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• W2141323033 abstract "Half of hereditary nonpolyposis colon cancer kindreds harbor mutations that inactivate MutLα (MLH1•PMS2 heterodimer). MutLα is required for mismatch repair, but its function in this process is unclear. We show that human MutLα is a latent endonuclease that is activated in a mismatch-, MutSα-, RFC-, PCNA-, and ATP-dependent manner. Incision of a nicked mismatch-containing DNA heteroduplex by this four-protein system is strongly biased to the nicked strand. A mismatch-containing DNA segment spanned by two strand breaks is removed by the 5′-to-3′ activity of MutSα-activated exonuclease I. The probable endonuclease active site has been localized to a PMS2 DQHA(X)2E(X)4E motif. This motif is conserved in eukaryotic PMS2 homologs and in MutL proteins from a number of bacterial species but is lacking in MutL proteins from bacteria that rely on d(GATC) methylation for strand discrimination in mismatch repair. Therefore, the mode of excision initiation may differ in these organisms. Half of hereditary nonpolyposis colon cancer kindreds harbor mutations that inactivate MutLα (MLH1•PMS2 heterodimer). MutLα is required for mismatch repair, but its function in this process is unclear. We show that human MutLα is a latent endonuclease that is activated in a mismatch-, MutSα-, RFC-, PCNA-, and ATP-dependent manner. Incision of a nicked mismatch-containing DNA heteroduplex by this four-protein system is strongly biased to the nicked strand. A mismatch-containing DNA segment spanned by two strand breaks is removed by the 5′-to-3′ activity of MutSα-activated exonuclease I. The probable endonuclease active site has been localized to a PMS2 DQHA(X)2E(X)4E motif. This motif is conserved in eukaryotic PMS2 homologs and in MutL proteins from a number of bacterial species but is lacking in MutL proteins from bacteria that rely on d(GATC) methylation for strand discrimination in mismatch repair. Therefore, the mode of excision initiation may differ in these organisms. Inactivation of the human mismatch repair system increases the mutation rate several hundred-fold and is the primary cause of hereditary nonpolyposis colon cancer (HNPCC). Genetic stabilization afforded by this system has been attributed to its function in the correction of DNA biosynthetic errors, its role in ensuring the fidelity of genetic recombination, and its participation in the checkpoint and apoptotic responses to several classes of DNA damage (reviewed in Surtees et al., 2004Surtees J.A. Argueso J.L. Alani E. Mismatch repair proteins: key regulators of genetic recombination.Cytogenet. Genome Res. 2004; 107: 146-159Crossref PubMed Scopus (123) Google Scholar, Stojic et al., 2004Stojic L. Brun R. Jiricny J. Mismatch repair and DNA damage signalling.DNA Repair (Amst.). 2004; 3: 1091-1101Crossref PubMed Scopus (312) Google Scholar, Kunkel and Erie, 2005Kunkel T.A. Erie D.A. DNA mismatch repair.Annu. Rev. Biochem. 2005; 74: 681-710Crossref PubMed Scopus (941) Google Scholar, Iyer et al., 2006Iyer R.R. Pluciennik A. Burdett V. Modrich P. DNA mismatch repair: functions and mechanisms.Chem. Rev. 2006; 106: 302-323Crossref PubMed Scopus (630) Google Scholar). The reaction responsible for correction of replication errors is the best understood in molecular terms. The mechanism of mismatch repair has been most extensively studied in E. coli, and the E. coli reaction has been reconstituted in a purified system (Kunkel and Erie, 2005Kunkel T.A. Erie D.A. DNA mismatch repair.Annu. Rev. Biochem. 2005; 74: 681-710Crossref PubMed Scopus (941) Google Scholar, Iyer et al., 2006Iyer R.R. Pluciennik A. Burdett V. Modrich P. DNA mismatch repair: functions and mechanisms.Chem. Rev. 2006; 106: 302-323Crossref PubMed Scopus (630) Google Scholar). Repair is directed to the daughter strand at the replication fork by virtue of the transient absence of d(GATC) methylation in newly synthesized DNA. Repair is initiated by binding of MutS to a mismatch, and MutL is recruited to the heteroduplex DNA in a MutS- and ATP-dependent manner. Assembly of the MutL•MutS•heteroduplex ternary complex is sufficient to activate the d(GATC) endonuclease activity of MutH, which incises the unmethylated strand. The ensuing strand break is the actual signal that directs repair to the new DNA strand and serves as an entry point for the excision system, comprised of DNA helicase II and an appropriate single-strand exonuclease. A 3′-to-5′ exonuclease is required when the MutH nick is introduced 3′ to the mismatch, while a 5′-to-3′ hydrolytic activity is necessary when the MutH strand break is 5′ to the mispair. DNA polymerase III holoenzyme repairs the ensuing gap, and ligase restores covalent integrity to the helix. Mammalian cell extracts support a similar reaction in which repair is directed by a strand discontinuity (a nick or gap) that may also reside either 3′ or 5′ to the mismatch (Kunkel and Erie, 2005Kunkel T.A. Erie D.A. DNA mismatch repair.Annu. Rev. Biochem. 2005; 74: 681-710Crossref PubMed Scopus (941) Google Scholar, Iyer et al., 2006Iyer R.R. Pluciennik A. Burdett V. Modrich P. DNA mismatch repair: functions and mechanisms.Chem. Rev. 2006; 106: 302-323Crossref PubMed Scopus (630) Google Scholar). The key proteins for initiation of eukaryotic mismatch repair are homologs of bacterial MutS and MutL. Eukaryotes harbor two mismatch recognition activities, MutSα (MSH2•MSH6 heterodimer) and MutSβ (MSH2•MSH3 heterodimer), although MutSα is probably responsible for most mismatch repair events in mammalian cells. Eukaryotic MutL homologs also function as heterodimers, with MLH1 serving as a common subunit. The best characterized of these has been MutLα, isolated from both human (MLH1•PMS2 heterodimer) and yeast (MLH1•PMS1 complex) (Prolla et al., 1994Prolla T.A. Pang Q. Alani E. Kolodner R.D. Liskay R.M. MLH1, PMS1, and MSH2 interactions during the initiation of DNA mismatch repair in yeast.Science. 1994; 265: 1091-1093Crossref PubMed Scopus (273) Google Scholar, Li and Modrich, 1995Li G.-M. Modrich P. Restoration of mismatch repair to nuclear extracts of H6 colorectal tumor cells by a heterodimer of human MutL homologs.Proc. Natl. Acad. Sci. USA. 1995; 92: 1950-1954Crossref PubMed Scopus (344) Google Scholar, Habraken et al., 1998Habraken Y. Sung P. Prakash L. Prakash S. ATP-dependent assembly of a ternary complex consisting of a DNA mismatch and the yeast MSH2–MSH6 and MLH1–PMS1 protein complexes.J. Biol. Chem. 1998; 273: 9837-9841Crossref PubMed Scopus (106) Google Scholar, Bowers et al., 2001Bowers J. Tran P.T. Joshi A. Liskay R.M. Alani E. MSH-MLH complexes formed at a DNA mismatch are disrupted by the PCNA sliding clamp.J. Mol. Biol. 2001; 306: 957-968Crossref PubMed Scopus (63) Google Scholar, Raschle et al., 2002Raschle M. Dufner P. Marra G. Jiricny J. Mutations within the hMLH1 and hPMS2 subunits of the human MutLalpha mismatch repair factor affect its ATPase activity, but not its ability to interact with hMutSalpha.J. Biol. Chem. 2002; 277: 21810-21820Crossref PubMed Scopus (92) Google Scholar, Tomer et al., 2002Tomer G. Buermeyer A.B. Nguyen M.M. Liskay R.M. Contribution of human mlh1 and pms2 ATPase activities to DNA mismatch repair.J. Biol. Chem. 2002; 277: 21801-21809Crossref PubMed Scopus (45) Google Scholar). Study of the mammalian extract reaction has implicated six activities in addition to MutSα, MutSβ, and MutLα in nick-directed mismatch repair: the PCNA replication clamp, the RFC clamp loader, the single-strand DNA binding protein RPA, exonuclease I (ExoI), DNA polymerase δ, and the DNA binding protein HMGB1 (Kunkel and Erie, 2005Kunkel T.A. Erie D.A. DNA mismatch repair.Annu. Rev. Biochem. 2005; 74: 681-710Crossref PubMed Scopus (941) Google Scholar, Iyer et al., 2006Iyer R.R. Pluciennik A. Burdett V. Modrich P. DNA mismatch repair: functions and mechanisms.Chem. Rev. 2006; 106: 302-323Crossref PubMed Scopus (630) Google Scholar). Surprisingly, ExoI, which hydrolyzes duplex DNA with 5′-to-3′ polarity in the absence of other proteins, is required for both 5′- and 3′-directed repair of G-T or G-G mismatches in extracts of human and mouse cells (Genschel et al., 2002Genschel J. Bazemore L.R. Modrich P. Human exonuclease I is required for 5′ and 3′ mismatch repair.J. Biol. Chem. 2002; 277: 13302-13311Crossref PubMed Scopus (189) Google Scholar, Wei et al., 2003Wei K. Clark A.B. Wong E. Kane M.F. Mazur D.J. Parris T. Kolas N.K. Russell R. Hou Jr., H. Kneitz B. et al.Inactivation of Exonuclease 1 in mice results in DNA mismatch repair defects, increased cancer susceptibility, and male and female sterility.Genes Dev. 2003; 17: 603-614Crossref PubMed Scopus (243) Google Scholar). However, extracts of an ExoI−/− mouse cell line retain significant activity on single-nucleotide and dinucleotide insertion/deletion heteroduplexes, and HPRT mutability of such cells is elevated 30-fold, substantially less than the 150-fold increase conferred by MSH2 deficiency (Wei et al., 2003Wei K. Clark A.B. Wong E. Kane M.F. Mazur D.J. Parris T. Kolas N.K. Russell R. Hou Jr., H. Kneitz B. et al.Inactivation of Exonuclease 1 in mice results in DNA mismatch repair defects, increased cancer susceptibility, and male and female sterility.Genes Dev. 2003; 17: 603-614Crossref PubMed Scopus (243) Google Scholar). Although the spectrum of HPRT mutations was not established in the Wei et al. study, these findings indicate that ExoI plays a major role in the MutSα-dependent repair of base-base mispairs, but also that alternate excision activities may function in insertion/deletion mismatch correction. These observations have led to several purified systems that support nick-directed mismatch-provoked excision and repair. The simplest of these consists of MutSα, MutLα, ExoI, and RPA (±HMGB1) (Genschel and Modrich, 2003Genschel J. Modrich P. Mechanism of 5′-directed excision in human mismatch repair.Mol. Cell. 2003; 12: 1077-1086Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, Zhang et al., 2005Zhang Y. Yuan F. Presnell S.R. Tian K. Gao Y. Tomkinson A.E. Gu L. Li G.M. Reconstitution of 5′-directed human mismatch repair in a purified system.Cell. 2005; 122: 693-705Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). Hydrolysis in this system is mismatch provoked but always proceeds 5′ to 3′ from the nick that directs excision. Although MutLα is not required in this system, it does enhance the mismatch dependence of the reaction by suppressing ExoI hydrolysis of mismatch-free DNA (Genschel and Modrich, 2003Genschel J. Modrich P. Mechanism of 5′-directed excision in human mismatch repair.Mol. Cell. 2003; 12: 1077-1086Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). Supplementation of MutSα, MutLα, ExoI, and RPA with PCNA and RFC yields a system that supports bidirectional excision, i.e., excision directed by a nick located either 3′ or 5′ to the mismatch (Dzantiev et al., 2004Dzantiev L. Constantin N. Genschel J. Iyer R.R. Burgers P.M. Modrich P. A defined human system that supports bidirectional mismatch-provoked excision.Mol. Cell. 2004; 15: 31-41Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). In contrast to the simpler 5′-to-3′ reaction, 3′-directed excision is absolutely dependent on MutLα, RFC, and PCNA. RFC apparently plays two roles in the activation of 3′-directed excision. It functions as a PCNA loader, with the loaded form of PCNA necessary to activate 3′-directed excision, but it also acts directly to suppress ExoI-mediated 5′-to-3′ hydrolysis from a nick or gap located 3′ to the mismatch (Dzantiev et al., 2004Dzantiev L. Constantin N. Genschel J. Iyer R.R. Burgers P.M. Modrich P. A defined human system that supports bidirectional mismatch-provoked excision.Mol. Cell. 2004; 15: 31-41Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar; N.C. and P.M., unpublished data). Since the activities other than ExoI used in this study were free of exonuclease activity and because an ExoI active-site mutant did not support 3′-directed excision, hydrolysis in this system was attributed to ExoI (Dzantiev et al., 2004Dzantiev L. Constantin N. Genschel J. Iyer R.R. Burgers P.M. Modrich P. A defined human system that supports bidirectional mismatch-provoked excision.Mol. Cell. 2004; 15: 31-41Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). Addition of DNA polymerase δ to these six components yields a system that supports mismatch repair in a reaction that can be directed by a strand break located 3′ or 5′ to the mismatch (Constantin et al., 2005Constantin N. Dzantiev L. Kadyrov F.A. Modrich P. Human mismatch repair: Reconstitution of a nick-directed bidirectional reaction.J. Biol. Chem. 2005; 280: 39752-39761Crossref PubMed Scopus (173) Google Scholar). As observed for 5′-directed excision, 5′-directed repair in this system is MutLα independent but requires RFC and PCNA for the repair synthesis step of the reaction. The work described here clarifies the functions of MutLα and ExoI in human mismatch repair. We show that MutLα harbors a latent endonuclease that is activated in a mismatch-, MutSα-, RFC-, PCNA-, and ATP-dependent manner. Incision of a nicked heteroduplex by this four-protein system is strongly biased to the nicked strand. A mismatch-containing segment spanned by two strand breaks is then excised by the 5′-to-3′ action of MutSα-activated ExoI. Biochemical analysis of human mismatch repair has relied on the use of circular substrates containing a mismatch and a strand discontinuity (a nick or a gap) that directs the reaction. Because mismatch-provoked excision in nuclear extracts is restricted to the shorter path linking the two DNA sites (Fang and Modrich, 1993Fang W.-h. Modrich P. Human strand-specific mismatch repair occurs by a bidirectional mechanism similar to that of the bacterial reaction.J. Biol. Chem. 1993; 268: 11838-11844Abstract Full Text PDF PubMed Google Scholar), circular DNAs of this form are referred to as 3′ or 5′ heteroduplexes depending on whether the strand discontinuity resides 3′ or 5′ to the mispair, respectively, as viewed along the shorter path (see Figure 1 and Figure 2).Figure 2MutSα, MutLα, PCNA, and RFC Incise the Nicked Strand of 5′ Heteroduplex DNAShow full captionReactions and analysis were as in Figure 1, except that substrates were nicked 5′ G-T heteroduplex or 5′ G•C homoduplex. DNA products were cleaved with ClaI, resolved by alkaline electrophoresis, and transferred to nylon membranes, which were probed with 32P-labeled oligonucleotides corresponding to f1MR1 (Su et al., 1988Su S.-S. Lahue R.S. Au K.G. Modrich P. Mispair specificity of methyl-directed DNA mismatch correction in vitro.J. Biol. Chem. 1988; 263: 6829-6835Abstract Full Text PDF PubMed Google Scholar) viral strand coordinates 5732–5755 (A), viral strand coordinates 2531–2552 (B), viral strand coordinates 2505–2526 (C), or complementary strand coordinates 2531–2552 (D).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Reactions and analysis were as in Figure 1, except that substrates were nicked 5′ G-T heteroduplex or 5′ G•C homoduplex. DNA products were cleaved with ClaI, resolved by alkaline electrophoresis, and transferred to nylon membranes, which were probed with 32P-labeled oligonucleotides corresponding to f1MR1 (Su et al., 1988Su S.-S. Lahue R.S. Au K.G. Modrich P. Mispair specificity of methyl-directed DNA mismatch correction in vitro.J. Biol. Chem. 1988; 263: 6829-6835Abstract Full Text PDF PubMed Google Scholar) viral strand coordinates 5732–5755 (A), viral strand coordinates 2531–2552 (B), viral strand coordinates 2505–2526 (C), or complementary strand coordinates 2531–2552 (D). We have previously shown that a system comprised of MutSα, MutLα, RFC, PCNA, ExoI, and RPA supports 3′-nick-directed mismatch-provoked excision (Dzantiev et al., 2004Dzantiev L. Constantin N. Genschel J. Iyer R.R. Burgers P.M. Modrich P. A defined human system that supports bidirectional mismatch-provoked excision.Mol. Cell. 2004; 15: 31-41Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). Subsequent experiments suggested that this reaction might involve MutSα-, MutLα-, RFC-, and PCNA-dependent endonucleolytic attack on the incised heteroduplex strand (data not shown). This was confirmed by Southern analysis after restriction endonuclease cleavage and resolution of DNA products on denaturing gels. The production of new DNA termini on a 3′ heteroduplex, as visualized by this method, is illustrated in Figure 1. Use of a radiolabeled probe complementary to the 3′ terminus of the nicked strand demonstrated the production of new DNA termini 5′ to the site of probe hybridization (Figure 1A). A mismatch, MutSα, MutLα, RFC, PCNA, and ATP were required for this effect, but RPA was not (lanes 1–7; see also Table S1 in the Supplemental Data available with this article online). Fifty percent of the products shown in Figure 1A range in size from 0.10 to 0.56 kb, although new termini as far as 3 kb from the original strand break were detectable (lanes 2 and 7). Because the nick-mismatch separation distance in this 3′ heteroduplex is 141 bp, these results imply that incision of this DNA occurred preferentially on the 5′ side of the mispair, although some incision 3′ to the mismatch also occurred. By contrast, the yield of these products was greatly reduced if ExoI was included in the reaction, as was the signal corresponding to the original heteroduplex 3′ terminus (Figure 1A, compare lane 8 with lanes 2 and 7). The nature of endonucleolytic incision by this system was also addressed using probes that hybridize to the nicked heteroduplex strand on either side of the ClaI site (Figures 1B and 1C). The experiment shown in Figure 1B probes the other end of the DNA fragment analyzed in Figure 1A, i.e., for the production of new 3′ termini within this DNA segment. Incision products obtained in the presence of MutSα, MutLα, RFC, and PCNA (±RPA) and visualized with this probe were consistent with those observed in Figure 1A. As noted above, the occurrence of new 5′ termini within the region bracketed by the 3′ heteroduplex strand break and the ClaI site was abolished when ExoI was included in the reaction (Figure 1A, lane 8). However, generation of new 3′ termini within this region was demonstrable under these conditions (Figure 1B, lane 8). Because the MutSα, MutLα, RFC, PCNA, and RPA preparations used in these experiments were free of detectable exonuclease activity (Dzantiev et al., 2004Dzantiev L. Constantin N. Genschel J. Iyer R.R. Burgers P.M. Modrich P. A defined human system that supports bidirectional mismatch-provoked excision.Mol. Cell. 2004; 15: 31-41Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar; Table S1), the simplest explanation for these results is that 5′ termini produced by the endonucleolytic action of the MutSα, MutLα, RFC, PCNA system serve as entry sites for 5′-to-3′ hydrolysis by MutSα-activated ExoI (Genschel and Modrich, 2003Genschel J. Modrich P. Mechanism of 5′-directed excision in human mismatch repair.Mol. Cell. 2003; 12: 1077-1086Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, Dzantiev et al., 2004Dzantiev L. Constantin N. Genschel J. Iyer R.R. Burgers P.M. Modrich P. A defined human system that supports bidirectional mismatch-provoked excision.Mol. Cell. 2004; 15: 31-41Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar), which excises the DNA segment between this site and the original heteroduplex strand break, thus eliminating the hybridization target of the oligonucleotide probe. Incubation of the 3′ heteroduplex with MutSα, MutLα, RFC, and PCNA (±RPA) also produced new termini on the 5′ side of the heteroduplex strand break (Figure 1C). However this effect was attenuated by ExoI, which led to the preferential elimination of those termini most distant from the mismatch (compare lane 8 with 2 and 7). A similar ExoI effect is evident in Figure 1B (lane 8 versus 2 and 7), and the products observed in the presence of ExoI in these two instances are similar to those observed previously in the defined 3′ excision system (Dzantiev et al., 2004Dzantiev L. Constantin N. Genschel J. Iyer R.R. Burgers P.M. Modrich P. A defined human system that supports bidirectional mismatch-provoked excision.Mol. Cell. 2004; 15: 31-41Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). We think it unlikely that this preferential loss of distal termini is due to ExoI hydrolysis of hybridization target sequences in these cases. Rather, we attribute this effect to hydrolytic removal of the mismatch, which leads to cis inactivation of the endonucleolytic system. When inactivation in this manner does not occur, MutSα-, MutLα-, RFC-, and PCNA-dependent endonucleolytic incision is an ongoing process, and endonucleolytic events can occur several thousand bp from the mismatch. Additional evidence for this view is presented below. Endonucleolytic attack on the nicked 3′ heteroduplex DNA by this system was strongly biased to the nicked strand; incision of the continuous strand was limited to about 10% of that occurring on the nicked strand (Figure 1D; Table S1; see also below). Furthermore, covalently closed circular heteroduplex and homoduplex DNAs were resistant to incision by this system (Figure S1). The nicked strand of a 5′ G-T heteroduplex (nick and mismatch separated by 128 bp) was also subjected to incision in the presence of MutSα, MutLα, RFC, and PCNA (Figure 2). As observed with 3′ substrates, incision was mismatch dependent and occurred in the absence of RPA (Figures 2A–2C), and the continuous heteroduplex strand was resistant (incision was about 6% of that occurring on the nicked strand; compare Figure 2D with Figures 2A–2C; Table S1). Furthermore, supplementation of these proteins with ExoI abolished endonucleolytic product signals when the oligonucleotide used for end labeling was complementary to 5′ terminus at the nick that directs the reaction (Figure 2A, compare lane 8 with lanes 2 and 7). Presence of the exonuclease also attenuated incision events occurring in the vicinity of the ClaI site distal from the mismatch (Figures 2B and 2C, compare lane 8 with lanes 2 and 7). Table S1 quantifies the results of the incision reactions presented in Figure 1 and Figure 2. As can be seen, 5′ and 3′ heteroduplexes are incised with similar efficiency and in a manner that is highly dependent on a mismatch, MutSα, MutLα, RFC, PCNA, and ATP. We have previously shown that MutSα, MutLα, ExoI, RFC, PCNA, and RPA are sufficient to support ATP-dependent excision directed by a strand break located 3′ to a mismatch. The experiments described above suggested that the endonucleolytic products produced in the presence of MutSα, MutLα, RFC, and PCNA are intermediates in this process. Indeed, the MutSα and MutLα dependence of the endonucleolytic reaction is similar to that of reconstituted 3′-directed excision (Figure S2), and homogeneous yeast RFC effectively substituted for human RFC in this system (Table S1), as it does in 3′-directed excision (Dzantiev et al., 2004Dzantiev L. Constantin N. Genschel J. Iyer R.R. Burgers P.M. Modrich P. A defined human system that supports bidirectional mismatch-provoked excision.Mol. Cell. 2004; 15: 31-41Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). Additional evidence for endonucleolytic involvement in 3′-directed excision was provided by the experiment shown in Figure 3A, which used an oligonucleotide probe complementary to that portion of the nicked heteroduplex strand spanning the mismatch. As can be seen, endonucleolytic products containing the mismatched base appeared and disappeared when ExoI was included in the reaction, as expected for a reaction intermediate (compare lanes 2–5 with lanes 7–10). Results of this kinetic analysis were also consistent with the above suggestion that hydrolytic removal of the mismatch inactivates the endonuclease system in cis to prevent incision events from occurring at sites distal from the mispair. As can be seen in Figure 3B, incision in the vicinity of the ClaI site was also suppressed when the endonucleolytic system was supplemented with ExoI. Whereas MutSα, ExoI, and RPA are sufficient for excision of a mismatch when the nick that directs hydrolysis is located 5′ to the mispair (Genschel and Modrich, 2003Genschel J. Modrich P. Mechanism of 5′-directed excision in human mismatch repair.Mol. Cell. 2003; 12: 1077-1086Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, Zhang et al., 2005Zhang Y. Yuan F. Presnell S.R. Tian K. Gao Y. Tomkinson A.E. Gu L. Li G.M. Reconstitution of 5′-directed human mismatch repair in a purified system.Cell. 2005; 122: 693-705Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar), mismatch excision directed by a 3′ strand break additionally requires MutLα, RFC, and PCNA (Dzantiev et al., 2004Dzantiev L. Constantin N. Genschel J. Iyer R.R. Burgers P.M. Modrich P. A defined human system that supports bidirectional mismatch-provoked excision.Mol. Cell. 2004; 15: 31-41Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). Figure 3C demonstrates that the activity requirements for 3′-directed excision can be resolved in a two-stage reaction. Mismatch removal from the 3′ heteroduplex occurred in a stage 2 incubation lacking MutLα and PCNA provided that the heteroduplex was previously incubated with MutSα, MutLα, RFC, PCNA, and RPA, components sufficient for endonucleolytic incision of the DNA (lanes 1–5). Because MutSα, RPA, and a mismatch are sufficient to activate 5′-to-3′ hydrolysis by ExoI (Genschel and Modrich, 2003Genschel J. Modrich P. Mechanism of 5′-directed excision in human mismatch repair.Mol. Cell. 2003; 12: 1077-1086Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar), we attribute 3′-directed excision in this system to the 5′-to-3′ hydrolytic function of ExoI initiating at a strand break introduced on the 5′ side of the mispair by the action of MutSα, MutLα, RFC, and PCNA. Mismatch-dependent production of endonucleolytic intermediates can also be detected in nuclear extracts of human cells. In order to reduce heteroduplex ligation in extracts, these experiments employed a circular A-C heteroduplex (or A•T homoduplex) containing a 150 nucleotide gap located 51 bp 3′ to the mismatch (Figure 3D). The gapped heteroduplex supported mismatch-dependent endonuclease in the presence of MutSα, MutLα, RFC, and PCNA (Figure 3D, compare lanes 2 and 9). DNA fragments spanning the mismatch were also produced in HeLa nuclear extracts, and production of this species was also mismatch dependent (lanes 7 and 14). By contrast, fragments spanning the mispair were not produced in extracts derived from MLH1−/− H6 tumor cells, although supplementation of H6 extract with near homogeneous MutLα led to production of this species in a manner similar to that observed in HeLa extracts (lanes 5–7). As in the purified system, DNA fragments produced in nuclear extracts were derived from the discontinuous heteroduplex strand (Figure S3A). However, heteroduplex incision in nuclear extracts was more restricted to the vicinity of the mismatch than endonucleolytic events occurring in the purified system were (Figure S3B, lane 2 versus lanes 6 and 7). A similar effect has been noted previously with respect to termini produced in the MutSα-, MutLα-, ExoI-, RFC-, PCNA-, and RPA-dependent 3′-directed excision system (Dzantiev et al., 2004Dzantiev L. Constantin N. Genschel J. Iyer R.R. Burgers P.M. Modrich P. A defined human system that supports bidirectional mismatch-provoked excision.Mol. Cell. 2004; 15: 31-41Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). These purified systems thus lack one or more activities that function to restrict action of the endonuclease component to the vicinity of the mispair. Two completely independent sets of near homogeneous proteins were used in this work, and individual preparations were free of detectable nonspecific endonuclease activity in presence of ATP and Mg2+ at the salt concentration used for mismatch repair assay (125 mM KCl) (Figure 1, Figure 2, and Figure S2). However, MutSα, MutLα, RFC, or PCNA must harbor the active site of the endonuclease observed in this system. Studies in our and other laboratories have failed to reveal such an activity associated with PCNA or RFC, and we have been unable to detect endonuclease activity in MutSα preparations. However, we have found that MutLα preparations display endonuclease activity under certain conditions. A weak endonuclease activity that incises closed circular supercoiled homoduplex DNA was detectable at low KCl concentration (23 mM) in the presence of 1 mM Mn2+ but was not detectable in the presence of Mg2+ (Figures 4A and 4B). The Mn2+-dependent activity was stimulated by 0.5 mM ATP (Figure 4A, compare lanes 2 and 3), consistent with the known involvement of MutLα ATP hydrolytic centers in mismatch repair (Tomer et al., 2002Tomer G. Buermeyer A.B. Nguyen M.M. Liskay R.M. Contribution of human mlh1 and pms2 ATPase activities to DNA mismatch repair.J. Biol. Chem. 2002; 277: 21801-21809Crossref PubMed Scopus (45) Google Scholar, Raschle et al., 2002Raschle M. Dufner P. Marra G. Jiricny J. Mutations within the hMLH1 and hPMS2 subunits of the human MutLalpha mismatch repair factor affect its ATPase activity, but not its ability to interact with hMutSalpha.J. Biol. Chem. 2002; 277: 21810-21820Crossref PubMed Scopus (92) Google Scholar), and was further activated by RFC and PCNA, an effect dependent on the presence of both proteins (Figure 4C). Mn2+-dependent incision of supercoiled circular DNA was independent of the p" @default.
• W2141323033 created "2016-06-24" @default.
• W2141323033 creator A5026876597 @default.
• W2141323033 creator A5032560166 @default.
• W2141323033 creator A5038911075 @default.
• W2141323033 creator A5060309783 @default.
• W2141323033 date "2006-07-01" @default.
• W2141323033 modified "2023-10-12" @default.
• W2141323033 title "Endonucleolytic Function of MutLα in Human Mismatch Repair" @default.
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