The Interaction of the Human MutL Homologues in Hereditary Nonpolyposis Colon Cancer

Germline mutations in two human mismatch repair (MMR) genes, hMSH2 and hMLH1, appear to account for approximately 70% of the common cancer susceptibility syndrome hereditary nonpolyposis colorectal cancer (HNPCC). Although the hMLH1 protein has been found to copurify with another MMR protein hPMS2 as a heterodimer, their function in MMR is unknown. In this study, we have identified the physical interaction regions of both hMLH1 with hPMS2. We then examined the effects of hMLH1 missense alterations found in HNPCC kindreds for their interaction with hPMS2. Four of these missense alterations (L574P, K616Δ, R659P, and A681T) displayed >95% reduction in binding to hPMS2. Two additional missense alterations (K618A and K618T) displayed a >85% reduction in binding to hPMS2, whereas three missense alterations (S44F, V506A, and E578G) displayed 25–65% reduction in binding to hPMS2. Interestingly, two HNPCC missense alterations (Q542L and L582V) contained within the consensus interaction region displayed no effect on interaction with hPMS2, suggesting that they may affect other functions of hMLH1. These data confirm that functional deficiencies in the interaction of hMLH1 with hPMS2 are associated with HNPCC as well as suggest that other unknown functional alteration of the human MutL homologues may lead to tumorigenesis in HNPCC kindreds. Germline mutations in two human mismatch repair (MMR) genes, hMSH2 and hMLH1, appear to account for approximately 70% of the common cancer susceptibility syndrome hereditary nonpolyposis colorectal cancer (HNPCC). Although the hMLH1 protein has been found to copurify with another MMR protein hPMS2 as a heterodimer, their function in MMR is unknown. In this study, we have identified the physical interaction regions of both hMLH1 with hPMS2. We then examined the effects of hMLH1 missense alterations found in HNPCC kindreds for their interaction with hPMS2. Four of these missense alterations (L574P, K616Δ, R659P, and A681T) displayed >95% reduction in binding to hPMS2. Two additional missense alterations (K618A and K618T) displayed a >85% reduction in binding to hPMS2, whereas three missense alterations (S44F, V506A, and E578G) displayed 25–65% reduction in binding to hPMS2. Interestingly, two HNPCC missense alterations (Q542L and L582V) contained within the consensus interaction region displayed no effect on interaction with hPMS2, suggesting that they may affect other functions of hMLH1. These data confirm that functional deficiencies in the interaction of hMLH1 with hPMS2 are associated with HNPCC as well as suggest that other unknown functional alteration of the human MutL homologues may lead to tumorigenesis in HNPCC kindreds. Mismatch repair is characterized by the recognition and repair of mispaired nucleotides that may arise from misincorporation during DNA replication, recombination between DNA lacking perfect homology, and physical damage to DNA such as deamination of a methylated cytosine (1Friedberg E.C. Walker G.C. Siede W. DNA Repair and Mutagenesis. American Society for Microbiology, Washington, D. C.1995Google Scholar). The best studied MMR 1The abbreviations used are: MMR, mismatch repair; h-, human; HNPCC, hereditary nonpolyposis colorectal cancer; IVTT, in vitro transcription and translation; DAM, DNA adenine methylation; MSH, MutS homologue; MLH, MutL homologue; PMS, post meioticsegregant; k assoc, relative association constant; Intrel, relative interaction; IRm, mutant interaction ratio; IRwt, wild type interaction ratio; GST, glutathione S-transferase; mutH , mutL, mutS, MutHLS; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis.1The abbreviations used are: MMR, mismatch repair; h-, human; HNPCC, hereditary nonpolyposis colorectal cancer; IVTT, in vitro transcription and translation; DAM, DNA adenine methylation; MSH, MutS homologue; MLH, MutL homologue; PMS, post meioticsegregant; k assoc, relative association constant; Intrel, relative interaction; IRm, mutant interaction ratio; IRwt, wild type interaction ratio; GST, glutathione S-transferase; mutH , mutL, mutS, MutHLS; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis.system is the MutHLS or Dam-instructed pathway in Escherichia coli where mismatch repair is initiated at sites of transient undermethylation of DNA adeninemethylation (Dam) GATC sequences (for review, see Ref. 2Modrich P. Annu. Rev. Genet. 1991; 25: 229-253Crossref PubMed Scopus (770) Google Scholar). Dam-instructed mismatch repair has been shown to require themutH, mutL, mutS, anduvrD(mutU) genes (3Rydberg B. Mutat. Res. 1978; 52: 11-24Crossref PubMed Scopus (77) Google Scholar, 4Glickman B.W. Radman M. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 1063-1067Crossref PubMed Scopus (270) Google Scholar). The mutL gene was first identified in the enteric bacteria Salmonella typhimuriumLT7 as a mutator (mutator LT7 or MutL) (5Demerec M. Lahr E.L. Miyake T. Galehran E. Balbinder E. Baric S. Hashimoto K. Glanville E.V. Gross J.D. Carnegie Inst. Wash. Year Book. 1957; 370: 390-406Google Scholar, 6Miyake T. Genetics. 1960; 45: 755-762Crossref PubMed Google Scholar) and later assigned as a member of the post-replication mismatch repair machinery (3Rydberg B. Mutat. Res. 1978; 52: 11-24Crossref PubMed Scopus (77) Google Scholar, 4Glickman B.W. Radman M. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 1063-1067Crossref PubMed Scopus (270) Google Scholar). The MutL protein was purified based on its ability to complement E. coli mutL extracts for mismatch repair (7Grilley M. Welsh K.M. Su S.S. Modrich P. J. Biol. Chem. 1989; 264: 1000-1004Abstract Full Text PDF PubMed Google Scholar). Although a specific biochemical function has not yet been identified for MutL, it does appear to interact with the mismatch recognition protein MutS (7Grilley M. Welsh K.M. Su S.S. Modrich P. J. Biol. Chem. 1989; 264: 1000-1004Abstract Full Text PDF PubMed Google Scholar), leading to subsequent activation of the endonucleolytic activity of MutH (8Welsh K.M. Lu A.L. Clark S. Modrich P. J. Biol. Chem. 1987; 262: 15624-15629Abstract Full Text PDF PubMed Google Scholar), which initiates excision repair by introducing a strand scission on the unmodified strand of hemimethylated GATC sequences (9Lahue R.S. Au K.G. Modrich P. Science. 1989; 245: 160-164Crossref PubMed Scopus (443) Google Scholar). This mechanism of methyl-directed mismatch repair appears to be unique to Gram-negative bacteria, because homologues of MutH have not been found in either Gram-positive bacteria or eukaryotes. The gene coding for MutL has been highly conserved throughout evolution (for review, see Refs. 10Fishel R. Wilson T. Curr. Opin. Genet. Dev. 1997; 7: 105-113Crossref PubMed Scopus (147) Google Scholar and 11Kolodner R. Genes Dev. 1996; 10: 1433-1442Crossref PubMed Scopus (539) Google Scholar). Homologous genes have been found in a number of organisms including the yeast Saccharomyces cerevisiae where there are at least fourMutLhomologues (MLH): MLH1, MLH2, MLH3, and PMS1 (Post MeioticSegregation) (Ref. 11Kolodner R. Genes Dev. 1996; 10: 1433-1442Crossref PubMed Scopus (539) Google Scholar; Stanford yeast sequence data base). Similar homologues of MutL have also been found in humans (12Bronner C.E. Baker S.M. Morrison P.T. Warren G. Smith L.G. Lescoe M.K. Kane M. Earabino C. Lipford J. Lindblom A. Tannergard P. Bollag R.J. Godwin A.R. Ward D.C. Nordenskjeld M. Fishel R. Kolodner R. Liskay R. Nature. 1994; 368: 258-261Crossref PubMed Scopus (1913) Google Scholar, 13Nicolaides N.C. Papadopoulos N. Liu B. Wei Y.F. Carter K.C. Ruben S.M. Rosen C.A. Haseltine W.A. Fleischmann R.D. Fraser C.M. Adams M.D. Venter J.C. Dunlop M.G. Hamilton S.R. Peterson G.M. de la Chapelle A. Vogelstein B. Kinzler K.W. Nature. 1994; 371: 75-80Crossref PubMed Scopus (1429) Google Scholar). Unfortunately the nomenclature has become complicated. Although human hMLH1 is most closely related to yeast MLH1, the human hPMS1 appears most closely related to yeast MLH2 and/or MLH3, and human hPMS2 is most closely related to yeast PMS1. Both genetic and biochemical evidence have suggested that the S. cerevisiae MLH1 and PMS1 form a heterodimer, and both genes have been shown to be essential for MMR (14Prolla T.A. Christie D.-M. Liskay R.M. Mol. Cell. Biol. 1994; 14: 407-415Crossref PubMed Google Scholar, 15Prolla T.A. Pang Q. Alani E. Kolodner R.D. Liskay R.M. Science. 1994; 265: 1091-1093Crossref PubMed Scopus (279) Google Scholar). Copurification of the human hMLH1-hPMS2, based on its ability to complement MMR in extracts derived from a cell line that was genetically deficient for hMLH1, has confirmed the evolutionary conservation of this heterodimer (16Li G.-M. Modrich P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1950-1954Crossref PubMed Scopus (349) Google Scholar). The function and interaction of hPMS1 as well as the yeast homologues MLH2 and MLH3 are unknown. Defects in the human MMR pathway have been strongly implicated in the etiology of hereditary nonpolyposis colorectal cancer (HNPCC) (12Bronner C.E. Baker S.M. Morrison P.T. Warren G. Smith L.G. Lescoe M.K. Kane M. Earabino C. Lipford J. Lindblom A. Tannergard P. Bollag R.J. Godwin A.R. Ward D.C. Nordenskjeld M. Fishel R. Kolodner R. Liskay R. Nature. 1994; 368: 258-261Crossref PubMed Scopus (1913) Google Scholar, 13Nicolaides N.C. Papadopoulos N. Liu B. Wei Y.F. Carter K.C. Ruben S.M. Rosen C.A. Haseltine W.A. Fleischmann R.D. Fraser C.M. Adams M.D. Venter J.C. Dunlop M.G. Hamilton S.R. Peterson G.M. de la Chapelle A. Vogelstein B. Kinzler K.W. Nature. 1994; 371: 75-80Crossref PubMed Scopus (1429) Google Scholar,17Fishel R. Lescoe M.K. Rao M.R. Copeland N.G. Jenkins N.A. Garber J. Kane M. Kolodner R. Cell. 1993; 75: 1027-1038Abstract Full Text PDF PubMed Scopus (2578) Google Scholar, 18Akiyama Y. Sato H. Yamada T. Nagasaki H. Tsuchiya A. Abe R. Yuasa Y. Cancer Res. 1997; 57: 3920-3923PubMed Google Scholar, 19Miyaki M. Konishi M. Tanaka K. Kikuchi Yanoshita R. Muraoka M. Yasuno M. Igari T. Koike M. Chiba M. Mori T. Nat. Genet. 1997; 17: 271-272Crossref PubMed Scopus (585) Google Scholar). Germline mutations in hMSH2 (humanMutS homologue) and hMLH1 account for approximately 70% of the HNPCC kindreds (20Peltomaki P. Vasen H.F. Gastroenterology. 1997; 113: 1146-1158Abstract Full Text Full Text PDF PubMed Scopus (676) Google Scholar). Mutations in the other MMR genes hPMS1, hPMS2, and hMSH6 appear rare; however, they have been reported in a few atypical families (13Nicolaides N.C. Papadopoulos N. Liu B. Wei Y.F. Carter K.C. Ruben S.M. Rosen C.A. Haseltine W.A. Fleischmann R.D. Fraser C.M. Adams M.D. Venter J.C. Dunlop M.G. Hamilton S.R. Peterson G.M. de la Chapelle A. Vogelstein B. Kinzler K.W. Nature. 1994; 371: 75-80Crossref PubMed Scopus (1429) Google Scholar, 18Akiyama Y. Sato H. Yamada T. Nagasaki H. Tsuchiya A. Abe R. Yuasa Y. Cancer Res. 1997; 57: 3920-3923PubMed Google Scholar, 19Miyaki M. Konishi M. Tanaka K. Kikuchi Yanoshita R. Muraoka M. Yasuno M. Igari T. Koike M. Chiba M. Mori T. Nat. Genet. 1997; 17: 271-272Crossref PubMed Scopus (585) Google Scholar). Biochemical characterization and structure/function analysis of the MMR proteins has contributed to our understanding of their contribution(s) to hereditary and sporadic carcinogenesis. In S. cerevisiae, an interaction region between MLH1 and PMS1 was localized to the carboxyl termini of both proteins using a two-hybrid in vivoassay system (21Pang Q. Prolla T.A. Liskay R.M. Mol. Cell. Biol. 1997; 17: 4465-4473Crossref PubMed Scopus (115) Google Scholar). In this study, we have localized the biochemical interaction region of hMLH1 and hPMS2 to similar, but not identical, carboxyl-terminal regions of both proteins. We found that the interaction region of hMLH1 lies between amino acids 506 and 675, whereas the interaction region of hPMS2 lies between amino acids 675 and 850. To evaluate the biochemical interactions of hMLH1 mutations found in HNPCC in vitro, we constructed several derivatives of the hMLH1 protein that contained mutations previously reported to cosegregate with cancer susceptibility. We found that the L574P, K616Δ, K618A, K618T, R659P, and A681T missense mutations of hMLH1 displayed a >80% loss of interaction with hPMS2, whereas the S44F, V506A, and E578G missense mutations displayed a 20–80% reduction in interaction with hPMS2. Two missense alterations (Q542L and L582V) showed no reduction in hPMS2 interaction, and one of those (Q542L) was also found to have no effect in the dominant mutator assay (22Shimodaira H. Filosi N. Shibata H. Suzuki T. Radice P. Kanamaru R. Friend S.H. Kolodner R.D. Ishioka C. Nat. Genet. 1998; 19: 384-389Crossref PubMed Scopus (121) Google Scholar). These data indicate that loss or reduced interaction between hMLH1 and hPMS2 may play a causative role in the development of HNPCC. However, other as yet undefined functional alterations also appear to contribute to cancer susceptibility. These data are largely consistent with anin vivo dominant mutator assay developed in yeast that was used to examine mutations of hMLH1 found in HNPCC (22Shimodaira H. Filosi N. Shibata H. Suzuki T. Radice P. Kanamaru R. Friend S.H. Kolodner R.D. Ishioka C. Nat. Genet. 1998; 19: 384-389Crossref PubMed Scopus (121) Google Scholar). Restriction endonucleases and T4 DNA ligase from using the from on an and DNA purified using DNA In vitro transcription and using the used to proteins was from from and form other used of the The of hMLH1 and hPMS2 has been previously (12Bronner C.E. Baker S.M. Morrison P.T. Warren G. Smith L.G. Lescoe M.K. Kane M. Earabino C. Lipford J. Lindblom A. Tannergard P. Bollag R.J. Godwin A.R. Ward D.C. Nordenskjeld M. Fishel R. Kolodner R. Liskay R. Nature. 1994; 368: 258-261Crossref PubMed Scopus (1913) Google Scholar). Both hMLH1 and hPMS2 using and proteins using the system of was as The DNA was with and and gel and the was by this is to as and for using and sites in which the is with the This also a of the and was both hMLH1 and hPMS2 at the by a that was to the of an in hMSH2 R. A. Lescoe M.K. Cancer Res. 1994; Google Scholar, J. Mol. Cell. Biol. 1997; 17: PubMed Google Scholar). and constructed by the and the of hMLH1 and hPMS2 with and the genes the The hMLH1 constructed using by using a that a in the first and the nucleotides to the The by the first nucleotides of the strand to the a and a was using as The and and and The hPMS2 constructed in the The by using a with a in the first and the nucleotides to the by the first nucleotides of the strand to the The and with and gel and of these in their to The HNPCC of hMLH1 constructed by using the J. Mol. Cell. Biol. 1997; 17: PubMed Google Scholar). The and These to and then The then in their to the of of or was in with of with was with and to an at of was to a of and in a at for and in of and was to a of and on for and then to of and and the was and two to the was to a of and the was on for was by at in a for and the was to a with such that approximately of protein to of for of protein The at on a at for the at in an for the was and the in of binding and The was three to the of most proteins. The was then to a with binding to approximately of glutathione and at on a for to to the of of these glutathione was then protein by binding the to glutathione by and of protein on using as a (14Prolla T.A. Christie D.-M. Liskay R.M. Mol. Cell. Biol. 1994; 14: 407-415Crossref PubMed Google Scholar). with with or using purified DNA to the to the relative of This was using the specific activity of the for the number of in and using and a with to to of the protein was to such that relative of that used as the was to the of in for at least at on a The three with the binding and then in of and The on an and then using a The interaction assay system is not and is to on the relative association of the hMLH1 mutant proteins with hPMS2. in the relative of may the of To between for such we the of the protein and the protein and and interaction was to a of the interaction with hPMS2 by missense mutant hMLH1 protein was as the of the mutant interaction by the wild type interaction The mutant interaction was by the of with and this number by the of protein with This was for which a of the mutant proteins to be as well as the wild type protein on a The wild type interaction was by the of wild type protein in an interaction and it by the In this interaction was to the of protein by and the interaction are as the and of These appear the assay system in The hMLH1 and hPMS2 proteins purified as a heterodimer based on of MMR in a human cell (16Li G.-M. Modrich P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1950-1954Crossref PubMed Scopus (349) Google Scholar). To the interaction between these we developed an assay that on the of a protein coli as a and in vitro and protein as This assay to be for of the that be for this and The interaction for of these was specific for the proteins because we binding as by and of the protein with the glutathione E. coli glutathione and glutathione as and of the with the hMLH1 or that the to in this assay These suggested that this system was to the interaction of the the we the interaction of hMLH1 with hPMS2 In a between of is to the the is to of hMLH1 not appear to interact with and However, interaction was identified in the carboxyl of the MLH1 protein acids This interaction region was by this carboxyl-terminal interaction in amino We found that amino of hMLH1 with whereas hMLH1 amino showed no interaction hMLH1 amino displayed interaction with whereas hMLH1 amino not and These suggest that the most hPMS2 interaction region of hMLH1 is between amino 506 and is that there is a reduced interaction with hPMS2 one hMLH1 amino with hMLH1 amino These suggest that the interaction region of hMLH1 with hPMS2 may the carboxyl from amino We also the interaction of hPMS2 with hMLH1 suggested a between of was to the the is to The first of hPMS2 not binding to and In amino of hPMS2 to the as a mechanism to the interaction we found that amino of hPMS2 to and that the interaction was with of amino acids amino of hPMS2 with and the interaction was with of amino acids These suggest that the hMLH1 interaction region of hPMS2 is between amino 675 and 850. missense mutations of hMLH1 have been reported to cosegregate with HNPCC and are found to be within the region of hMLH1 that we to interact with hPMS2 (20Peltomaki P. Vasen H.F. Gastroenterology. 1997; 113: 1146-1158Abstract Full Text Full Text PDF PubMed Scopus (676) Google Scholar). a first in the functional of these we have examined hMLH1 the V506A, L574P, K616Δ, K618A, K618T, R659P, and A681T missense mutations for their interaction with hPMS2. We also the because alteration of the in S. cerevisiae has been reported to effect the interaction of yeast with (21Pang Q. Prolla T.A. Liskay R.M. Mol. Cell. Biol. 1997; 17: 4465-4473Crossref PubMed Scopus (115) Google Scholar). In of we the relative protein and of protein to of for interaction To of mutant we the of the missense mutant proteins in the glutathione by the proteins for interaction interaction is and then an by We found no of the mutant during this not Interestingly, this of be for the protein as well as the that the of the it is that reduction in interaction with hPMS2 is because of either or of the proteins. suggested that several of the hMLH1 missense mutant proteins alterations in binding to hPMS2. To this we developed a of hMLH1 interaction with hPMS2 and is shown in B. of the hMLH1 missense mutant proteins (L574P, K616Δ, K618A, K618T, R659P, and A681T) showed >85% reduction in their interaction with hPMS2. The S44F, and MLH1 missense mutant proteins displayed 25–65% of that with wild type interaction with hPMS2 and Two hMLH1 missense mutant and to have effect on interaction with hPMS2 and of wild type interaction, is to that two hMLH1 missense mutations at (K618A and K618T) effects on their interaction with hPMS2 and of wild the that the interactions Mutations in the human MutL hMLH1, appear to account for approximately of HNPCC (20Peltomaki P. Vasen H.F. Gastroenterology. 1997; 113: 1146-1158Abstract Full Text Full Text PDF PubMed Scopus (676) Google Scholar). The hMLH1 protein was purified as a heterodimer with hPMS2 protein by biochemical of MMR in an cell (16Li G.-M. Modrich P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1950-1954Crossref PubMed Scopus (349) Google Scholar). Although the role of the heterodimer in MMR has not been it is to be an of the MMR These suggested that a of the heterodimer the of MMR and as HNPCC. To of hMLH1 and hPMS2 interaction be a of cancer susceptibility in HNPCC, we first the interaction of hMLH1 and hPMS2. We found that this interaction was to a region in the carboxyl of both proteins. This is to that reported for the homologues and in S. of the data and with the yeast homologues suggest that these interact regions on of these proteins. of the hMLH1 and hPMS2 interaction regions is in Interestingly, sequence of the MLH1 homologues does not conserved within the interaction region amino However, sequence of the PMS1 homologues hPMS1, which is largely within the consensus interaction region amino We found a between HNPCC mutations and a loss of hMLH1 interaction with hPMS2. We hMLH1 missense alterations reported in HNPCC kindreds and in the consensus interaction of hMLH1. of these missense alterations displayed reduced interaction with hPMS2. Four of these alterations (L574P, R659P, and A681T) displayed interaction with hPMS2 whereas two alterations (K618A and K618T) displayed a loss of interaction alteration of protein not be and is to be for several of these missense our with the and alterations for the that protein is not the to heterodimer protein This is by the that the alteration is in the first of three that it is appears that one or three of these form a between hMLH1 and hPMS2. Although the alteration of hMLH1 was found to be of the consensus hPMS2 interaction we this missense alteration in our system because the alteration in S. cerevisiae displayed reduced interaction with PMS1 (21Pang Q. Prolla T.A. Liskay R.M. Mol. Cell. Biol. 1997; 17: 4465-4473Crossref PubMed Scopus (115) Google Scholar). We found a reduction in the ability of hMLH1 to interact with hPMS2 of wild We three The assay for these regions may have to a interaction at the amino of hMLH1, where the is the amino may be in to the consensus hPMS2 interaction or of with a the protein such that the interaction region is largely The and E578G hMLH1 missense to consistent reduced binding to hPMS2. Although it is that these alterations not interaction with it is to that a functional has been confirmed for these alterations using a yeast dominant mutator assay that appear to on interactions (22Shimodaira H. Filosi N. Shibata H. Suzuki T. Radice P. Kanamaru R. Friend S.H. Kolodner R.D. Ishioka C. Nat. Genet. 1998; 19: 384-389Crossref PubMed Scopus (121) Google Scholar). Two hMLH1 missense and not appear to affect interaction with hPMS2. These alterations have been identified in the for HNPCC H.F. 1991; PubMed Scopus Google M. S. Y. Mol. Genet. 1995; PubMed Scopus Google Scholar). We not the that an interaction by these amino is by the of these However, are that the and may be either or that these alterations affect a function of the hMLH1 protein other interaction with hPMS2. Interestingly, the missense alteration has no effect in an yeast dominant mutator assay system (22Shimodaira H. Filosi N. Shibata H. Suzuki T. Radice P. Kanamaru R. Friend S.H. Kolodner R.D. Ishioka C. Nat. Genet. 1998; 19: 384-389Crossref PubMed Scopus (121) Google Scholar). these alterations are from well HNPCC these appear to the that there are other functional alterations of the human MutL homologues that may contribute to cancer susceptibility. The hMLH1 proteins missense alterations appear to a between HNPCC and loss of interaction with hPMS2. is to that there have been no of in hPMS2, which are in the hMLH1 interaction This may suggest that there is another for hMLH1 that has a function with hPMS2 to the with and hPMS1 be a for this We have previously identified the interaction of hMSH2 with and hMSH2 with hMSH6 S. Wilson T. S. Fishel R. Mol. Cell. Biol. 1998; PubMed Scopus Google Scholar). However, we found no effect of missense alterations found in HNPCC kindreds on the interaction with either and This suggested that alteration of a interaction between hMSH2 with or hMSH6 was to play a causative role in HNPCC. we confirm the between interaction between hMLH1 and hPMS2 and susceptibility to HNPCC. We and the of the for and for to the for this and and for