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Mechanisms in Eukaryotic Mismatch Repair

2006; Elsevier BV; Volume: 281; Issue: 41 Linguagem: Inglês

10.1074/jbc.r600022200

1083-351X

Paul Modrich,

Cancer Genomics and Diagnostics

Inactivation of the human mismatch repair system confers a large increase in spontaneous mutability and a strong predisposition to tumor development. Mismatch repair provides several genetic stabilization functions; it corrects DNA biosynthetic errors, ensures the fidelity of genetic recombination, and participates in the earliest steps of checkpoint and apoptotic responses to several classes of DNA damage (see Refs. 1Kunkel T.A. Erie D.A. Annu. Rev. Biochem. 2005; 74: 681-710Crossref PubMed Scopus (1051) Google Scholar, 2Iyer R.R. Pluciennik A. Burdett V. Modrich P.L. Chem. Rev. 2006; 106: 302-323Crossref PubMed Scopus (708) Google Scholar, 3Jiricny J. Nat. Rev. Mol. Cell Biol. 2006; 7: 335-346Crossref PubMed Scopus (995) Google Scholar for recent reviews). Defects in this pathway are the cause of typical and atypical hereditary nonpolyposis colon cancer (4Peltomaki P. Fam. Cancer. 2005; 4: 227-232Crossref PubMed Scopus (204) Google Scholar) but may also play a role in the development of 15–25% of sporadic tumors that occur in a number of tissues (5Peltomaki P. J. Clin. Oncol. 2003; 21: 1174-1179Crossref PubMed Scopus (603) Google Scholar). The system is also of biomedical interest because mismatch repair-deficient tumor cells are resistant to certain cytotoxic chemotherapeutic drugs (2Iyer R.R. Pluciennik A. Burdett V. Modrich P.L. Chem. Rev. 2006; 106: 302-323Crossref PubMed Scopus (708) Google Scholar, 3Jiricny J. Nat. Rev. Mol. Cell Biol. 2006; 7: 335-346Crossref PubMed Scopus (995) Google Scholar), a manifestation of its involvement in the DNA damage response. Of the several mutation avoidance functions of mismatch repair, the reaction responsible for replication error correction has been the most thoroughly studied, and the discussion that follows is restricted to this pathway. Correction of DNA biosynthetic errors requires targeting of mismatch repair to the newly synthesized strand at the replication fork. In contrast to Escherichia coli, where mismatch repair is directed by the transient absence of adenine methylation at d(GATC) sites within newly synthesized DNA, the strand signals that direct replication error correction in eukaryotes have not been identified. However, the function of the hemimethylated d(GATC) strand signal in E. coli mismatch repair is provision of a nick on the unmethylated strand, which serves as the actual signal that directs the reaction (2Iyer R.R. Pluciennik A. Burdett V. Modrich P.L. Chem. Rev. 2006; 106: 302-323Crossref PubMed Scopus (708) Google Scholar). Similarly, a strand-specific nick or gap is sufficient to direct mismatch repair in extracts of mammalian and Drosophila cells as well as Xenopus egg extracts (1Kunkel T.A. Erie D.A. Annu. Rev. Biochem. 2005; 74: 681-710Crossref PubMed Scopus (1051) Google Scholar, 2Iyer R.R. Pluciennik A. Burdett V. Modrich P.L. Chem. Rev. 2006; 106: 302-323Crossref PubMed Scopus (708) Google Scholar, 3Jiricny J. Nat. Rev. Mol. Cell Biol. 2006; 7: 335-346Crossref PubMed Scopus (995) Google Scholar). These findings, coupled with the observation that mismatch repair is more efficient on the lagging strand at the replication fork (6Pavlov Y.I. Mian I.M. Kunkel T.A. Curr. Biol. 2003; 13: 744-748Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar), suggest that DNA termini that occur as natural intermediates during replication (3′ terminus on the leading strand; 3′ and 5′ termini on the lagging strand) may suffice as strand signals to direct the correction of DNA biosynthetic errors in eukaryotic cells. Available information on the mechanism of eukaryotic mismatch repair is largely derived from analysis of the nick-directed repair of circular heteroduplexes in mammalian cell extracts. As shown in Fig. 1, the strand break that directs repair may reside either 3′ or 5′ to the mispair as viewed along the shorter path linking the two sites in the circular substrate, and processing of such molecules in extracts is largely restricted to this region. Examination of intermediates produced in HeLa nuclear extracts when repair DNA synthesis is blocked has demonstrated that mismatch-provoked excision removes that portion of the incised strand spanning the shorter path between the nick and the mismatch (Fig. 1) with excision tracts extending from the strand break to a number of sites within a region ≈90–170 nucleotides beyond the mispair (7Fang W.-h. Modrich P. J. Biol. Chem. 1993; 268: 11838-11844Abstract Full Text PDF PubMed Google Scholar, 8Wang H. Hays J.B. J. Biol. Chem. 2002; 277: 26136-26142Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Radiolabeling of repair DNA synthesis tracts is also consistent with this view (9Thomas D.C. Roberts J.D. Kunkel T.A. J. Biol. Chem. 1991; 266: 3744-3751Abstract Full Text PDF PubMed Google Scholar). The mammalian repair system thus displays a bidirectional capability in the sense that it responds to both 3′- and 5′-heteroduplex orientations, and functionality is retained at nick mismatch separation distances as large as 1000 bp (7Fang W.-h. Modrich P. J. Biol. Chem. 1993; 268: 11838-11844Abstract Full Text PDF PubMed Google Scholar). The nicks that direct the E. coli mismatch repair also serve as sites for initiation of excision (2Iyer R.R. Pluciennik A. Burdett V. Modrich P.L. Chem. Rev. 2006; 106: 302-323Crossref PubMed Scopus (708) Google Scholar), and function of the strand break in the eukaryotic reaction has generally been interpreted in a similar manner (1Kunkel T.A. Erie D.A. Annu. Rev. Biochem. 2005; 74: 681-710Crossref PubMed Scopus (1051) Google Scholar, 2Iyer R.R. Pluciennik A. Burdett V. Modrich P.L. Chem. Rev. 2006; 106: 302-323Crossref PubMed Scopus (708) Google Scholar, 3Jiricny J. Nat. Rev. Mol. Cell Biol. 2006; 7: 335-346Crossref PubMed Scopus (995) Google Scholar). However, a distinct mechanism for mismatch-provoked excision has been proposed based on radiolabeling of repair DNA synthesis tracts in Xenopus egg extracts. In contrast to results obtained with the human system (9Thomas D.C. Roberts J.D. Kunkel T.A. J. Biol. Chem. 1991; 266: 3744-3751Abstract Full Text PDF PubMed Google Scholar), radiolabeling of repair products in Xenopus extracts was significantly higher near the mismatch than the strand break (10Varlet I. Canard B. Brooks P. Cerovic G. Radman M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10156-10161Crossref PubMed Scopus (25) Google Scholar). Based on this analysis, Varlet et al. (10Varlet I. Canard B. Brooks P. Cerovic G. Radman M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10156-10161Crossref PubMed Scopus (25) Google Scholar) suggested that the nick that directs repair does not correspond to the site of excision initiation. Rather, a mismatch-activated strand-specific endonuclease is postulated to introduce a second nick near the mispair, with this nick serving as an entry site for the excision system. As described below, recent experiments suggest that the mammalian repair system supports both of these modes of excision. The activities responsible for initiation of E. coli mismatch repair are MutS and MutL, which function as homo-oligomers (2Iyer R.R. Pluciennik A. Burdett V. Modrich P.L. Chem. Rev. 2006; 106: 302-323Crossref PubMed Scopus (708) Google Scholar). MutS is responsible for mismatch recognition and MutL serves to interface mismatch recognition by MutS to activation of downstream activities. Mammalian cells possess two MutS activities that function as heterodimers and share MSH2 as a common subunit (11Drummond J.T. Li G.-M. Longley M.J. Modrich P. Science. 1995; 268: 1909-1912Crossref PubMed Scopus (541) Google Scholar, 12Palombo F. Gallinari P. Iaccarino I. Lettieri T. Hughes M. D'Arrigo A. Truong O. Hsuan J.J. Jiricny J. Science. 1995; 268: 1912-1914Crossref PubMed Scopus (483) Google Scholar, 13Palombo F. Iaccarino I. Nakajima E. Ikejima M. Shimada T. Jiricny J. Curr. Biol. 1996; 6: 1181-1184Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar, 14Acharya S. Wilson T. Gradia S. Kane M.F. Guerrette S. Marsischky G.T. Kolodner R. Fishel R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13629-13634Crossref PubMed Scopus (476) Google Scholar): MutSα (MSH2·MSH6 heterodimer) and MutSβ (MSH2·MSH3 heterodimer). MutSα, which represents 80–90% of the cellular MSH2, preferentially recognizes base-base mismatches and insertion/deletion (ID) 2The abbreviations used are: ID, insertion/deletion; PCNA, proliferating cell nuclear antigen; RPA, replication protein A; RFC, replication factor C. mispairs in which one strand contains 1 or 2 unpaired nucleotides but is also capable of recognition of larger ID heterologies with reduced affinity (11Drummond J.T. Li G.-M. Longley M.J. Modrich P. Science. 1995; 268: 1909-1912Crossref PubMed Scopus (541) Google Scholar, 12Palombo F. Gallinari P. Iaccarino I. Lettieri T. Hughes M. D'Arrigo A. Truong O. Hsuan J.J. Jiricny J. Science. 1995; 268: 1912-1914Crossref PubMed Scopus (483) Google Scholar, 13Palombo F. Iaccarino I. Nakajima E. Ikejima M. Shimada T. Jiricny J. Curr. Biol. 1996; 6: 1181-1184Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar, 15Genschel J. Littman S.J. Drummond J.T. Modrich P. J. Biol. Chem. 1998; 273: 19895-19901Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar). MutSβ recognizes ID mismatches of 2 to about 10 nucleotides, weakly recognizes single-nucleotide ID mispairs, and is essentially inert on base-base mismatches (12Palombo F. Gallinari P. Iaccarino I. Lettieri T. Hughes M. D'Arrigo A. Truong O. Hsuan J.J. Jiricny J. Science. 1995; 268: 1912-1914Crossref PubMed Scopus (483) Google Scholar, 15Genschel J. Littman S.J. Drummond J.T. Modrich P. J. Biol. Chem. 1998; 273: 19895-19901Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar). MSH2 and MSH6 defects have been implicated in tumor development, but the cancer predisposition conferred by MSH6 inactivation is less severe (4Peltomaki P. Fam. Cancer. 2005; 4: 227-232Crossref PubMed Scopus (204) Google Scholar, 16Edelmann W. Umar A. Yang K. Heyer J. Kucherlapati M. Lia M. Kneitz B. Avdievich E. Fan K. Wong E. Crouse G. Kunkel T. Lipkin M. Kolodner R.D. Kucherlapati R. Cancer Res. 2000; 60: 803-807PubMed Google Scholar). The association of MSH3 defects with tumor development appears to be limited (4Peltomaki P. Fam. Cancer. 2005; 4: 227-232Crossref PubMed Scopus (204) Google Scholar, 5Peltomaki P. J. Clin. Oncol. 2003; 21: 1174-1179Crossref PubMed Scopus (603) Google Scholar, 16Edelmann W. Umar A. Yang K. Heyer J. Kucherlapati M. Lia M. Kneitz B. Avdievich E. Fan K. Wong E. Crouse G. Kunkel T. Lipkin M. Kolodner R.D. Kucherlapati R. Cancer Res. 2000; 60: 803-807PubMed Google Scholar). Three eukaryotic MutL activities have been identified and, like eukaryotic MutS activities, function as heterodimeric complexes with MLH1 serving as a common subunit. MutLα, a heterodimer of MLH1 and PMS2, is the primary MutL activity in human mitotic cells and supports repair initiated by either MutSα or MutSβ (17Li G.-M. Modrich P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1950-1954Crossref PubMed Scopus (357) Google Scholar). MutLα accounts for ≈90% of the MLH1 in human cells (18Raschle M. Marra G. Nystrom-Lahti M. Schar P. Jiricny J. J. Biol. Chem. 1999; 274: 32368-32375Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 19Cannavo E. Marra G. Sabates-Bellver J. Menigatti M. Lipkin S.M. Fischer F. Cejka P. Jiricny J. Cancer Res. 2005; 65: 10759-10766Crossref PubMed Scopus (97) Google Scholar), but two low abundance complexes involving MLH1 have also been identified. A human MLH1·PMS1 heterodimer (MutLβ) has been isolated, but involvement in mismatch repair has not been demonstrated (18Raschle M. Marra G. Nystrom-Lahti M. Schar P. Jiricny J. J. Biol. Chem. 1999; 274: 32368-32375Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). However, the MutLγ MLH1·MLH3 complex has been reported to support modest levels of base-base and single-nucleotide ID mismatch repair in vitro, events that are presumably initiated by MutSα (19Cannavo E. Marra G. Sabates-Bellver J. Menigatti M. Lipkin S.M. Fischer F. Cejka P. Jiricny J. Cancer Res. 2005; 65: 10759-10766Crossref PubMed Scopus (97) Google Scholar). Genetic inactivation of MLH1 or PMS2 confers cancer predisposition, but mutations in PMS1 do not (4Peltomaki P. Fam. Cancer. 2005; 4: 227-232Crossref PubMed Scopus (204) Google Scholar, 16Edelmann W. Umar A. Yang K. Heyer J. Kucherlapati M. Lia M. Kneitz B. Avdievich E. Fan K. Wong E. Crouse G. Kunkel T. Lipkin M. Kolodner R.D. Kucherlapati R. Cancer Res. 2000; 60: 803-807PubMed Google Scholar). Involvement of MLH3 defects in tumor development is uncertain (4Peltomaki P. Fam. Cancer. 2005; 4: 227-232Crossref PubMed Scopus (204) Google Scholar, 5Peltomaki P. J. Clin. Oncol. 2003; 21: 1174-1179Crossref PubMed Scopus (603) Google Scholar). Yeast genetic studies and analysis of the mammalian extract reaction have implicated six additional activities in eukaryotic mismatch repair. The key finding that culminated in the reconstitution studies described below was the demonstration that exonuclease 1 (Exo1) participates in the reaction. Genetic evidence for Exo1 involvement in yeast mismatch repair (20Szankasi P. Smith G.R. Science. 1995; 267: 1166-1169Crossref PubMed Scopus (184) Google Scholar, 21Tishkoff D.X. Boerger A.L. Bertrand P. Filosi N. Gaida G.M. Kane M.F. Kolodner R.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7487-7492Crossref PubMed Scopus (335) Google Scholar) led to the subsequent demonstration that Exo1 is required for repair of base-base and single-nucleotide ID mismatches in mammalian cell extracts (22Genschel J. Bazemore L.R. Modrich P. J. Biol. Chem. 2002; 277: 13302-13311Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 23Wei K. Clark A.B. Wong E. Kane M.F. Mazur D.J. Parris T. Kolas N.K. Russell R. Hou Jr., H. Kneitz B. Yang G. Kunkel T.A. Kolodner R.D. Cohen P.E. Edelmann W. Genes Dev. 2003; 17: 603-614Crossref PubMed Scopus (273) Google Scholar). Because Exo1 hydrolyzes duplex DNA with 5′ to 3′ polarity (24Wilson III, D.M. Carney J.P. Coleman M.A. Adamson A.W. Christensen M. Lamerdin J.E. Nucleic Acids Res. 1998; 26: 3762-3768Crossref PubMed Scopus (97) Google Scholar, 25Lee B.I. Wilson III, D.M. J. Biol. Chem. 1999; 274: 37763-37769Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar), the surprising feature of this requirement is that the enzyme is necessary for excision and repair directed by strand break located either 5′ or 3′ to the mismatch. This paradoxical requirement led to the suggestion that Exo1 might play a positive regulatory role in 3′ excision or that the reaction may be mediated by a cryptic Exo1 3′ to 5′ hydrolytic activity (22Genschel J. Bazemore L.R. Modrich P. J. Biol. Chem. 2002; 277: 13302-13311Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). As discussed below, this issue has been recently resolved, and it is not necessary to invoke either of these possibilities. Exo1–/– mice display modest cancer predisposition, and Exo1 deficiency is associated with a 30-fold elevation of hypoxanthine-guanine phosphoribosyltransferase mutability, substantially less than the 150-fold increase observed with Msh2–/– cells (23Wei K. Clark A.B. Wong E. Kane M.F. Mazur D.J. Parris T. Kolas N.K. Russell R. Hou Jr., H. Kneitz B. Yang G. Kunkel T.A. Kolodner R.D. Cohen P.E. Edelmann W. Genes Dev. 2003; 17: 603-614Crossref PubMed Scopus (273) Google Scholar). Although extracts of Exo1–/– mouse cells are virtually devoid of repair activity on base-base mismatches, they retain significant activity on one- or two-nucleotide ID mispairs (23Wei K. Clark A.B. Wong E. Kane M.F. Mazur D.J. Parris T. Kolas N.K. Russell R. Hou Jr., H. Kneitz B. Yang G. Kunkel T.A. Kolodner R.D. Cohen P.E. Edelmann W. Genes Dev. 2003; 17: 603-614Crossref PubMed Scopus (273) Google Scholar). These findings imply the existence of one or more alternate excision activities, and several possibilities have been suggested. Involvement of the 3′ to 5′ editing exonuclease functions of DNA polymerases δ and ϵ in mismatch repair has been proposed on genetic and biochemical grounds (26Tran H.T. Gordenin D.A. Resnick M.A. Mol. Cell. Biol. 1999; 19: 2000-2007Crossref PubMed Scopus (182) Google Scholar, 27Wang H. Hays J.B. J. Biol. Chem. 2002; 277: 26143-26148Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 28Jin Y.H. Garg P. Stith C.M. Al-Refai H. Sterling J.F. Murray L.J. Kunkel T.A. Resnick M.A. Burgers P.M. Gordenin D.A. Mol. Cell. Biol. 2005; 25: 461-471Crossref PubMed Scopus (63) Google Scholar), but this idea has been questioned (29Datta A. Schmeits J.L. Amin N.S. Lau P.J. Myung K. Kolodner R.D. Mol. Cell. 2000; 6: 593-603Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 30Constantin N. Dzantiev L. Kadyrov F.A. Modrich P. J. Biol. Chem. 2005; 280: 39752-39761Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). Using a small interfering RNA knockdown approach, Vo et al. (31Vo A.T. Zhu F. Wu X. Yuan F. Gao Y. Gu L. Li G.M. Lee T.H. Her C. EMBO Rep. 2005; 6: 438-444Crossref PubMed Scopus (49) Google Scholar) have suggested that the Mre11 3′ to 5′-exonuclease participates in 3′-directed mismatch repair. Mre11 depletion was shown to reduce the efficiency of 3′-directed repair by ≈40%, and repair was restored to normal levels by the addition of partially purified Mre11. However, the involvement of other activities in repair restoration was not excluded because the Mre11 fraction tested was relatively crude. This is of concern because down-regulation of Mre11 also leads to relatively rapid chromosome breakage and can interfere with cell proliferation (32Yamaguchi-Iwai Y. Sonoda E. Sasaki M.S. Morrison C. Haraguchi T. Hiraoka Y. Yamashita Y.M. Yagi T. Takata M. Price C. Kakazu N. Takeda S. EMBO J. 1999; 18: 6619-6629Crossref PubMed Scopus (244) Google Scholar). Experiments in human cell extracts and partially purified fractions have also indicated involvement of several DNA binding proteins in eukaryotic mismatch repair. The extract reaction is abolished by antibody against the single-stranded DNA binding protein RPA (33Lin Y.L. Shivji M.K. Chen C. Kolodner R. Wood R.D. Dutta A. J. Biol. Chem. 1998; 273: 1453-1461Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), which stimulates excision, stabilizes the ensuing gap against endonuclease attack, and promotes repair DNA synthesis (34Ramilo C. Gu L. Guo S. Zhang X. Patrick S.M. Turchi J.J. Li G.M. Mol. Cell. Biol. 2002; 22: 2037-2046Crossref PubMed Scopus (67) Google Scholar). The non-histone chromatin protein HMGB1, which interacts with MutSα, may also play an important role in the initiation of mismatch-provoked excision in nuclear extracts (35Yuan F. Gu L. Guo S. Wang C. Li G.M. J. Biol. Chem. 2004; 279: 20935-20940Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). The PCNA replication clamp and DNA polymerase δ have also been implicated in mismatch repair in human cell extracts (36Umar A. Buermeyer A.B. Simon J.A. Thomas D.C. Clark A.B. Liskay R.M. Kunkel T.A. Cell. 1996; 87: 65-73Abstract Full Text Full Text PDF PubMed Scopus (503) Google Scholar, 37Longley M.J. Pierce A.J. Modrich P. J. Biol. Chem. 1997; 272: 10917-10921Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 38Gu L. Hong Y. McCulloch S. Watanabe H. Li G.M. Nucleic Acids Res. 1998; 26: 1173-1178Crossref PubMed Scopus (185) Google Scholar, 39Genschel J. Modrich P. Mol. Cell. 2003; 12: 1077-1086Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). PCNA, which confers processivity on polymerase δ (40Johnson A. O'Donnell M. Annu. Rev. Biochem. 2005; 74: 283-315Crossref PubMed Scopus (446) Google Scholar), plays multiple roles in mismatch repair. As might be expected, PCNA is necessary for repair DNA synthesis (38Gu L. Hong Y. McCulloch S. Watanabe H. Li G.M. Nucleic Acids Res. 1998; 26: 1173-1178Crossref PubMed Scopus (185) Google Scholar, 39Genschel J. Modrich P. Mol. Cell. 2003; 12: 1077-1086Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar), but it is also required for mismatch-provoked excision (36Umar A. Buermeyer A.B. Simon J.A. Thomas D.C. Clark A.B. Liskay R.M. Kunkel T.A. Cell. 1996; 87: 65-73Abstract Full Text Full Text PDF PubMed Scopus (503) Google Scholar). The most compelling evidence for PCNA involvement in the excision step of mismatch repair has been provided by p21 inhibition studies. By forming a stable complex with DNA-bound PCNA, p21 interferes with downstream PCNA-dependent events (41Waga S. Stillman B. Mol. Cell. Biol. 1998; 18: 4177-4187Crossref PubMed Scopus (69) Google Scholar). Although p21 abolishes 3′-directed mismatch-provoked excision in HeLa cell extracts, only 40–50% of 5′-directed excision events are subject to p21 inhibition, implying occurrence of at least two hydrolytic modes on 5′-heteroduplexes (36Umar A. Buermeyer A.B. Simon J.A. Thomas D.C. Clark A.B. Liskay R.M. Kunkel T.A. Cell. 1996; 87: 65-73Abstract Full Text Full Text PDF PubMed Scopus (503) Google Scholar, 39Genschel J. Modrich P. Mol. Cell. 2003; 12: 1077-1086Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 42Guo S. Presnell S.R. Yuan F. Zhang Y. Gu L. Li G.M. J. Biol. Chem. 2004; 279: 16912-16917Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). These findings have led to several reconstituted systems that rely on near homogeneous proteins and support mismatch-provoked excision and repair. The simplest excision system depends on MutSα, MutLα, Exo1, RPA, and ATP (39Genschel J. Modrich P. Mol. Cell. 2003; 12: 1077-1086Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar), and similar results have been obtained in a system that also contains HMGB1 (43Zhang Y. Yuan F. Presnell S.R. Tian K. Gao Y. Tomkinson A.E. Gu L. Li G.M. Cell. 2005; 122: 693-705Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar). As illustrated in Fig. 2, 5′-directed excision in this system is mismatch-provoked and terminates upon mismatch removal. Analysis of this reaction has revealed several features of the hydrolytic mechanism. MutSα activates Exo1 hydrolysis on a 5′-heteroduplex in a mismatch- and ATP-dependent manner. In the absence of other proteins, 5′ to 3′ hydrolysis by Exo1 occurs by a distributive mechanism, but MutSα renders the enzyme highly processive, resulting in removal of ≈2,000 nucleotides prior to dissociation (39Genschel J. Modrich P. Mol. Cell. 2003; 12: 1077-1086Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar), an effect attributed to formation of a MutSα ·Exo1 complex. Hydrolysis by the MutSα·Exo1 complex is controlled in part by RPA, which reduces processivity of the MutSα·Exo1 complex to ≈250 nucleotides, and by binding to gaps, RPA controls access of Exo1 to 5′ termini in excision intermediates/products (39Genschel J. Modrich P. Mol. Cell. 2003; 12: 1077-1086Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). Although an RPA-filled gap is a very poor substrate for Exo1, MutSα promotes Exo1 loading at such sites provided that the gapped molecule contains a mismatched base pair. The ramifications of these RPA effects are 2-fold. Excision on 5′-heteroduplexes proceeds via a set of pseudo-discrete hydrolytic intermediates, which differ in size by about 250 nucleotides, an effect attributed to multiple reloading of MutSα and Exo1 (39Genschel J. Modrich P. Mol. Cell. 2003; 12: 1077-1086Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 43Zhang Y. Yuan F. Presnell S.R. Tian K. Gao Y. Tomkinson A.E. Gu L. Li G.M. Cell. 2005; 122: 693-705Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar). Second, hydrolysis is dramatically attenuated upon mismatch removal because MutSα can no longer promote Exo1 loading at the RPA-filled gap in the excision product. RPA thus has both negative and positive regulatory effects on this reaction; by suppressing processive behavior of the MutSα·Exo1 complex and by restricting hydrolytic activity on excision products, it promotes turnover of the system after mismatch removal, allowing other heteroduplex molecules to participate in the reaction. MutLα is not required for mismatch- and MutSα-dependent activation of Exo1, but it does play a significant role in excision. By acting in concert with MutSα to suppress Exo1 hydrolysis on DNA that lacks a mispair, MutLα enhances the mismatch dependence of the reaction (39Genschel J. Modrich P. Mol. Cell. 2003; 12: 1077-1086Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 44Nielsen F.C. Jager A.C. Lutzen A. Bundgaard J.R. Rasmussen L.J. Oncogene. 2004; 23: 1457-1468Crossref PubMed Scopus (65) Google Scholar). MutLα also participates in excision termination in this system, but two different mechanisms have been proposed to account for its function in this regard. Genschel et al. (39Genschel J. Modrich P. Mol. Cell. 2003; 12: 1077-1086Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar) have attributed MutLα involvement in termination to its role in suppressing Exo1 activity on mismatch-free DNA. In this mechanism MutLα simply stabilizes excision products against nonspecific hydrolysis by Exo1. By contrast, Zhang et al. (43Zhang Y. Yuan F. Presnell S.R. Tian K. Gao Y. Tomkinson A.E. Gu L. Li G.M. Cell. 2005; 122: 693-705Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar) have concluded that MutLα, acting in concert with RPA, plays an active role in excision termination upon mismatch removal. This issue has not been resolved. MutSα, MutLα, Exo1, and RPA also support mismatch-provoked excision on a 3′-heteroduplex. As in the case of a 5′-substrate, hydrolysis on a 3′-heteroduplex proceeds 5′ to 3′ from the strand break (Fig. 2), which is the wrong polarity for mismatch removal (22Genschel J. Bazemore L.R. Modrich P. J. Biol. Chem. 2002; 277: 13302-13311Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 45Dzantiev L. Constantin N. Genschel J. Iyer R.R. Burgers P.M. Modrich P. Mol. Cell. 2004; 15: 31-41Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). The 5′ to 3′ directionality of this system has been referred to as a default polarity (2Iyer R.R. Pluciennik A. Burdett V. Modrich P.L. Chem. Rev. 2006; 106: 302-323Crossref PubMed Scopus (708) Google Scholar). Although PCNA has no significant effect on the restricted directionality of this system, supplementation with both PCNA and RFC (RFC loads PCNA onto the helix (40Johnson A. O'Donnell M. Annu. Rev. Biochem. 2005; 74: 283-315Crossref PubMed Scopus (446) Google Scholar)) yields a system that supports mismatch removal from both 5′ and 3′-heteroduplexes (45Dzantiev L. Constantin N. Genschel J. Iyer R.R. Burgers P.M. Modrich P. Mol. Cell. 2004; 15: 31-41Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). Excision products obtained from a 5′-heteroduplex in this six-component system are similar to those produced by MutSα, MutLα, Exo1, and RPA. However, when the nick is located 3′ to the mismatch, Exo1 5′ to 3′ hydrolysis initiating at the nick is largely repressed by RFC, and excision occurs with apparent 3′ to 5′ polarity resulting in mismatch removal. Although excision in this six-component system displays similarities to the bidirectional reaction that has been studied in nuclear extracts, the distribution of excision products in the purified system is more disperse than that observed in extracts. This purified system therefore lacks one or more activities that play significant roles in mismatch repair (45Dzantiev L. Constantin N. Genschel J. Iyer R.R. Burgers P.M. Modrich P. Mol. Cell. 2004; 15: 31-41Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). Because an Exo1 active site mutant failed to support both 5′- and 3′-directed excision in this system, mismatch removal in both cases was attributed to this exonuclease (45Dzantiev L. Constantin N. Genschel J. Iyer R.R. Burgers P.M. Modrich P. Mol. Cell. 2004; 15: 31-41Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). It was suggested that a cryptic Exo1 3′ to 5′ hydrolytic function is responsible for 3′-directed excision. However, the necessity for a 3′ to 5′-exonuclease in this purified system was rendered moot by the demonstration that MutSα, RFC, and PCNA activate a latent MutLα endonuclease in an ATP- and mismatch-dependent manner (46Kadyrov F.A. Dzantiev L. Constantin N. Modrich P. Cell. 2006; 126: 297-308Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar). As shown in Fig. 3 incision by activated MutLα endonuclease occurs on both 3′- and 5′-heteroduplexes and is strongly biased to the nicked heteroduplex strand. For heteroduplexes with a nick mismatch separation distance of ≈100 bp, incision tends to occur on the distal side of the mismatch relative to the strand break but at larger separation distances readily occurs between the two DNA sites. 3F. Kadyrov and P. Modrich, unpublished data. In the case of a 3′-heteroduplex, incision distal to the mismatch provides an initiation site for mismatch removal by the 5′ to 3′ action of MutSα-activated Exo1 (Fig. 3). Inasmuch as PCNA-dependent and independent modes of 5′-directed excision have been invoked in nuclear extracts (39Genschel J. Modrich P. Mol. Cell. 2003; 12: 1077-1086Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 42Guo S. Presnell S.R. Yuan F. Zhang Y. Gu L. Li G.M. J. Biol. Chem. 2004; 279: 16912-16917Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), it is noteworthy that this PCNA-dependent endonucleolytic system also incises 5′-heteroduplexes (46Kadyrov F.A. Dzantiev L. Constantin N. Modrich P. Cell. 2006; 126: 297-308Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar). The endonucleolytic mode of action of this system is reminiscent of the mechanism for mismatch repair in Xenopus egg extracts proposed by Varlet et al. (10Varlet I. Canard B. Brooks P. Cerovic G. Radman M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10156-10161Crossref PubMed Scopus (25) Google Scholar). As discussed above, this model posits that the nick that directs repair serves as a strand signal but not as a site for excision initiation, which actually occurs at a strand break produced by a mismatch-activated endonuclease. This mode of excision is fundamentally different from that used by the E. coli methyl-directed pathway, where hydrolysis initiates at a 3′ or 5′ strand break that directs repair (2Iyer R.R. Pluciennik A. Burdett V. Modrich P.L. Chem. Rev. 2006; 106: 302-323Crossref PubMed Scopus (708) Google Scholar). The probable active site of the MutLα endonuclease has been localized to a divalent metal binding site within the PMS2 subunit that is defined by a DQHA(X)2E(X)4E motif (46Kadyrov F.A. Dzantiev L. Constantin N. Modrich P. Cell. 2006; 126: 297-308Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar). Amino acid substitution mutations within this motif abolish MutLα endonuclease activity as well as the ability of the protein to support mismatch repair in nuclear extracts. This motif is conserved in homologs of eukaryotic PMS2 and in archaeal and eubacterial MutL proteins but is conspicuously absent in MutL proteins from bacteria like E. coli that rely on d(GATC) methylation to direct mismatch repair. The presence or absence of this MutL motif may therefore define two distinct classes of mismatch repair systems. Several defined systems that support mismatch correction have also been described, but these differ in several respects. Zhang et al. (43Zhang Y. Yuan F. Presnell S.R. Tian K. Gao Y. Tomkinson A.E. Gu L. Li G.M. Cell. 2005; 122: 693-705Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar) have shown that MutSα, MutLα, Exo1, RPA, HMGB1, and DNA polymerase δ are sufficient to support repair of 5′-heteroduplexes containing a G-T mismatch or a 3-nucleotide ID mispair and that covalently closed repair products are obtained upon supplementation of these proteins with DNA ligase I. As observed for 5′-directed excision (39Genschel J. Modrich P. Mol. Cell. 2003; 12: 1077-1086Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar), MutLα is not required for repair in this system (43Zhang Y. Yuan F. Presnell S.R. Tian K. Gao Y. Tomkinson A.E. Gu L. Li G.M. Cell. 2005; 122: 693-705Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar). The functions of RPA and HMGB1 in this reconstituted reaction appear to be largely redundant because either protein is sufficient to support reconstituted repair, and excision in the presence of RPA is only modestly enhanced by the addition of HMGB1. Interestingly, substitution of MutSβ for MutSα yields a system that supports 5′-directed excision and repair of a 3-nucleotide ID mismatch, implying that like MutSα, MutSβ can activate Exo1. One surprising feature of this reconstituted system is that repair is independent of RFC and PCNA. This is unexpected because the DNA synthesis step of 5′-heteroduplex repair in nuclear extracts is PCNA-dependent (38Gu L. Hong Y. McCulloch S. Watanabe H. Li G.M. Nucleic Acids Res. 1998; 26: 1173-1178Crossref PubMed Scopus (185) Google Scholar, 39Genschel J. Modrich P. Mol. Cell. 2003; 12: 1077-1086Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). Furthermore, in contrast to its activity on a 5′-heteroduplex, this purified system does not support 3′-directed excision or repair when supplemented with RFC and PCNA (43Zhang Y. Yuan F. Presnell S.R. Tian K. Gao Y. Tomkinson A.E. Gu L. Li G.M. Cell. 2005; 122: 693-705Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar). A reconstituted repair system with somewhat different properties has been described by Constantin et al. (30Constantin N. Dzantiev L. Kadyrov F.A. Modrich P. J. Biol. Chem. 2005; 280: 39752-39761Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). In contrast to the 5′-directed repair system described above (43Zhang Y. Yuan F. Presnell S.R. Tian K. Gao Y. Tomkinson A.E. Gu L. Li G.M. Cell. 2005; 122: 693-705Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar), this system supports bidirectional mismatch repair dependent on MutSα, MutLα, Exo1, RPA, DNA polymerase δ, RFC, and PCNA (30Constantin N. Dzantiev L. Kadyrov F.A. Modrich P. J. Biol. Chem. 2005; 280: 39752-39761Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). MutLα is dispensable for 5′ repair in this system but required for 3′-heteroduplex repair. The RFC and PCNA requirement for 5′-directed correction is because of their involvement in the repair DNA synthesis step, whereas both proteins are also required for excision on a 3′-heteroduplex. The different results obtained by Zhang et al. as compared with those of Constantin et al. (30Constantin N. Dzantiev L. Kadyrov F.A. Modrich P. J. Biol. Chem. 2005; 280: 39752-39761Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar) and Dzantiev et al. (45Dzantiev L. Constantin N. Genschel J. Iyer R.R. Burgers P.M. Modrich P. Mol. Cell. 2004; 15: 31-41Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar) have been attributed to activity differences between the RFC preparations used (43Zhang Y. Yuan F. Presnell S.R. Tian K. Gao Y. Tomkinson A.E. Gu L. Li G.M. Cell. 2005; 122: 693-705Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar, 46Kadyrov F.A. Dzantiev L. Constantin N. Modrich P. Cell. 2006; 126: 297-308Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar). Whereas Zhang et al. (43Zhang Y. Yuan F. Presnell S.R. Tian K. Gao Y. Tomkinson A.E. Gu L. Li G.M. Cell. 2005; 122: 693-705Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar) employed recombinant human RFC, Constantin et al. (30Constantin N. Dzantiev L. Kadyrov F.A. Modrich P. J. Biol. Chem. 2005; 280: 39752-39761Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar) used native human RFC. Dzantiev et al. (45Dzantiev L. Constantin N. Genschel J. Iyer R.R. Burgers P.M. Modrich P. Mol. Cell. 2004; 15: 31-41Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar) obtained similar results using either native human or recombinant yeast RFC. The recent establishment of defined systems that support mismatch-provoked excision and repair reactions should facilitate study of this elaborate reaction. However, because the reconstituted reactions described to date are minimal systems, it is premature to assume that they can account for mismatch repair as it occurs in the eukaryotic cell. There is excellent evidence for participation of hydrolytic activities in addition to Exo1 (23Wei K. Clark A.B. Wong E. Kane M.F. Mazur D.J. Parris T. Kolas N.K. Russell R. Hou Jr., H. Kneitz B. Yang G. Kunkel T.A. Kolodner R.D. Cohen P.E. Edelmann W. Genes Dev. 2003; 17: 603-614Crossref PubMed Scopus (273) Google Scholar), but these have not been convincingly identified. Activities that regulate the action of MutLα endonuclease and control 3′-directed excision have also been invoked (45Dzantiev L. Constantin N. Genschel J. Iyer R.R. Burgers P.M. Modrich P. Mol. Cell. 2004; 15: 31-41Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 46Kadyrov F.A. Dzantiev L. Constantin N. Modrich P. Cell. 2006; 126: 297-308Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar), but these have not been characterized. The defining feature of the replication error correction reaction is its strand-specific character, an effect that depends on the interaction of a mismatch and strand break that can be separated by hundreds of base pairs. Several models, which attempt to explain the molecular nature of this interaction, have been thoroughly debated in the literature (1Kunkel T.A. Erie D.A. Annu. Rev. Biochem. 2005; 74: 681-710Crossref PubMed Scopus (1051) Google Scholar, 2Iyer R.R. Pluciennik A. Burdett V. Modrich P.L. Chem. Rev. 2006; 106: 302-323Crossref PubMed Scopus (708) Google Scholar, 3Jiricny J. Nat. Rev. Mol. Cell Biol. 2006; 7: 335-346Crossref PubMed Scopus (995) Google Scholar), but the mechanism responsible for communication between the two DNA sites has not been established. However, the recent finding that mismatch-dependent incision by activated MutLα endonuclease is strongly biased to the nicked heteroduplex strand (46Kadyrov F.A. Dzantiev L. Constantin N. Modrich P. Cell. 2006; 126: 297-308Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar) suggests that interaction of the mismatch and strand break may involve keeping track of a DNA strand. The sequence of events during the course of nick-directed mismatch repair is presumably dictated by the temporal course of protein-protein interactions that occur on the heteroduplex. A number of protein-protein interactions have been documented in this system, including MutSα-MutLα, MutSα-PCNA, MutSβ-PCNA, MutLα-PCNA, MutSα-Exo1, MutLα-Exo1, Exo1-PCNA, and PCNA-polymerase δ (1Kunkel T.A. Erie D.A. Annu. Rev. Biochem. 2005; 74: 681-710Crossref PubMed Scopus (1051) Google Scholar, 2Iyer R.R. Pluciennik A. Burdett V. Modrich P.L. Chem. Rev. 2006; 106: 302-323Crossref PubMed Scopus (708) Google Scholar, 3Jiricny J. Nat. Rev. Mol. Cell Biol. 2006; 7: 335-346Crossref PubMed Scopus (995) Google Scholar, 40Johnson A. O'Donnell M. Annu. Rev. Biochem. 2005; 74: 283-315Crossref PubMed Scopus (446) Google Scholar). With the exception of interactions between MutSα and Exo1 and between PCNA and polymerase δ, the significance of these protein-protein interactions in nick-directed mismatch repair has not been established. I thank Vickers Burdett, Leo Dzantiev, Jochen Genschel, Ravi Iyer, and Anna Pluciennik for valuable comments and suggestions.