Portal de Periódicos da CAPES

A Mutation in the MSH6 Subunit of the Saccharomyces cerevisiae MSH2-MSH6 Complex Disrupts Mismatch Recognition

1999; Elsevier BV; Volume: 274; Issue: 23 Linguagem: Inglês

10.1074/jbc.274.23.16115

1083-351X

Jayson Bowers, Tanya Sokolsky, Tony Quach, Eric Alani,

Fungal and yeast genetics research

In yeast, MSH2 interacts with MSH6 to repair base pair mismatches and single nucleotide insertion/deletion mismatches and with MSH3 to recognize small loop insertion/deletion mismatches. We identified a msh6 mutation (msh6-F337A) that when overexpressed in wild type strains conferred a defect in both MSH2-MSH6- and MSH2-MSH3-dependent mismatch repair pathways. Genetic analysis suggested that this phenotype was due to msh6-F337A sequestering MSH2 and preventing it from interacting with MSH3 and MSH6. In UV cross-linking, filter binding, and gel retardation assays, the MSH2-msh6-F337A complex displayed a mismatch recognition defect. These observations, in conjunction with ATPase and dissociation rate analysis, suggested that MSH2-msh6-F337A formed an unproductive complex that was unable to stably bind to mismatch DNA. In yeast, MSH2 interacts with MSH6 to repair base pair mismatches and single nucleotide insertion/deletion mismatches and with MSH3 to recognize small loop insertion/deletion mismatches. We identified a msh6 mutation (msh6-F337A) that when overexpressed in wild type strains conferred a defect in both MSH2-MSH6- and MSH2-MSH3-dependent mismatch repair pathways. Genetic analysis suggested that this phenotype was due to msh6-F337A sequestering MSH2 and preventing it from interacting with MSH3 and MSH6. In UV cross-linking, filter binding, and gel retardation assays, the MSH2-msh6-F337A complex displayed a mismatch recognition defect. These observations, in conjunction with ATPase and dissociation rate analysis, suggested that MSH2-msh6-F337A formed an unproductive complex that was unable to stably bind to mismatch DNA. DNA mismatches can arise through DNA replication errors, physical damage, and heteroduplex formation during genetic recombination. If left unrepaired, these mismatches become fixed in the genome as mutations. The best understood mismatch repair system is theEscherichia coli mutHLS long-patch repair pathway (1Modrich P. Lahue R.S. Annu. Rev. Biochem. 1996; 65: 101-133Crossref PubMed Scopus (1318) Google Scholar). A model for the initiation of mismatch repair by MutH, MutL, and MutS immediately after passage of a DNA replication fork has been developed based upon in vivo studies and an in vitromismatch repair system reconstituted from purified components (1Modrich P. Lahue R.S. Annu. Rev. Biochem. 1996; 65: 101-133Crossref PubMed Scopus (1318) Google Scholar, 2Lahue R.S. Au K.G. Modrich P. Science. 1989; 245: 160-164Crossref PubMed Scopus (444) Google Scholar, 3Au K.G. Welsh K. Modrich P. J. Biol. Chem. 1992; 267: 12142-12148Abstract Full Text PDF PubMed Google Scholar, 4Su S. Modrich P. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 5057-5061Crossref PubMed Scopus (258) Google Scholar, 5Haber L.T. Walker G.C. EMBO J. 1991; 10: 2707-2715Crossref PubMed Scopus (134) Google Scholar, 6Welsh K.M. Lu A.-L. Clark S. Modrich P. J. Biol. Chem. 1987; 262: 15624-15629Abstract Full Text PDF PubMed Google Scholar). In this model, a mismatch is first recognized and bound by a dimer of MutS that displays an intrinsic ATPase activity. In a reaction that requires ATP, a dimer of MutL binds to MutS and then activates the methylation sensitive endonuclease MutH. Activation of MutH results in cleavage of the unmethylated DNA strand at hemimethylated d(GATC) sites that are transiently present after replication fork passage, providing an entry point for excision and replication proteins to remove the mismatch and repair the resulting DNA gap using the parental DNA strand as a template.The ability of MutS to both recognize base pair mismatches and trigger downstream events that can be located several kilobases away from a mismatch site suggests that it can bind to DNA in at least two different modes. The first mode allows mismatch recognition, and the second mode allows MutS protein to use the energy of ATP hydrolysis to translocate along DNA with MutL so that it can activate MutH at GATC sites (1Modrich P. Lahue R.S. Annu. Rev. Biochem. 1996; 65: 101-133Crossref PubMed Scopus (1318) Google Scholar, 7Allen D.J. Makhov A. Grilley M. Taylor J. Thresher R. Modrich P. Griffith J.D. EMBO J. 1997; 16: 4467-4476Crossref PubMed Scopus (268) Google Scholar, 8Grilley M. Welsh K.M. Su S.-S. Modrich P. J. Biol. Chem. 1989; 264: 1000-1004Abstract Full Text PDF PubMed Google Scholar). Support for the presence of multiple MutS DNA binding modes was initially obtained from DNA binding assays showing that MutS protein can specifically recognize base pair mismatches in the absence of ATP and that the addition of ATP resulted in the loss of mismatch binding specificity (8Grilley M. Welsh K.M. Su S.-S. Modrich P. J. Biol. Chem. 1989; 264: 1000-1004Abstract Full Text PDF PubMed Google Scholar). An elegant electron microscopy analysis performed by the Griffith laboratory (7Allen D.J. Makhov A. Grilley M. Taylor J. Thresher R. Modrich P. Griffith J.D. EMBO J. 1997; 16: 4467-4476Crossref PubMed Scopus (268) Google Scholar) and the Modrich laboratory (9Blackwell L.J. Martik D. Bjornson K.P. Bjornson E.S. Modrich P. J. Biol. Chem. 1998; 273: 32055-32062Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar) provides further evidence for this idea. They showed that MutS can form ATP-dependent loop structures on DNA substrates that contain a mismatch site. The size of loop was dependent on incubation time and the presence of a mismatch site. They hypothesized that MutS can bind to a mismatch substrate in an ATP independent step. After recognition, a second binding mode is activated through an ATP-dependent conformational change in MutS resulting in the loss of its mismatch recognition functions and a shift into a mode that allows it to bidirectionally translocate along DNA away from a mismatch site. This activity results in the formation of loop structures that are thought to serve as topological intermediates for strand excision and allow MutS to scan along DNA until it identifies and activates MutH bound at GATC sites.At present, little is known about which domains in MutS are important for mismatch recognition. Recently, a DNA cross-linking analysis performed by Malkov et al. (10Malkov V.A. Biswas I. Camerini-Otero R.D. Hsieh P. J. Biol Chem. 1997; 272: 23811-23817Crossref PubMed Scopus (91) Google Scholar) revealed that the phenylalanine 39 residue in Thermus aquaticus MutS was critical for photocross-linking of MutS to a mismatch substrate. They also showed that a mutant derivative of MutS, mutS-F39A, displayed a reduced affinity for mismatch substrate. Although this study identified a domain that is important for mismatch recognition, it did not address whether this domain functions in general DNA binding and/or in mismatch binding and whether it functions during one or more DNA binding modes.Whereas studies in bacteria have greatly increased our understanding of mismatch repair, analogous repair systems in eukaryotes appear to be more complex (1Modrich P. Lahue R.S. Annu. Rev. Biochem. 1996; 65: 101-133Crossref PubMed Scopus (1318) Google Scholar, 11Kolodner R. Genes Dev. 1996; 10: 1433-1442Crossref PubMed Scopus (540) Google Scholar, 12Crouse G.F. Nickoloff J. Hoekstra M. DNA Damage and Repair: Biochemistry, Genetics and Cell Biology. Humana Press, Totowa, NJ1996: 411-448Google Scholar). Several homologs of mutS andmutL have been identified in eukaryotic organisms. A feature of the MutS homologs is they all contain a highly conserved ATP binding domain. In the yeast Saccharomyces cerevisiae, sixmutS homologs and four mutL homologs have been identified, and the gene products of the MSH2, MSH3, MSH6, MLH1, and PMS1 genes have been identified as components of nuclear mismatch repair. Several studies have suggested that during mismatch recognition, MSH2 acts a scaffold for mismatch binding, whereas MSH6 and MSH3 act as specificity factors (13Alani E. Mol. Cell. Biol. 1996; 16: 5604-5615Crossref PubMed Scopus (141) Google Scholar, 14Marsischky G.T. Filosi N. Kane M.F. Kolodner R. Genes Dev. 1996; 10: 407-420Crossref PubMed Scopus (495) Google Scholar, 15Studamire B. Quach T. Alani E. Mol. Cell. Biol. 1998; 18: 7590-7601Crossref PubMed Scopus (81) Google Scholar, 16Johnson R.E. Kovvali G.K. Prakash L. Prakash S. J. Biol. Chem. 1996; 271: 7285-7288Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 17Iaccarino I. Marra G. Palombo F. Jiricny J. EMBO J. 1998; 17: 2677-2686Crossref PubMed Scopus (144) Google Scholar). This hypothesis was based on genetic studies showing that msh2mutants are defective in the repair of both base pair and loop insertion/deletion mismatches, whereas msh3 mutants are principally defective in the repair of 2–4-nucleotide loop mismatches and msh6 mutants are principally defective in the repair of base-base and single nucleotide insertion/deletion mismatches (14Marsischky G.T. Filosi N. Kane M.F. Kolodner R. Genes Dev. 1996; 10: 407-420Crossref PubMed Scopus (495) Google Scholar, 16Johnson R.E. Kovvali G.K. Prakash L. Prakash S. J. Biol. Chem. 1996; 271: 7285-7288Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar,18Strand M. Earley M.C. Crouse G.F. Petes T.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10418-10421Crossref PubMed Scopus (126) Google Scholar). Interestingly, MSH3 and MSH6 functions have been shown to overlap, as msh3 and msh6 single mutants do not display as strong a defect in mismatch repair in their respective mismatch repair assays as do msh2 mutants. msh3 msh6 double mutants, however, display a mutator phenotype that is identical to that observed in msh2 mutants (14Marsischky G.T. Filosi N. Kane M.F. Kolodner R. Genes Dev. 1996; 10: 407-420Crossref PubMed Scopus (495) Google Scholar, 16Johnson R.E. Kovvali G.K. Prakash L. Prakash S. J. Biol. Chem. 1996; 271: 7285-7288Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). Subsequent biochemical studies have shown that MSH2 can interact with MSH3 and MSH6 to form distinct complexes that bind subsets of mismatches predicted by the genetic studies and that an MLH1-PMS1 heterodimer can interact with these complexes when they are bound to a mismatch site (13Alani E. Mol. Cell. Biol. 1996; 16: 5604-5615Crossref PubMed Scopus (141) Google Scholar, 17Iaccarino I. Marra G. Palombo F. Jiricny J. EMBO J. 1998; 17: 2677-2686Crossref PubMed Scopus (144) Google Scholar,19Iaccarino I. Palombo F. Drummond J. Totty N.F. Hsuan J.J. Modrich P. Jiricny J. Curr. Biol. 1996; 6: 484-486Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 20Habraken Y. Sung P. Prakash L. Prakash S. Curr. Biol. 1996; 6: 1185-1187Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 21Habraken Y. Sung P. Prakash L. Prakash S. Curr. Biol. 1997; 7: 790-793Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 22Habraken Y. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1998; 273: 9837-9841Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar).In this study, we used genetic and biochemical assays to investigate the mismatch recognition properties of MSH2-MSH6. Based on the pioneering studies performed by Malkov et al. (10Malkov V.A. Biswas I. Camerini-Otero R.D. Hsieh P. J. Biol Chem. 1997; 272: 23811-23817Crossref PubMed Scopus (91) Google Scholar), we created site-specific mutations in putative DNA binding domains in the MSH2 and MSH6 subunits and examined the effect of these mutations in UV cross-linking and DNA binding studies. Our analysis, described below, is consistent with MSH2 acting primarily as a scaffold for interactions with MSH6 and MSH3 that confer specificity to mismatch recognition. Interestingly, a mutation in MSH6 (msh6-F337A) was identified that conferred a dominant negative phenotype that was consistent with msh6-F337A sequestering MSH2 from functioning in mismatch recognition. This analysis also suggested that subunit interactions, mismatch recognition functions, and ATPase activities are coordinated to allow for the recognition and repair of DNA mismatches. DNA mismatches can arise through DNA replication errors, physical damage, and heteroduplex formation during genetic recombination. If left unrepaired, these mismatches become fixed in the genome as mutations. The best understood mismatch repair system is theEscherichia coli mutHLS long-patch repair pathway (1Modrich P. Lahue R.S. Annu. Rev. Biochem. 1996; 65: 101-133Crossref PubMed Scopus (1318) Google Scholar). A model for the initiation of mismatch repair by MutH, MutL, and MutS immediately after passage of a DNA replication fork has been developed based upon in vivo studies and an in vitromismatch repair system reconstituted from purified components (1Modrich P. Lahue R.S. Annu. Rev. Biochem. 1996; 65: 101-133Crossref PubMed Scopus (1318) Google Scholar, 2Lahue R.S. Au K.G. Modrich P. Science. 1989; 245: 160-164Crossref PubMed Scopus (444) Google Scholar, 3Au K.G. Welsh K. Modrich P. J. Biol. Chem. 1992; 267: 12142-12148Abstract Full Text PDF PubMed Google Scholar, 4Su S. Modrich P. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 5057-5061Crossref PubMed Scopus (258) Google Scholar, 5Haber L.T. Walker G.C. EMBO J. 1991; 10: 2707-2715Crossref PubMed Scopus (134) Google Scholar, 6Welsh K.M. Lu A.-L. Clark S. Modrich P. J. Biol. Chem. 1987; 262: 15624-15629Abstract Full Text PDF PubMed Google Scholar). In this model, a mismatch is first recognized and bound by a dimer of MutS that displays an intrinsic ATPase activity. In a reaction that requires ATP, a dimer of MutL binds to MutS and then activates the methylation sensitive endonuclease MutH. Activation of MutH results in cleavage of the unmethylated DNA strand at hemimethylated d(GATC) sites that are transiently present after replication fork passage, providing an entry point for excision and replication proteins to remove the mismatch and repair the resulting DNA gap using the parental DNA strand as a template. The ability of MutS to both recognize base pair mismatches and trigger downstream events that can be located several kilobases away from a mismatch site suggests that it can bind to DNA in at least two different modes. The first mode allows mismatch recognition, and the second mode allows MutS protein to use the energy of ATP hydrolysis to translocate along DNA with MutL so that it can activate MutH at GATC sites (1Modrich P. Lahue R.S. Annu. Rev. Biochem. 1996; 65: 101-133Crossref PubMed Scopus (1318) Google Scholar, 7Allen D.J. Makhov A. Grilley M. Taylor J. Thresher R. Modrich P. Griffith J.D. EMBO J. 1997; 16: 4467-4476Crossref PubMed Scopus (268) Google Scholar, 8Grilley M. Welsh K.M. Su S.-S. Modrich P. J. Biol. Chem. 1989; 264: 1000-1004Abstract Full Text PDF PubMed Google Scholar). Support for the presence of multiple MutS DNA binding modes was initially obtained from DNA binding assays showing that MutS protein can specifically recognize base pair mismatches in the absence of ATP and that the addition of ATP resulted in the loss of mismatch binding specificity (8Grilley M. Welsh K.M. Su S.-S. Modrich P. J. Biol. Chem. 1989; 264: 1000-1004Abstract Full Text PDF PubMed Google Scholar). An elegant electron microscopy analysis performed by the Griffith laboratory (7Allen D.J. Makhov A. Grilley M. Taylor J. Thresher R. Modrich P. Griffith J.D. EMBO J. 1997; 16: 4467-4476Crossref PubMed Scopus (268) Google Scholar) and the Modrich laboratory (9Blackwell L.J. Martik D. Bjornson K.P. Bjornson E.S. Modrich P. J. Biol. Chem. 1998; 273: 32055-32062Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar) provides further evidence for this idea. They showed that MutS can form ATP-dependent loop structures on DNA substrates that contain a mismatch site. The size of loop was dependent on incubation time and the presence of a mismatch site. They hypothesized that MutS can bind to a mismatch substrate in an ATP independent step. After recognition, a second binding mode is activated through an ATP-dependent conformational change in MutS resulting in the loss of its mismatch recognition functions and a shift into a mode that allows it to bidirectionally translocate along DNA away from a mismatch site. This activity results in the formation of loop structures that are thought to serve as topological intermediates for strand excision and allow MutS to scan along DNA until it identifies and activates MutH bound at GATC sites. At present, little is known about which domains in MutS are important for mismatch recognition. Recently, a DNA cross-linking analysis performed by Malkov et al. (10Malkov V.A. Biswas I. Camerini-Otero R.D. Hsieh P. J. Biol Chem. 1997; 272: 23811-23817Crossref PubMed Scopus (91) Google Scholar) revealed that the phenylalanine 39 residue in Thermus aquaticus MutS was critical for photocross-linking of MutS to a mismatch substrate. They also showed that a mutant derivative of MutS, mutS-F39A, displayed a reduced affinity for mismatch substrate. Although this study identified a domain that is important for mismatch recognition, it did not address whether this domain functions in general DNA binding and/or in mismatch binding and whether it functions during one or more DNA binding modes. Whereas studies in bacteria have greatly increased our understanding of mismatch repair, analogous repair systems in eukaryotes appear to be more complex (1Modrich P. Lahue R.S. Annu. Rev. Biochem. 1996; 65: 101-133Crossref PubMed Scopus (1318) Google Scholar, 11Kolodner R. Genes Dev. 1996; 10: 1433-1442Crossref PubMed Scopus (540) Google Scholar, 12Crouse G.F. Nickoloff J. Hoekstra M. DNA Damage and Repair: Biochemistry, Genetics and Cell Biology. Humana Press, Totowa, NJ1996: 411-448Google Scholar). Several homologs of mutS andmutL have been identified in eukaryotic organisms. A feature of the MutS homologs is they all contain a highly conserved ATP binding domain. In the yeast Saccharomyces cerevisiae, sixmutS homologs and four mutL homologs have been identified, and the gene products of the MSH2, MSH3, MSH6, MLH1, and PMS1 genes have been identified as components of nuclear mismatch repair. Several studies have suggested that during mismatch recognition, MSH2 acts a scaffold for mismatch binding, whereas MSH6 and MSH3 act as specificity factors (13Alani E. Mol. Cell. Biol. 1996; 16: 5604-5615Crossref PubMed Scopus (141) Google Scholar, 14Marsischky G.T. Filosi N. Kane M.F. Kolodner R. Genes Dev. 1996; 10: 407-420Crossref PubMed Scopus (495) Google Scholar, 15Studamire B. Quach T. Alani E. Mol. Cell. Biol. 1998; 18: 7590-7601Crossref PubMed Scopus (81) Google Scholar, 16Johnson R.E. Kovvali G.K. Prakash L. Prakash S. J. Biol. Chem. 1996; 271: 7285-7288Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 17Iaccarino I. Marra G. Palombo F. Jiricny J. EMBO J. 1998; 17: 2677-2686Crossref PubMed Scopus (144) Google Scholar). This hypothesis was based on genetic studies showing that msh2mutants are defective in the repair of both base pair and loop insertion/deletion mismatches, whereas msh3 mutants are principally defective in the repair of 2–4-nucleotide loop mismatches and msh6 mutants are principally defective in the repair of base-base and single nucleotide insertion/deletion mismatches (14Marsischky G.T. Filosi N. Kane M.F. Kolodner R. Genes Dev. 1996; 10: 407-420Crossref PubMed Scopus (495) Google Scholar, 16Johnson R.E. Kovvali G.K. Prakash L. Prakash S. J. Biol. Chem. 1996; 271: 7285-7288Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar,18Strand M. Earley M.C. Crouse G.F. Petes T.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10418-10421Crossref PubMed Scopus (126) Google Scholar). Interestingly, MSH3 and MSH6 functions have been shown to overlap, as msh3 and msh6 single mutants do not display as strong a defect in mismatch repair in their respective mismatch repair assays as do msh2 mutants. msh3 msh6 double mutants, however, display a mutator phenotype that is identical to that observed in msh2 mutants (14Marsischky G.T. Filosi N. Kane M.F. Kolodner R. Genes Dev. 1996; 10: 407-420Crossref PubMed Scopus (495) Google Scholar, 16Johnson R.E. Kovvali G.K. Prakash L. Prakash S. J. Biol. Chem. 1996; 271: 7285-7288Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). Subsequent biochemical studies have shown that MSH2 can interact with MSH3 and MSH6 to form distinct complexes that bind subsets of mismatches predicted by the genetic studies and that an MLH1-PMS1 heterodimer can interact with these complexes when they are bound to a mismatch site (13Alani E. Mol. Cell. Biol. 1996; 16: 5604-5615Crossref PubMed Scopus (141) Google Scholar, 17Iaccarino I. Marra G. Palombo F. Jiricny J. EMBO J. 1998; 17: 2677-2686Crossref PubMed Scopus (144) Google Scholar,19Iaccarino I. Palombo F. Drummond J. Totty N.F. Hsuan J.J. Modrich P. Jiricny J. Curr. Biol. 1996; 6: 484-486Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 20Habraken Y. Sung P. Prakash L. Prakash S. Curr. Biol. 1996; 6: 1185-1187Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 21Habraken Y. Sung P. Prakash L. Prakash S. Curr. Biol. 1997; 7: 790-793Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 22Habraken Y. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1998; 273: 9837-9841Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). In this study, we used genetic and biochemical assays to investigate the mismatch recognition properties of MSH2-MSH6. Based on the pioneering studies performed by Malkov et al. (10Malkov V.A. Biswas I. Camerini-Otero R.D. Hsieh P. J. Biol Chem. 1997; 272: 23811-23817Crossref PubMed Scopus (91) Google Scholar), we created site-specific mutations in putative DNA binding domains in the MSH2 and MSH6 subunits and examined the effect of these mutations in UV cross-linking and DNA binding studies. Our analysis, described below, is consistent with MSH2 acting primarily as a scaffold for interactions with MSH6 and MSH3 that confer specificity to mismatch recognition. Interestingly, a mutation in MSH6 (msh6-F337A) was identified that conferred a dominant negative phenotype that was consistent with msh6-F337A sequestering MSH2 from functioning in mismatch recognition. This analysis also suggested that subunit interactions, mismatch recognition functions, and ATPase activities are coordinated to allow for the recognition and repair of DNA mismatches. We thank Elizabeth Evans, Richard Kolodner, Chris Roberts, Jeff Roberts, Barbara Studamire, and Yali Xie for technical advice and helpful discussions and Elizabeth Evans, Jeff Roberts, and members of the Alani laboratory for their insightful comments on the manuscript.