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      Activation of Saccharomyces cerevisiae Mlh1-Pms1 Endonuclease in a Reconstituted Mismatch Repair System*

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          Abstract

          Background: Biochemical analysis of S. cerevisiae MMR mutants has been limited by a lack of reconstituted MMR reactions.

          Results: 3′ nick-directed Mlh1-Pms1-dependent endonuclease and reconstituted MMR reactions were developed.

          Conclusion: 3′ nick-directed MMR required the Mlh1-Pms1 endonuclease and was eliminated by mutations inactivating Exo1-independent MMR.

          Significance: The reconstituted MMR reactions facilitated analysis of uncharacterized MMR mutants and the mechanism of Exo1-independent MMR.

          Abstract

          Previous studies reported the reconstitution of an Mlh1-Pms1-independent 5′ nick-directed mismatch repair (MMR) reaction using Saccharomyces cerevisiae proteins. Here we describe the reconstitution of a mispair-dependent Mlh1-Pms1 endonuclease activation reaction requiring Msh2-Msh6 (or Msh2-Msh3), proliferating cell nuclear antigen (PCNA), and replication factor C (RFC) and a reconstituted Mlh1-Pms1-dependent 3′ nick-directed MMR reaction requiring Msh2-Msh6 (or Msh2-Msh3), exonuclease 1 (Exo1), replication protein A (RPA), RFC, PCNA, and DNA polymerase δ. Both reactions required Mg 2+ and Mn 2+ for optimal activity. The MMR reaction also required two reaction stages in which the first stage required incubation of Mlh1-Pms1 with substrate DNA, with or without Msh2-Msh6 (or Msh2-Msh3), PCNA, and RFC but did not require nicking of the substrate, followed by a second stage in which other proteins were added. Analysis of different mutant proteins demonstrated that both reactions required a functional Mlh1-Pms1 endonuclease active site, as well as mispair recognition and Mlh1-Pms1 recruitment by Msh2-Msh6 but not sliding clamp formation. Mutant Mlh1-Pms1 and PCNA proteins that were defective for Exo1-independent but not Exo1-dependent MMR in vivo were partially defective in the Mlh1-Pms1 endonuclease and MMR reactions, suggesting that both reactions reflect the activation of Mlh1-Pms1 seen in Exo1-independent MMR in vivo. The availability of this reconstituted MMR reaction should now make it possible to better study both Exo1-independent and Exo1-dependent MMR.

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          Most cited references82

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          Mechanisms and functions of DNA mismatch repair.

          Guo-Min Li (2008)
          DNA mismatch repair (MMR) is a highly conserved biological pathway that plays a key role in maintaining genomic stability. The specificity of MMR is primarily for base-base mismatches and insertion/deletion mispairs generated during DNA replication and recombination. MMR also suppresses homeologous recombination and was recently shown to play a role in DNA damage signaling in eukaryotic cells. Escherichia coli MutS and MutL and their eukaryotic homologs, MutSalpha and MutLalpha, respectively, are key players in MMR-associated genome maintenance. Many other protein components that participate in various DNA metabolic pathways, such as PCNA and RPA, are also essential for MMR. Defects in MMR are associated with genome-wide instability, predisposition to certain types of cancer including hereditary non-polyposis colorectal cancer, resistance to certain chemotherapeutic agents, and abnormalities in meiosis and sterility in mammalian systems.
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            DNA mismatch repair: functions and mechanisms.

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              Endonucleolytic function of MutLalpha in human mismatch repair.

              Half of hereditary nonpolyposis colon cancer kindreds harbor mutations that inactivate MutLalpha (MLH1*PMS2 heterodimer). MutLalpha is required for mismatch repair, but its function in this process is unclear. We show that human MutLalpha is a latent endonuclease that is activated in a mismatch-, MutSalpha-, 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 MutSalpha-activated exonuclease I. The probable endonuclease active site has been localized to a PMS2 DQHA(X)(2)E(X)(4)E 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.
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                Author and article information

                Journal
                J Biol Chem
                J. Biol. Chem
                jbc
                jbc
                JBC
                The Journal of Biological Chemistry
                American Society for Biochemistry and Molecular Biology (11200 Rockville Pike, Suite 302, Rockville, MD 20852-3110, U.S.A. )
                0021-9258
                1083-351X
                28 August 2015
                13 July 2015
                13 July 2015
                : 290
                : 35
                : 21580-21590
                Affiliations
                From the []Ludwig Institute for Cancer Research,
                the [§ ]Department of Cellular and Molecular Medicine,
                []Moores-UCSD Cancer Center, and
                the []Institute of Genomic Medicine, University of California, San Diego School of Medicine, La Jolla, California 92093
                Author notes
                [1 ] To whom correspondence should be addressed: Ludwig Inst. for Cancer Research, University of California, San Diego School of Medicine, La Jolla, CA 92093. Tel.: 858-534-7804; Fax: 858-534-7750; E-mail: rkolodner@ 123456ucsd.edu .
                Article
                M115.662189
                10.1074/jbc.M115.662189
                4571882
                26170454
                5e25955c-7fcf-416e-9a5a-7510905ab9c3
                © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

                Author's Choice—Final version free via Creative Commons CC-BY license.

                History
                : 28 April 2015
                : 10 July 2015
                Funding
                Funded by: National Institutes of Health
                Award ID: R01 GM50006
                Award ID: P01 CA92584
                Award ID: F32 GM106598
                Categories
                DNA and Chromosomes

                Biochemistry
                dna recombination,dna repair,dna replication,mutagenesis,proliferating cell nuclear antigen (pcna),replication factor c (rfc),yeast genetics,exonuclease 1,genome instability,msh2-msh6

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