membrane protein whose activity is dependent on phospholipids, was purified to near homogeneity (Green, P. R., Merrill, A. H., Jr., and Bell, R. M., (1981) J. Biol. Chem. 256, 11151-11159). Determination of a partial NH2-terminal sequence and the COOH terminus permitted alignment of the polypeptide on the sequenced sn-glycerol-3-phosphate acyltransferase structural gene (Lightner, V. A., Bell, R. M., and Modrich, P. (1983) J. Biol. Chem. 258, 10856-10861). Processing of the sn-glycerol-3-phosphate acyltransferase is apparently limited to the removal of the NH2-terminal formylmethionine. Thirteen of 27 possible cyanogen bromide peptides predicted from the DNA sequence were purified, characterized, and assigned to their location in the primary structure. Three peptides located at positions throughout the sequence were partially sequenced by automated Edman degradation. The partial sequence analysis of the homogeneous sn-glycerol-3-phosphate acyltransferase is fully in accord with the primary structure inferred from the DNA sequence.

Authors
Green, PR; Vanaman, TC; Modrich, P; Bell, RM
MLA Citation
Green, PR, Vanaman, TC, Modrich, P, and Bell, RM. "Partial NH2- and COOH-terminal sequence and cyanogen bromide peptide analysis of Escherichia coli sn-glycerol-3-phosphate acyltransferase." Journal of Biological Chemistry 258.18 (1983): 10862-10866.
Source
scival
Published In
Journal of Biological Chemistry
Volume
258
Issue
18
Publish Date
1983
Start Page
10862
End Page
10866

The DNA sequences encoding plsB and dgk loci of Escherichia coli.

We have determined the sequence of a 3865-base pair DNA segment from Escherichia coli containing plsB, the structural gene for the sn-glycerol-3-phosphate acyltransferase, and the dgk locus, believed to encode diglyceride kinase. The 806-amino acid sequence encoded within the longest open reading frame is in agreement with NH2-terminal sequences of the sn-glycerol-3-phosphate acyltransferase (Green, P., Vanaman, T. C., Modrich, P., and Bell, R. M. (1983) J. Biol. Chem. 258, 10862-10866), indicating that this is the structural gene for this protein. Furthermore, an open reading frame encoding a 122-residue polypeptide consistent with the size of diglyceride kinase has been identified and coincides with the position of dgk determined by deletion analysis.

Authors
Lightner, VA; Bell, RM; Modrich, P
MLA Citation
Lightner, VA, Bell, RM, and Modrich, P. "The DNA sequences encoding plsB and dgk loci of Escherichia coli." Journal of Biological Chemistry 258.18 (1983): 10856-10861.
Source
scival
Published In
Journal of Biological Chemistry
Volume
258
Issue
18
Publish Date
1983
Start Page
10856
End Page
10861

Methyl-directed repair of DNA base-pair mismatches in vitro.

An assay has been developed that permits analysis of DNA mismatch repair in cell-free extracts of Escherichia coli. The method relies on repair of heteroduplex molecules of f1 R229 DNA, which contain a base-pair mismatch within the single EcoRI site of the molecule. As observed with mismatch heteroduplexes of lambda DNA [Pukkila, P. J., Peterson, J., Herman, G., Modrich, P. & Meselson, M. (1983) Genetics, in press], in vivo mismatch correction of f1 heteroduplexes is directed by the state of dam methylation of d(G-A-T-C) sequences within the DNA duplex. Thus, the heteroduplex (formula: see book) is repaired in vivo to an EcoRI-sensitive form if the strand bearing the wild-type EcoRI sequence carries the dam modification and the other does not. Such molecules are also subject to mismatch repair by E. coli extracts. The in vitro activity is also dependent on ATP, the state of dam methylation of mismatch heteroduplexes, and products of mutH, mutL, mutS, and uvrE loci. However, crude fractions deficient in these gene products do complement in the cell-free system, thus providing assays for their isolation. The in vitro reaction is accompanied by repair synthesis on the unmethylated DNA strand.

Authors
Lu, AL; Clark, S; Modrich, P
MLA Citation
Lu, AL, Clark, S, and Modrich, P. "Methyl-directed repair of DNA base-pair mismatches in vitro." Proceedings of the National Academy of Sciences of the United States of America 80.15 (1983): 4639-4643.
PMID
6308634
Source
scival
Published In
Proceedings of the National Academy of Sciences of the United States of America
Volume
80
Issue
15
Publish Date
1983
Start Page
4639
End Page
4643

Thermodynamic parameters governing interaction of EcoRI endonuclease with specific and nonspecific DNA sequences.

Equilibrium binding of EcoRI endonuclease to DNA has been analyzed by nitrocellulose filter and preferential DNA cleavage methods. Association constants for pBR322 and a 34-base pair molecule containing the EcoRI site of this plasmid in a central position were determined to be 1.9 X 10(11) M-1 and 1.0 X 10(11) M-1 at 37 degrees C, respectively, with the stoichiometry of binding being 0.8 +/- 0.1 mol of endonuclease dimer per mol of DNA. In contrast, the affinity of the enzyme for a pBR322 derivative from which the EcoRI site has been deleted is 3.2 X 10(9) M-1 as judged by competitive binding experiments. If it is assumed that each base pair can define the beginning of a nonspecific binding site, this value corresponds to an affinity for nonspecific sites of 7.4 X 10(5) M-1. Furthermore, the affinity of the endonuclease for the EcoRI-methylated sequence is at least three orders of magnitude less than that for the unmodified recognition site. The dependence on temperature and ionic strength of the equilibrium constant governing specific interactions has also been examined. The temperature dependence of the reaction indicates that entropy increase accounts for 70% of the free energy of specific binding at 37 degrees C. Affinity of the endonuclease for the EcoRI site is highly dependent on NaCl concentration. Analysis of this dependence according to the theory of Record and colleagues (Record, T. M., Jr., Lohman, T. M., and deHaseth, P. (1976) J. Mol. Biol. 107, 145-158) has implicated 8 ion pairs in the stability of specific complexes, a value identical with the number of phosphate contacts determined by ethylation interference analysis (Lu, A. L., Jack, W. E., and Modrich, P. (1981) J. Biol. Chem. 256, 13200-13206). Extrapolation to 1 M NaCl suggests that nonelectrostatic interactions account for 40% of the free energy change associated with specific complex formation.

Authors
Terry, BJ; Jack, WE; Rubin, RA; Modrich, P
MLA Citation
Terry, BJ, Jack, WE, Rubin, RA, and Modrich, P. "Thermodynamic parameters governing interaction of EcoRI endonuclease with specific and nonspecific DNA sequences." Journal of Biological Chemistry 258.16 (1983): 9820-9825.
PMID
6309785
Source
scival
Published In
Journal of Biological Chemistry
Volume
258
Issue
16
Publish Date
1983
Start Page
9820
End Page
9825

Effects of high levels of DNA adenine methylation on methyl-directed mismatch repair in Escherichia coli.

Two methods were used in an attempt to increase the efficiency and strand selectivity of methyl-directed mismatch repair of bacteriophage lambda heteroduplexes in E. coli. Previous studies of such repair used lambda DNA that was only partially methylated as the source of methylated chains. Also, transfection was carried out in methylating strains. Either of these factors might have been responsible for the incompleteness of the strand selectivity observed previously. In the first approach to increasing strand selectivity, heteroduplexes were transfected into a host deficient in methylation, but no changes in repair frequencies were observed. In the second approach, heteroduplexes were prepared using DNA that had been highly methylated in vitro with purified DNA adenine methylase as the source of methylated chains. In heteroduplexes having a repairable cI/+ mismatch, strand selectivity was indeed enhanced. In heteroduplexes with one chain highly methylated and the complementary chain unmethylated, the frequency of repair on the unmethylated chain increased to nearly 100%. Heteroduplexes with both chains highly methylated were not repaired at a detectable frequency. Thus, chains highly methylated by DNA adenine methylase were refractory to mismatch repair by this system, regardless of the methylation of the complementary chain. These results support the hypothesis that methyl-directed mismatch repair acts to correct errors of replication, thus lowering the mutation rate.

Authors
Pukkila, PJ; Peterson, J; Herman, G; Modrich, P; Meselson, M
MLA Citation
Pukkila, PJ, Peterson, J, Herman, G, Modrich, P, and Meselson, M. "Effects of high levels of DNA adenine methylation on methyl-directed mismatch repair in Escherichia coli." Genetics 104.4 (1983): 571-582.
PMID
6225697
Source
scival
Published In
Genetics
Volume
104
Issue
4
Publish Date
1983
Start Page
571
End Page
582

T7-induced DNA polymerase. Requirement for thioredoxin sulfhydryl groups.

Bacteriophage T7-induced DNA polymerase is composed of a 1:1 complex of phage-induced gene 5 protein and Escherichia coli thioredoxin. Preparation of active subunits in the absence of sulfhydryl reagents indicates the reduced form of thioredoxin is sufficient for formation of the active holoenzyme. The oxidized form of thioredoxin, thioredoxin modified at one active site sulfhydryl by iodoacetate or methyl iodide, or thioredoxin modified at both active site sulfhydryls by N-ethylmaleimide, are all inactive, being defective in complex formation with gene 5 protein. Thioredoxin sulfhydryl groups present in native T7 DNA polymerase do not appear to be involved in an intersubunit disulfide bond; one and probably both sulfhydryls are available in the native holoenzyme for modification by N-ethylmaleimide. Furthermore, DNA substrates alter the reactivity of thioredoxin cysteines within the holoenzyme with respect to this reagent. Substrates for the single strand exonuclease enhance the reactivity of thioredoxin sulfhydryl groups while those for the polymerase or double strand exonuclease functions afford protection. It, therefore, seems likely that thioredoxin sulfhydryl groups are present in the reduced state within the native polymerase.

Authors
Adler, S; Modrich, P
MLA Citation
Adler, S, and Modrich, P. "T7-induced DNA polymerase. Requirement for thioredoxin sulfhydryl groups." Journal of Biological Chemistry 258.11 (1983): 6956-6962.
PMID
6343383
Source
scival
Published In
Journal of Biological Chemistry
Volume
258
Issue
11
Publish Date
1983
Start Page
6956
End Page
6962

Positive-selection cloning vehicle useful for overproduction of hybrid proteins

Plasmid pSCC31 contains the EcoRI endonuclease gene downstream from lambda p(L). It does not yield transformants upon introduction into Escherichia coli unless the structural integrity of the endonuclease is destroyed. This makes it useful as a positive-selection cloning vehicle which can be employed for regulated overproduction of hybrid proteins.

Authors
Cheng, SC; Modrich, P
MLA Citation
Cheng, SC, and Modrich, P. "Positive-selection cloning vehicle useful for overproduction of hybrid proteins." Journal of Bacteriology 154.2 (1983): 1005-1008.
PMID
6302074
Source
scival
Published In
Journal of Bacteriology
Volume
154
Issue
2
Publish Date
1983
Start Page
1005
End Page
1008

Studies on sequence recognition by type II restriction and modification enzymes.

Type II DNA restriction and modification systems are ideally suited for analysis of mechanisms by which proteins specifically recognize unique DNA sequences. Each system is comprised of a unique DNA recognition site and two enzymes, which in those cases examined in detail, are comprised of distinct polypeptide chains. Thus, not only are the DNA substrates extremely well defined, but each system affords the opportunity to compare distinct proteins which interact with a common DNA sequence. This review will focus only on those Type II systems which have been examined in sufficient molecular detail to permit some insight into modes of specific DNA-protein interaction.

Authors
Modrich, P
MLA Citation
Modrich, P. "Studies on sequence recognition by type II restriction and modification enzymes." CRC critical reviews in biochemistry 13.3 (1982): 287-323.
PMID
6293768
Source
scival
Published In
CRC critical reviews in biochemistry
Volume
13
Issue
3
Publish Date
1982
Start Page
287
End Page
323

Involvement of outside DNA sequences in the major kinetic path by which EcoRI endonuclease locates and leaves its recognition sequence.

We have examined the kinetics of the interaction between endodeoxyribonuclease EcoRI (EC 3.1.23.13) and nine linear DNA fragments that range in size between 34 and 6,200 base pairs and contain the EcoRI site of plasmid pBR322 in a central location. The kinetic parameters governing both formation and decay of specific endonuclease . DNA complexes increase 8-fold with increasing chain length over this size range. In contrast, equilibrium competition experiments demonstrated that the intrinsic affinity of endonuclease for its recognition sequence is independent of DNA chain length over this range. Thus, DNA sequences outside the recognition site enhance the rate at which EcoRI endonuclease locates or leaves its recognition site without affecting the intrinsic thermodynamic parameters of site-specific interaction. These results are consistent with a facilitated diffusion mechanism for specific DNA site location by this enzyme.

Authors
Jack, WE; Terry, BJ; Modrich, P
MLA Citation
Jack, WE, Terry, BJ, and Modrich, P. "Involvement of outside DNA sequences in the major kinetic path by which EcoRI endonuclease locates and leaves its recognition sequence." Proceedings of the National Academy of Sciences of the United States of America 79.13 (1982): 4010-4014.
PMID
6287460
Source
scival
Published In
Proceedings of the National Academy of Sciences of the United States of America
Volume
79
Issue
13
Publish Date
1982
Start Page
4010
End Page
4014

Stereochemical course of nucleotidyl transfer catalyzed by bacteriophage T7 induced DNA polymerase

The bacteriophage T7 induced DNA polymerase, consisting of the phage specified gene 5 protein associated with Escherichia coli thioredoxin, catalyzes the copolymerization of SP-dATPαS with dTTP, producing the alternating copolymer poly[d(T-sA)] by a mechanism involving inversion of configuration at Pα. Degradation of poly[d(T-sA)] by the nucleolytic action of E. coli DNA polymerase I produced the dinucleotide pdTp-sdA, whose configuration at the phosphorothioate diester was assigned as R by comparison of the phosphorus-31 nuclear magnetic resonance chemical shift (55.0 ppm downfield from H3PO4) with that of an authentic sample. Further degradation by alkaline phosphatase to RP-dTp-sdA (55.6 ppm downfield from H3PO4) confirmed the configuration. The stereochemistry provides no evidence of a double displacement mechanism. © 1982 American Chemical Society.

Authors
Brody, RS; Adler, S; Modrich, P; Stec, WJ; Leznikowski, ZJ; Frey, PA
MLA Citation
Brody, RS, Adler, S, Modrich, P, Stec, WJ, Leznikowski, ZJ, and Frey, PA. "Stereochemical course of nucleotidyl transfer catalyzed by bacteriophage T7 induced DNA polymerase." Biochemistry 21.10 (1982): 2570-2572.
PMID
7093204
Source
scival
Published In
Biochemistry
Volume
21
Issue
10
Publish Date
1982
Start Page
2570
End Page
2572

Escherichia coli dam methylase. Physical and catalytic properties of the homogeneous enzyme

Authors
Herman, GE; Modrich, P
MLA Citation
Herman, GE, and Modrich, P. "Escherichia coli dam methylase. Physical and catalytic properties of the homogeneous enzyme." Journal of Biological Chemistry 257.5 (1982): 2605-2612.
PMID
7037767
Source
scival
Published In
Journal of Biological Chemistry
Volume
257
Issue
5
Publish Date
1982
Start Page
2605
End Page
2612

Preliminary X-ray diffraction studies of EcoRI restriction endonuclease-DNA complex

A complex between EcoRI restriction endonuclease and cognate DNA fragment, 5′-G-A-A-T-T-C C-T-T-A-A-G-5′, has been crystallized. The space group is P4212 with a = b = 183.2A ̊, c = 49.7A ̊, α = β = γ = 90 °. The unit cell contains four enzyme monomers plus two duplex DNA fragments in an asymmetric unit. High quality crystals of the enzyme alone have also been obtained. © 1981.

Authors
Young, T-S; Kim, S-H; Modrich, P; Beth, A; Jay, E
MLA Citation
Young, T-S, Kim, S-H, Modrich, P, Beth, A, and Jay, E. "Preliminary X-ray diffraction studies of EcoRI restriction endonuclease-DNA complex." Journal of Molecular Biology 145.3 (1981): 607-610.
PMID
6267292
Source
scival
Published In
Journal of Molecular Biology
Volume
145
Issue
3
Publish Date
1981
Start Page
607
End Page
610

In vitro studies on the bacteriophage P2 terminase system.

Authors
Bowden, DW; Modrich, P
MLA Citation
Bowden, DW, and Modrich, P. "In vitro studies on the bacteriophage P2 terminase system." Progress in clinical and biological research 64 (1981): 223-230.
PMID
6120523
Source
scival
Published In
Progress in clinical and biological research
Volume
64
Publish Date
1981
Start Page
223
End Page
230

DNA determinants important in sequence recognition by Eco RI endonuclease

Authors
Lu, AL; Jack, WE; Modrich, P
MLA Citation
Lu, AL, Jack, WE, and Modrich, P. "DNA determinants important in sequence recognition by Eco RI endonuclease." Journal of Biological Chemistry 256.24 (1981): 13200-13206.
PMID
6273420
Source
scival
Published In
Journal of Biological Chemistry
Volume
256
Issue
24
Publish Date
1981
Start Page
13200
End Page
13206

Structures and mechanisms of Eco RI DNA restriction and modification enzymes.

Authors
Jack, WE; Rubin, RA; Newman, A; Modrich, P
MLA Citation
Jack, WE, Rubin, RA, Newman, A, and Modrich, P. "Structures and mechanisms of Eco RI DNA restriction and modification enzymes." Gene amplification and analysis 1 (1981): 165-179.
PMID
6765638
Source
scival
Published In
Gene amplification and analysis
Volume
1
Publish Date
1981
Start Page
165
End Page
179

DNA sequences of structural genes for Eco RI DNA restriction and modification enzymes

Authors
Newman, AK; Rubin, RA; Kim, SH; Modrich, P
MLA Citation
Newman, AK, Rubin, RA, Kim, SH, and Modrich, P. "DNA sequences of structural genes for Eco RI DNA restriction and modification enzymes." Journal of Biological Chemistry 256.5 (1981): 2131-2139.
PMID
6257701
Source
scival
Published In
Journal of Biological Chemistry
Volume
256
Issue
5
Publish Date
1981
Start Page
2131
End Page
2139

Partial NH2- and COOH-terminal sequence analyses of Eco RI DNA restriction and modification enzymes

Authors
Rubin, RA; Modrich, P; Vanaman, TC
MLA Citation
Rubin, RA, Modrich, P, and Vanaman, TC. "Partial NH2- and COOH-terminal sequence analyses of Eco RI DNA restriction and modification enzymes." Journal of Biological Chemistry 256.5 (1981): 2140-2142.
PMID
6257702
Source
scival
Published In
Journal of Biological Chemistry
Volume
256
Issue
5
Publish Date
1981
Start Page
2140
End Page
2142

Escherichia coli K-12 clones that overproduce dam methylase are hypermutable

Authors
Herman, GE; Modrich, P
MLA Citation
Herman, GE, and Modrich, P. "Escherichia coli K-12 clones that overproduce dam methylase are hypermutable." Journal of Bacteriology 145.1 (1981): 644-646.
PMID
7007328
Source
scival
Published In
Journal of Bacteriology
Volume
145
Issue
1
Publish Date
1981
Start Page
644
End Page
646

[12] Purification and properties of EcoRI endonuclease

Authors
Rubin, RA; Modrich, P
MLA Citation
Rubin, RA, and Modrich, P. "[12] Purification and properties of EcoRI endonuclease." Methods in Enzymology 65.C (1980): 96-104.
PMID
6246382
Source
scival
Published In
Methods in Enzymology
Volume
65
Issue
C
Publish Date
1980
Start Page
96
End Page
104
DOI
10.1016/S0076-6879(80)65014-9

MEMBRANE PHOSPHOLIPID-SYNTHESIS IN ESCHERICHIA-COLI - CLONING OF A STRUCTURAL GENE (PLSB) OF THE SN-GLYCEROL-3-PHOSPHATE ACYLTRANSFERASE

Authors
LIGHTNER, VA; LARSON, TJ; TAILLEUR, P; KANTOR, GD; RAETZ, CRH; BELL, RM; MODRICH, P
MLA Citation
LIGHTNER, VA, LARSON, TJ, TAILLEUR, P, KANTOR, GD, RAETZ, CRH, BELL, RM, and MODRICH, P. "MEMBRANE PHOSPHOLIPID-SYNTHESIS IN ESCHERICHIA-COLI - CLONING OF A STRUCTURAL GENE (PLSB) OF THE SN-GLYCEROL-3-PHOSPHATE ACYLTRANSFERASE." JOURNAL OF BIOLOGICAL CHEMISTRY 255.19 (1980): 9413-9420.
PMID
6251087
Source
wos-lite
Published In
The Journal of biological chemistry
Volume
255
Issue
19
Publish Date
1980
Start Page
9413
End Page
9420

REGULATION OF PHOSPHOLIPID BIOSYNTHESIS IN ESCHERICHIA-COLI - CLONING OF THE STRUCTURAL GENE FOR THE BIOSYNTHETIC SN-GLYCEROL-3-PHOSPHATE DEHYDROGENASE

Authors
CLARK, D; LIGHTNER, V; EDGAR, R; MODRICH, P; CRONAN, JE; BELL, RM
MLA Citation
CLARK, D, LIGHTNER, V, EDGAR, R, MODRICH, P, CRONAN, JE, and BELL, RM. "REGULATION OF PHOSPHOLIPID BIOSYNTHESIS IN ESCHERICHIA-COLI - CLONING OF THE STRUCTURAL GENE FOR THE BIOSYNTHETIC SN-GLYCEROL-3-PHOSPHATE DEHYDROGENASE." JOURNAL OF BIOLOGICAL CHEMISTRY 255.2 (1980): 714-717.
PMID
6985897
Source
wos-lite
Published In
The Journal of biological chemistry
Volume
255
Issue
2
Publish Date
1980
Start Page
714
End Page
717

MEMBRANE PHOSPHOLIPID-SYNTHESIS IN ESCHERICHIA-COLI - IDENTIFICATION OF THE SN-GLYCEROL-3-PHOSPHATE ACYLTRANSFERASE POLYPEPTIDE AS THE PLSB GENE-PRODUCT

Authors
LARSON, TJ; LIGHTNER, VA; GREEN, PR; MODRICH, P; BELL, RM
MLA Citation
LARSON, TJ, LIGHTNER, VA, GREEN, PR, MODRICH, P, and BELL, RM. "MEMBRANE PHOSPHOLIPID-SYNTHESIS IN ESCHERICHIA-COLI - IDENTIFICATION OF THE SN-GLYCEROL-3-PHOSPHATE ACYLTRANSFERASE POLYPEPTIDE AS THE PLSB GENE-PRODUCT." JOURNAL OF BIOLOGICAL CHEMISTRY 255.19 (1980): 9421-9426.
PMID
6997313
Source
wos-lite
Published In
The Journal of biological chemistry
Volume
255
Issue
19
Publish Date
1980
Start Page
9421
End Page
9426

Recognition sequence of the dam methylase of Escherichia coli K12 and mode of cleavage of Dpn I endonuclease.

The recognition sequence for the dam methylase of Escherichia coli K12 has been determined directly by use of in vivo methylated ColE1 DNA or DNA methylated in vitro with purified enzyme. The methylase recognizes the symmetric tetranucleotide d(pG-A-T-C) and introduces two methyl groups per site in duplex DNA with the product of methylation being 6-methylaminopurine. This work has also demonstrated that Dpn I restriction endonuclease cleaves on the 3' side of the modified adenine within the methylated sequence to yield DNA fragments possessing fully base-paired termini. All sequences in ColE1 DNA methylated by the dam enzyme are subject to double strand cleavage by Dpn I endonuclease. Therefore, this restriction enzyme can be employed for mapping the location of sequences possessing the dam modification.

Authors
Geier, GE; Modrich, P
MLA Citation
Geier, GE, and Modrich, P. "Recognition sequence of the dam methylase of Escherichia coli K12 and mode of cleavage of Dpn I endonuclease." Journal of Biological Chemistry 254.4 (1979): 1408-1413.
PMID
368070
Source
scival
Published In
Journal of Biological Chemistry
Volume
254
Issue
4
Publish Date
1979
Start Page
1408
End Page
1413

T7-induced DNA polymerase. Characterization of associated exonuclease activities and resolution into biologically active subunits.

Bacteriophage T7-induced DNA polymerase has been isolated by a procedure suitable for large scale use and which yields near homogeneous enzyme. In addition to previously described DNA polymerase activity and 3' to 5' exonucleolytic activity on single stranded DNA (Grippo, P., and Richardson, C. C. (1971) J. Biol. Chem. 246, 6867-6873), the enzyme also possesses a highly active exonuclease which hydrolyzes duplex substrates with 3' to 5' directionality. The native polymerase has been dissociated using 6 M guanidine HCl and resolved into biologically active subunits: T7 gene 5 protein and Escherichia coli thioredoxin. The phage-specified subunit obtained by this procedure is deficient in DNA polymerase and double strand exonuclease activities, with deficiencies in these activities being apparent at the level of a single turnover. However, it possesses near normal levels of a single strand hydrolytic activity which is identical to that associated with the native polymerase with respect to substrate specificity and suppression of hydrolysis by low levels of deoxyribonucleoside 5'-triphosphates. Thioredoxin forms a molecular complex with the T7 gene 5 protein, and addition of the host protein restores restores DNA polymerase and double strand exonuclease activities to near normal levels.

Authors
Adler, S; Modrich, P
MLA Citation
Adler, S, and Modrich, P. "T7-induced DNA polymerase. Characterization of associated exonuclease activities and resolution into biologically active subunits." Journal of Biological Chemistry 254.22 (1979): 11605-11614.
PMID
387776
Source
scival
Published In
Journal of Biological Chemistry
Volume
254
Issue
22
Publish Date
1979
Start Page
11605
End Page
11614

Structures and mechanisms of DNA restriction and modification enzymes

Authors
Modrich, P
MLA Citation
Modrich, P. "Structures and mechanisms of DNA restriction and modification enzymes." Quarterly Reviews of Biophysics 12.3 (1979): 315-369.
PMID
232555
Source
scival
Published In
Quarterly Reviews of Biophysics
Volume
12
Issue
3
Publish Date
1979
Start Page
315
End Page
369

Substrate dependence of the mechanism of EcoRI endonuclease

Authors
Rubin, RA; Modrich, P
MLA Citation
Rubin, RA, and Modrich, P. "Substrate dependence of the mechanism of EcoRI endonuclease." Nucleic Acids Research 5.8 (1978): 2991-2997.
PMID
211492
Source
scival
Published In
Nucleic Acids Research
Volume
5
Issue
8
Publish Date
1978
Start Page
2991
End Page
2997

EcoRI methylase. Physical and catalytic properties of the homogeneous enzyme

Authors
Rubin, RA; Modrich, P
MLA Citation
Rubin, RA, and Modrich, P. "EcoRI methylase. Physical and catalytic properties of the homogeneous enzyme." Journal of Biological Chemistry 252.20 (1977): 7265-7272.
PMID
332688
Source
scival
Published In
Journal of Biological Chemistry
Volume
252
Issue
20
Publish Date
1977
Start Page
7265
End Page
7272

Role of the 2-amino group of deoxyguanosine in sequence recognition by EcoRI restriction and modification enzymes

Authors
Modrich, P; Rubin, RA
MLA Citation
Modrich, P, and Rubin, RA. "Role of the 2-amino group of deoxyguanosine in sequence recognition by EcoRI restriction and modification enzymes." Journal of Biological Chemistry 252.20 (1977): 7273-7278.
PMID
332689
Source
scival
Published In
Journal of Biological Chemistry
Volume
252
Issue
20
Publish Date
1977
Start Page
7273
End Page
7278

Modification of Escherichia coli DNA ligase by cleavage with trypsin

Authors
Panasenko, SM; Modrich, P; Lehman, IR
MLA Citation
Panasenko, SM, Modrich, P, and Lehman, IR. "Modification of Escherichia coli DNA ligase by cleavage with trypsin." Journal of Biological Chemistry 251.11 (1976): 3432-3435.
PMID
179997
Source
scival
Published In
Journal of Biological Chemistry
Volume
251
Issue
11
Publish Date
1976
Start Page
3432
End Page
3435

EcoRI endonuclease. Physical and catalytic properties of the homogeneous enzyme

A procedure for large scale isolation of E. coli RI endonuclease in high yield has been developed. The purified enzyme is homogeneous as judged by polyacrylamide gel electrophoresis and analytical sedimentation. The denatured and reduced form of the enzyme has a molecular weight of 28,500 ± 500. In solution the enzyme exists as a mixture of dimers and tetramers of molecular weights 57,000 and 114,000, respectively. We estimate the dissociation constant for tetramer to dimer transition to be less than or approximately equal to 1 x 10-7 M. Steady state kinetic analysis of the endonuclease with ColE1 DNA as substrate showed that the enzyme obeys Michaelis Menten kinetics. At 37° the turnover number is four double strand scissions per min, and the Km for ColE1 molecules is 8 x 10-9 M. At 0° the major product of endonuclease action contains only one single strand break in the RI site, and such molecules can dissociate from the enzyme. In contrast, at 30° or 37°, two single strand breaks are introduced into the RI sequence prior to dissociation of the enzyme. A transient enzyme bound intermediate containing only one break in the RI site was observed in studies of a single turnover at 30°. Kinetic analysis of this reaction indicates that the first break is introduced into the RI site with a first order rate constant of at least 40 min-1, while the second cleavage occurs with a rate constant of 14 min-1. Since the turnover number of the enzyme at 30° is only 0.72 min-1, these results indicate that the rate limiting step is release of endonuclease from its DNA product.

Authors
Modrich, P; Zabel, D
MLA Citation
Modrich, P, and Zabel, D. "EcoRI endonuclease. Physical and catalytic properties of the homogeneous enzyme." Journal of Biological Chemistry 251.19 (1976): 5866-5874.
PMID
786985
Source
scival
Published In
Journal of Biological Chemistry
Volume
251
Issue
19
Publish Date
1976
Start Page
5866
End Page
5874

Bacteriophage T7 deoxyribonucleic acid replication in vitro. A protein of Escherichia coli required for bacteriophage T7 DNA polymerase activity

Authors
Modrich, P; Richardson, CC
MLA Citation
Modrich, P, and Richardson, CC. "Bacteriophage T7 deoxyribonucleic acid replication in vitro. A protein of Escherichia coli required for bacteriophage T7 DNA polymerase activity." Journal of Biological Chemistry 250.14 (1975): 5508-5514.
PMID
1095578
Source
scival
Published In
Journal of Biological Chemistry
Volume
250
Issue
14
Publish Date
1975
Start Page
5508
End Page
5514

Bacteriophage T7 deoxyribonucleic acid replication in vitro. Bacteriophage T7 DNA polymerase: an enzyme composed of phage and host specified subunits

Authors
Modrich, P; Richardson, CC
MLA Citation
Modrich, P, and Richardson, CC. "Bacteriophage T7 deoxyribonucleic acid replication in vitro. Bacteriophage T7 DNA polymerase: an enzyme composed of phage and host specified subunits." Journal of Biological Chemistry 250.14 (1975): 5515-5522.
PMID
1095579
Source
scival
Published In
Journal of Biological Chemistry
Volume
250
Issue
14
Publish Date
1975
Start Page
5515
End Page
5522

DNA synthesis in strains of Escherichia coli K12 with temperature-sensitive DNA ligase and DNA polymerase I

The net DNA synthesis that persists at the restrictive temperature in the conditional lethal DNA ligase mutant Escherichia coli ligts7 is semiconservative, suggesting that although the rate of joining of 10 S "Okazaki fragments" in the mutant is greatly reduced, it is nevertheless sufficient to permit continued progression of the replication fork and the initiation of new rounds of replication. DNA synthesis in E. coli ligts7 of a phage (P2) that replicates its chromosomes unidirectionally is discontinuous on both strands. A double mutant of E. coli K12, (ligts7 polA12), has been constructed which bears a temperature-sensitive mutation (polA12) in the structural gene for DNA polymerase I in addition to the ligts7 mutation. Joining of the Okazaki fragments in the double mutant occurs at a slower rate than in either the ligts7 or polA12 parents. In contrast to the behavior of the single mutants, DNA synthesis in the double mutant stops abruptly upon shift from 25 °C to 42 °C. © 1974.

Authors
Konrad, EB; Modrich, P; Lehman, IR
MLA Citation
Konrad, EB, Modrich, P, and Lehman, IR. "DNA synthesis in strains of Escherichia coli K12 with temperature-sensitive DNA ligase and DNA polymerase I." Journal of Molecular Biology 90.1 (1974): 115-126.
PMID
4616094
Source
scival
Published In
Journal of Molecular Biology
Volume
90
Issue
1
Publish Date
1974
Start Page
115
End Page
126
DOI
10.1016/0022-2836(74)90260-5

Deoxyribonucleic acid ligase. Isolation and physical characterization of the homogeneous enzyme from Escherichia coli

DNA ligase of Escherichia coli has been purified to homogeneity as judged by polyacrylamide gel electrophoresis and by analytical ultracentrifugation. The molecular weights of the native, and the denatured and reduced forms of the enzyme are 74,000 ± 3,000, hence, ligase consists of a single polypeptide chain. When the unadenylylated form of the enzyme is incubated with diphosphopyridine nucleotide, approximately 1 mole of adenosine 5' monophosphate becomes covalently linked per 74,000 g of protein.

Authors
Modrich, P; Anraku, Y; Lehman, IR
MLA Citation
Modrich, P, Anraku, Y, and Lehman, IR. "Deoxyribonucleic acid ligase. Isolation and physical characterization of the homogeneous enzyme from Escherichia coli." Journal of Biological Chemistry 248.21 (1973): 7495-7501.
PMID
4355584
Source
scival
Published In
Journal of Biological Chemistry
Volume
248
Issue
21
Publish Date
1973
Start Page
7495
End Page
7501

Deoxyribonucleic acid ligase. A steady state kinetic analysis of the reaction catalyzed by the enzyme from Escherichia coli

The DNA joining reaction catalyzed by the Escherichia coli DNA ligase is markedly enhanced by low concentrations of monovalent cations, NH4+ being most effective. The K(m) for NH4+ is about 1 mM, and at saturating concentrations it increases the true V(max) by 20 fold. Under these conditions the K(m) for diphosphopyridine nucleotide is 7 μM, the K(m) for single strand breaks is 0.03 to 0.06 μM, and the turnover number is 25 sealing events per min. The DNA joining reaction obeys ping pong kinetics, thus providing kinetic evidence for the participation of a covalent intermediate, presumably ligase adenylate. The rate of the ligase catalyzed diphosphopyridine nucleotide nicotinamide mononucleotide exchange reaction is unaffected by NH4+, indicating that the activation occurs at a step subsequent to the formation of ligase adenylate; furthermore, the exchange reaction is faster than the rate of DNA joining, demonstrating that ligase adenylate can be formed at a rate sufficient to be an intermediate in the over all reaction. The rate of release of adenosine 5' monophosphate from synthetic DNA adenylate in the absence of NH4+ is greater than the rate of DNA joining, as expected for a kinetically significant intermediate. However, in the presence of NH4+, the rate of adenosine 5' monophosphate release is less than that of the over all reaction. Since NH4+ markedly increases the apparent rate of dissociation of ligase adenylate from DNA, the result may reflect reversal of the reaction to form ligase adenylate from ligase and DNA adenylate and dissociation of this form of the enzyme from the DNA.

Authors
Modrich, P; Lehman, IR
MLA Citation
Modrich, P, and Lehman, IR. "Deoxyribonucleic acid ligase. A steady state kinetic analysis of the reaction catalyzed by the enzyme from Escherichia coli." Journal of Biological Chemistry 248.21 (1973): 7502-7511.
PMID
4355585
Source
scival
Published In
Journal of Biological Chemistry
Volume
248
Issue
21
Publish Date
1973
Start Page
7502
End Page
7511

Genetic and enzymatic characterization of a conditional lethal mutant of Escherichia coli K12 with a temperature-sensitive DNA ligase

TAUts7 an Escherichia coli 15 strain with a thermolabile DNA ligase, has previously been shown to be a temperature-sensitive conditional lethal mutant that is sensitive to methyl methane sulfonate and to ultraviolet irradiation; it also accumulates 10 S DNA fragments to an abnormal extent. When the ligase mutation is transferred to a wild-type E. coli K12 strain, the strain becomes temperature sensitive for growth and displays the same characteristics as TAUts7. These findings show that a functional DNA ligase is essential for normal DNA replication and repair in E. coli. © 1973.

Authors
Konrad, EB; Modrich, P; Lehman, IR
MLA Citation
Konrad, EB, Modrich, P, and Lehman, IR. "Genetic and enzymatic characterization of a conditional lethal mutant of Escherichia coli K12 with a temperature-sensitive DNA ligase." Journal of Molecular Biology 77.4 (1973): 519-529.
PMID
4353283
Source
scival
Published In
Journal of Molecular Biology
Volume
77
Issue
4
Publish Date
1973
Start Page
519
End Page
529
DOI
10.1016/0022-2836(73)90220-9

Enzymatic joining of polynucleotides. XI. Reversal of Escherichia coli deoxyribonucleic acid ligase reaction.

Authors
Modrich, P; Lehman, IR; Wang, JC
MLA Citation
Modrich, P, Lehman, IR, and Wang, JC. "Enzymatic joining of polynucleotides. XI. Reversal of Escherichia coli deoxyribonucleic acid ligase reaction." Journal of Biological Chemistry 247.19 (1972): 6370-6372.
PMID
4568616
Source
scival
Published In
Journal of Biological Chemistry
Volume
247
Issue
19
Publish Date
1972
Start Page
6370
End Page
6372

Enzymatic characterization of a mutant of Escherichia coli with an altered DNA ligase.

Authors
Modrich, P; Lehman, IR
MLA Citation
Modrich, P, and Lehman, IR. "Enzymatic characterization of a mutant of Escherichia coli with an altered DNA ligase." Proceedings of the National Academy of Sciences of the United States of America 68.5 (1971): 1002-1005.
PMID
4995816
Source
scival
Published In
Proceedings of the National Academy of Sciences of the United States of America
Volume
68
Issue
5
Publish Date
1971
Start Page
1002
End Page
1005

Enzymatic joining of polynucleotides. IX. A simple and rapid assay of polynucleotide joining (ligase) activity by measurement of circle formation from linear deoxyadenylate-deoxythymidylate copolymer.

Authors
Modrich, P; Lehman, IR
MLA Citation
Modrich, P, and Lehman, IR. "Enzymatic joining of polynucleotides. IX. A simple and rapid assay of polynucleotide joining (ligase) activity by measurement of circle formation from linear deoxyadenylate-deoxythymidylate copolymer." Journal of Biological Chemistry 245.14 (1970): 3626-3631.
PMID
4919213
Source
scival
Published In
Journal of Biological Chemistry
Volume
245
Issue
14
Publish Date
1970
Start Page
3626
End Page
3631
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