Functions of MutLα, Replication Protein A (RPA), and HMGB1 in 5′-Directed Mismatch Repair
2009; Elsevier BV; Volume: 284; Issue: 32 Linguagem: Inglês
10.1074/jbc.m109.021287
ISSN1083-351X
AutoresJochen Genschel, Paul Modrich,
Tópico(s)Genomic variations and chromosomal abnormalities
ResumoA purified system comprised of MutSα, MutLα, exonuclease 1 (Exo1), and replication protein A (RPA) (in the absence or presence of HMGB1) supports 5′-directed mismatch-provoked excision that terminates after mismatch removal. MutLα is not essential for this reaction but enhances excision termination, although the basis of this effect has been uncertain. One model attributes the primary termination function in this system to RPA, with MutLα functioning in a secondary capacity by suppressing Exo1 hydrolysis of mismatch-free DNA (Genschel, J., and Modrich, P. (2003) Mol. Cell 12, 1077–1086). A second invokes MutLα as the primary effector of excision termination (Zhang, Y., Yuan, F., Presnell, S. R., Tian, K., Gao, Y., Tomkinson, A. E., Gu, L., and Li, G. M. (2005) Cell 122, 693–705). In the latter model, RPA provides a secondary termination function, but together with HMGB1, also participates in earlier steps of the reaction. To distinguish between these models, we have reanalyzed the functions of MutLα, RPA, and HMGB1 in 5′-directed mismatch-provoked excision using purified components as well as mammalian cell extracts. Analysis of extracts derived from A2780/AD cells, which are devoid of MutLα but nevertheless support 5′-directed mismatch repair, has demonstrated that 5′-directed excision terminates normally in the absence of MutLα. Experiments using purified components confirm a primary role for RPA in terminating excision by MutSα-activated Exo1 but are inconsistent with direct participation of MutLα in this process. While HMGB1 attenuates excision by activated Exo1, this effect is distinct from that mediated by RPA. Assay of extracts derived from HMGB1+/+ and HMGB1−/− mouse embryo fibroblast cells indicates that HMGB1 is not essential for mismatch repair. A purified system comprised of MutSα, MutLα, exonuclease 1 (Exo1), and replication protein A (RPA) (in the absence or presence of HMGB1) supports 5′-directed mismatch-provoked excision that terminates after mismatch removal. MutLα is not essential for this reaction but enhances excision termination, although the basis of this effect has been uncertain. One model attributes the primary termination function in this system to RPA, with MutLα functioning in a secondary capacity by suppressing Exo1 hydrolysis of mismatch-free DNA (Genschel, J., and Modrich, P. (2003) Mol. Cell 12, 1077–1086). A second invokes MutLα as the primary effector of excision termination (Zhang, Y., Yuan, F., Presnell, S. R., Tian, K., Gao, Y., Tomkinson, A. E., Gu, L., and Li, G. M. (2005) Cell 122, 693–705). In the latter model, RPA provides a secondary termination function, but together with HMGB1, also participates in earlier steps of the reaction. To distinguish between these models, we have reanalyzed the functions of MutLα, RPA, and HMGB1 in 5′-directed mismatch-provoked excision using purified components as well as mammalian cell extracts. Analysis of extracts derived from A2780/AD cells, which are devoid of MutLα but nevertheless support 5′-directed mismatch repair, has demonstrated that 5′-directed excision terminates normally in the absence of MutLα. Experiments using purified components confirm a primary role for RPA in terminating excision by MutSα-activated Exo1 but are inconsistent with direct participation of MutLα in this process. While HMGB1 attenuates excision by activated Exo1, this effect is distinct from that mediated by RPA. Assay of extracts derived from HMGB1+/+ and HMGB1−/− mouse embryo fibroblast cells indicates that HMGB1 is not essential for mismatch repair. DNA mismatch repair provides several genetic stabilization functions but is best known for its role in the correction of replication errors (reviewed in Refs. 1Kunkel T.A. Erie D.A. Annu. Rev. Biochem. 2005; 74: 681-710Crossref PubMed Scopus (995) Google Scholar, 2Iyer R.R. Pluciennik A. Burdett V. Modrich P.L. Chem. Rev. 2006; 106: 302-323Crossref PubMed Scopus (672) Google Scholar, 3Jiricny J. Nat. Rev. Mol. Cell Biol. 2006; 7: 335-346Crossref PubMed Scopus (927) Google Scholar, 4Modrich P. J. Biol. Chem. 2006; 281: 30305-30309Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, 5Li G.M. Cell Res. 2008; 18: 85-98Crossref PubMed Scopus (842) Google Scholar). When triggered by a mismatched base pair, removal of a DNA biosynthetic error by this system is targeted to the newly synthesized strand by secondary signals within the helix. Although the strand signals that direct eukaryotic mismatch repair have not been identified, a strand break in the form of a nick or gap is sufficient to direct repair to the discontinuous strand in cell extracts, and there is evidence that similar signals may function in vivo (6Pavlov Y.I. Newlon C.S. Kunkel T.A. Mol. Cell. 2002; 10: 207-213Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Analysis of the cell extract reaction has shown that the mammalian repair system possesses a bidirectional capability in the sense that the strand break that directs repair can be located either 3′ or 5′ to the mismatch (7Holmes Jr., J. Clark S. Modrich P. Proc. Natl. Acad. Sci. U.S.A. 1990; 87: 5837-5841Crossref PubMed Scopus (334) Google Scholar, 8Thomas D.C. Roberts J.D. Kunkel T.A. J. Biol. Chem. 1991; 266: 3744-3751Abstract Full Text PDF PubMed Google Scholar, 9Fang W.H. Modrich P. J. Biol. Chem. 1993; 268: 11838-11844Abstract Full Text PDF PubMed Google Scholar).Several purified mammalian systems have been described that support 3′ and/or 5′-directed mismatch-provoked excision or repair (10Genschel J. Modrich P. Mol. Cell. 2003; 12: 1077-1086Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 11Dzantiev 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 (198) Google Scholar, 12Zhang 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 (293) Google Scholar, 13Constantin N. Dzantiev L. Kadyrov F.A. Modrich P. J. Biol. Chem. 2005; 280: 39752-39761Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). The simplest of these depends on the mismatch recognition activities MutSα (MSH2·MSH6 heterodimer) or MutSΒ (MSH2·MSH3 heterodimer), MutLα (MLH1·PMS2 heterodimer), the single-stranded DNA-binding protein replication protein A (RPA), 2The abbreviations used are: RPAreplication protein ABSAbovine serum albuminPCNAproliferative cell nuclear antigenMEFmouse embryo fibroblast. 2The abbreviations used are: RPAreplication protein ABSAbovine serum albuminPCNAproliferative cell nuclear antigenMEFmouse embryo fibroblast. and the 5′ to 3′ double-strand hydrolytic activity Exo1 (exonuclease 1). These four activities support a mismatch-provoked excision reaction directed by a 5′ strand break, and excision terminates upon mismatch removal. MutLα is not essential for this excision reaction, but together with RPA has been implicated in excision termination (10Genschel J. Modrich P. Mol. Cell. 2003; 12: 1077-1086Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 12Zhang 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 (293) Google Scholar). In addition, the non-histone protein HMGB1 has been found to be required for mismatch repair in partially fractionated mammalian nuclear extracts (14Yuan F. Gu L. Guo S. Wang C. Li G.M. J. Biol. Chem. 2004; 279: 20935-20940Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). This small DNA binding protein has been postulated to functionally complement RPA in this 5′-directed system (12Zhang 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 (293) Google Scholar).Supplementation of MutSα, MutLα, RPA, and Exo1 with the replication clamp PCNA proliferative cell nuclear antigen (PCNA) and the clamp loader replication factor C (RFC) yields a system that supports mismatch-provoked excision directed by a 3′ or 5′ strand break (11Dzantiev 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 (198) Google Scholar). The basis of the bidirectional excision capability of this system was clarified with the demonstration that MutLα is a latent endonuclease that is activated in a mismatch-, MutSα-, RFC-, and ATP-dependent fashion (15Kadyrov F.A. Dzantiev L. Constantin N. Modrich P. Cell. 2006; 126: 297-308Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar). Incision by activated MutLα is restricted to the discontinuous strand of a nicked heteroduplex and tends to occur on the distal side of the mismatch. Thus, for a nicked heteroduplex in which the strand break resides 3′ to the mismatch, activated MutLα introduces an additional strand break 5′ to the mispair. This 5′ strand discontinuity provides the loading site for the 5′ to 3′ excision system described above, which removes the mismatch.MutSα has been shown to activate the 5′ to 3′ hydrolytic function of Exo1 on heteroduplex DNA, rendering the exonuclease highly processive, an effect attributed to physical interaction of the two activities (10Genschel J. Modrich P. Mol. Cell. 2003; 12: 1077-1086Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). However, there are differences in the literature with respect to the roles of RPA and MutLα in the control of this processive activity that leads to termination of 5′-directed excision (10Genschel J. Modrich P. Mol. Cell. 2003; 12: 1077-1086Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 12Zhang 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 (293) Google Scholar). One study has ascribed the primary excision termination function to RPA, which was shown to reduce the processive hydrolytic tracts of the MutSα·Exo1 complex from ≈2,000 to ≈250 nucleotides and by binding to gaps, to restrict Exo1 access to 5′ termini in excision intermediates and products (10Genschel J. Modrich P. Mol. Cell. 2003; 12: 1077-1086Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Analysis of excision intermediates as a function of nick-mismatch separation distance led to the conclusion that MutSα is able to reload Exo1 at an RPA-filled gap provided that a mismatch remains in the molecule; however, excision is attenuated upon mismatch removal because MutSα is no longer able to assist in this regard. In this mechanism MutLα functions in excision termination by suppressing nonspecific hydrolysis of the mismatch-free product, an effect attributed to its function as a general negative regulator of Exo1 (10Genschel J. Modrich P. Mol. Cell. 2003; 12: 1077-1086Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 16Nielsen F.C. Jäger A.C. Lützen A. Bundgaard J.R. Rasmussen L.J. Oncogene. 2004; 23: 1457-1468Crossref PubMed Scopus (65) Google Scholar). By contrast, a second study has attributed the primary termination function in this 5′-directed excision system to MutLα, with RPA playing a secondary role (12Zhang 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 (293) Google Scholar). In this mechanism RPA has several proposed functions. It is postulated to bind to the 5′ strand break where MutSα recruits HMGB1. RPA and HGB1 then partially melt the helix in the vicinity of the strand break leading to recruitment of Exo1, which initiates processive 5′ to 3′ hydrolysis. Binding of RPA to the ensuing gap results in displacement of MutSα and HMGB1 from the DNA and promotes physical interaction of the exonuclease with MutLα. This results in Exo1 inactivation and dissociation of the MutLα·Exo1 complex from the substrate. As in the other mechanism described above, Exo1 is reloaded if a mismatch remains within the DNA.To distinguish between these models, we have reassessed the roles of MutLα, RPA, and HMGB1 in mammalian mismatch repair. We demonstrate that 5′-directed excision terminates normally in extracts of A2780/AD cells, which support 5′-directed mismatch repair despite deficiency of MLH1 and PMS2 (17Drummond J.T. Anthoney A. Brown R. Modrich P. J. Biol. Chem. 1996; 271: 19645-19648Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar). Reanalysis of 5′-directed mismatch-provoked excision in the purified system described above has confirmed a direct role for RPA in terminating processive excision by MutSα-activated Exo1 but is inconsistent with direct participation of MutLα in this process. These experiments also indicate that while HMGB1 can attenuate Exo1 excision, this effect is distinct from that mediated by RPA. We also show that in contrast to results obtained with partially fractionated HeLa extracts (14Yuan F. Gu L. Guo S. Wang C. Li G.M. J. Biol. Chem. 2004; 279: 20935-20940Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), extracts derived from HMGB1−/− mouse embryo fibroblast cells are fully proficient in mismatch repair.DISCUSSIONSeveral drug-resistant human cell lines, in which the MLH1 loci are silenced (30Strathdee G. MacKean M.J. Illand M. Brown R. Oncogene. 1999; 18: 2335-2341Crossref PubMed Scopus (309) Google Scholar),3 have been shown to be defective in 3′- but not 5′-directed mismatch rectification (17Drummond J.T. Anthoney A. Brown R. Modrich P. J. Biol. Chem. 1996; 271: 19645-19648Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar). We have shown here that extracts derived from one of these cell lines, A2780/AD, supports mismatch-dependent 5′-directed excision, proving that the 5′-directed rectification that was previously documented with this line is mismatch-provoked and hence corresponds to bona fide mismatch repair. As observed previously with respect to repair (17Drummond J.T. Anthoney A. Brown R. Modrich P. J. Biol. Chem. 1996; 271: 19645-19648Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar), we have also found that MutLα supplementation modestly enhances 5′-directed excision in these extracts, suggesting the potential existence of MutLα-dependent and -independent modes of 5′-directed excision in mammalian cells. Interestingly, previous studies have documented two distinct modes of 5′-directed excision in HeLa nuclear extracts with respect to PCNA involvement. While essentially all 3′-directed excision events that occur in HeLa extracts are PCNA-dependent, both PCNA-dependent and -independent modes of 5′-directed excision have been documented in this system (supplemental data in Refs. 10Genschel J. Modrich P. Mol. Cell. 2003; 12: 1077-1086Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 31Guo 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 (45) Google Scholar).We have also found that 5′-directed excision tracts that occur A2780/AD extracts terminate within the same region as do those that occur in extracts derived from MutLα-proficient HeLa cells (Figs. 2 and supplemental S1). Thus, the postulated requirement for MutLα in the termination of mismatch-provoked excision (12Zhang 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 (293) Google Scholar) can be ruled out for the extract system. Analysis of MutLα effects on the termination of hydrolysis by MutSα-activated Exo1 in a purified system is also consistent with this view. We have demonstrated previously in the presence of MutLα (10Genschel J. Modrich P. Mol. Cell. 2003; 12: 1077-1086Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar) and confirm here in the absence or presence of MutLα, the dramatic effects of RPA on mismatch-provoked excision by MutSα-activated Exo1. Surprisingly, RPA has both positive and negative effector roles in this reaction. The single-strand-binding protein not only stimulates substrate turnover but also functions to stabilize reaction products after mismatch removal (compare right panels of Fig. 5 with the center panels of Fig. 3). Furthermore, the reaction products produced in the presence of RPA and MutLα are similar to those produced in the presence of RPA alone, although it is clear that MutLα does enhance their lifetime (compare right and center panels of Fig. 3). This substantiates the previous conclusion (10Genschel J. Modrich P. Mol. Cell. 2003; 12: 1077-1086Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar) that RPA functions as the primary termination activity in this system, with MutLα functioning after the fact to stabilize primary hydrolytic products against further Exo1 degradation.We have also confirmed here the previous finding that RPA functions as a negative regulator of the MutSα·Exo1 complex, reducing its processivity from about 2,000 to about 200 nucleotides (Figs. 6 and supplemental S2). By contrast, MutLα is without significant effect on the processive behavior of activated Exo1. As described previously (10Genschel J. Modrich P. Mol. Cell. 2003; 12: 1077-1086Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar), we attribute the activating effect of RPA on mismatch-provoked excision to its ability to regulate processive behavior of the MutSa·Exo1 complex. In particular, we suggest that RPA-mediated displacement of MutSα and/or Exo1 excision intermediates and products permits the system to turn over.We have also confirmed the ability of the nonhistone protein HMGB1 to stimulate excision by MutSα-activated Exo1 and have evaluated its effects on the processive behavior of this complex. In both cases HMGB1 behaved in a manner distinct from RPA. In amounts comparable to that present in 100 μg of nuclear extract (≈120 ng as judged by quantitative Western blot, data not shown), HMGB1 significantly stimulated mismatch-provoked excision (compare center and right panels of Fig. 5), but less effectively than RPA. As in the case of RPA, this effect may be due to the ability of HMGB1 to interfere with progression of the processive hydrolysis, forcing turnover of the hydrolytic complex. However, as can be seen in Fig. 6, HMGB1 was less effective than RPA with respect to its ability to disrupt processive excision.As noted above, the most compelling evidence for an HMGB1 requirement in mammalian mismatch repair has derived from analysis of partially fractionated HeLa cell extracts, a system in which repair was shown to be strongly dependent on HMGB1 addition (14Yuan F. Gu L. Guo S. Wang C. Li G.M. J. Biol. Chem. 2004; 279: 20935-20940Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Inasmuch as HMGB1-proficient and -null cell lines are available (18Calogero S. Grassi F. Aguzzi A. Voigtländer T. Ferrier P. Ferrari S. Bianchi M.E. Nat. Genet. 1999; 22: 276-280Crossref PubMed Scopus (439) Google Scholar, 19Lange S.S. Mitchell D.L. Vasquez K.M. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 10320-10325Crossref PubMed Scopus (157) Google Scholar), we have evaluated the mismatch repair requirement for this protein in whole cell extracts. In contrast to results obtained with partially fractionated extracts, we have found that unfractionated HMGB1-deficient extracts support robust mismatch repair that can be directed by either a 3′ or 5′ strand break. There are several possible explanations for these different findings. For example, mammalian cell extracts may possess a second activity, which provides a mismatch repair function that is redundant with respect to HMGB1; in this case, the fractionation method employed by Yuan et al. (14Yuan F. Gu L. Guo S. Wang C. Li G.M. J. Biol. Chem. 2004; 279: 20935-20940Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar) may have resolved these two activities. An alternate possibility is that the fractionation method employed by Yuan et al. enriched for an inhibitor of the reaction, the action of which is reversed by HMGB1. Distinction between these and other possibilities must await further studies, but we note that as in the case of the unfractionated extracts used here, several purified systems have been described that support highly efficient mismatch repair in the absence of HMGB1 (12Zhang 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 (293) Google Scholar, 13Constantin N. Dzantiev L. Kadyrov F.A. Modrich P. J. Biol. Chem. 2005; 280: 39752-39761Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar).Comparison of results obtained with the extract and purified systems described here has revealed significant differences in termination zones in the two cases. Excision occurring in extracts is characterized with a single primary termination zone centered about 130–140 nucleotides beyond the mispair. By contrast, two major termination zones occur in the purified system, located about 40–100 nucleotides or 220–350 nucleotides beyond the mismatch, with the former appearing to be a precursor to the latter. As described previously, we regard the reconstituted mismatch repair systems that have been described to date as minimal systems (4Modrich P. J. Biol. Chem. 2006; 281: 30305-30309Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar) and attribute the sort of difference noted above to as yet unidentified extract components that significantly modulate various steps of the reaction. DNA mismatch repair provides several genetic stabilization functions but is best known for its role in the correction of replication errors (reviewed in Refs. 1Kunkel T.A. Erie D.A. Annu. Rev. Biochem. 2005; 74: 681-710Crossref PubMed Scopus (995) Google Scholar, 2Iyer R.R. Pluciennik A. Burdett V. Modrich P.L. Chem. Rev. 2006; 106: 302-323Crossref PubMed Scopus (672) Google Scholar, 3Jiricny J. Nat. Rev. Mol. Cell Biol. 2006; 7: 335-346Crossref PubMed Scopus (927) Google Scholar, 4Modrich P. J. Biol. Chem. 2006; 281: 30305-30309Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, 5Li G.M. Cell Res. 2008; 18: 85-98Crossref PubMed Scopus (842) Google Scholar). When triggered by a mismatched base pair, removal of a DNA biosynthetic error by this system is targeted to the newly synthesized strand by secondary signals within the helix. Although the strand signals that direct eukaryotic mismatch repair have not been identified, a strand break in the form of a nick or gap is sufficient to direct repair to the discontinuous strand in cell extracts, and there is evidence that similar signals may function in vivo (6Pavlov Y.I. Newlon C.S. Kunkel T.A. Mol. Cell. 2002; 10: 207-213Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Analysis of the cell extract reaction has shown that the mammalian repair system possesses a bidirectional capability in the sense that the strand break that directs repair can be located either 3′ or 5′ to the mismatch (7Holmes Jr., J. Clark S. Modrich P. Proc. Natl. Acad. Sci. U.S.A. 1990; 87: 5837-5841Crossref PubMed Scopus (334) Google Scholar, 8Thomas D.C. Roberts J.D. Kunkel T.A. J. Biol. Chem. 1991; 266: 3744-3751Abstract Full Text PDF PubMed Google Scholar, 9Fang W.H. Modrich P. J. Biol. Chem. 1993; 268: 11838-11844Abstract Full Text PDF PubMed Google Scholar). Several purified mammalian systems have been described that support 3′ and/or 5′-directed mismatch-provoked excision or repair (10Genschel J. Modrich P. Mol. Cell. 2003; 12: 1077-1086Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 11Dzantiev 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 (198) Google Scholar, 12Zhang 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 (293) Google Scholar, 13Constantin N. Dzantiev L. Kadyrov F.A. Modrich P. J. Biol. Chem. 2005; 280: 39752-39761Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). The simplest of these depends on the mismatch recognition activities MutSα (MSH2·MSH6 heterodimer) or MutSΒ (MSH2·MSH3 heterodimer), MutLα (MLH1·PMS2 heterodimer), the single-stranded DNA-binding protein replication protein A (RPA), 2The abbreviations used are: RPAreplication protein ABSAbovine serum albuminPCNAproliferative cell nuclear antigenMEFmouse embryo fibroblast. 2The abbreviations used are: RPAreplication protein ABSAbovine serum albuminPCNAproliferative cell nuclear antigenMEFmouse embryo fibroblast. and the 5′ to 3′ double-strand hydrolytic activity Exo1 (exonuclease 1). These four activities support a mismatch-provoked excision reaction directed by a 5′ strand break, and excision terminates upon mismatch removal. MutLα is not essential for this excision reaction, but together with RPA has been implicated in excision termination (10Genschel J. Modrich P. Mol. Cell. 2003; 12: 1077-1086Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 12Zhang 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 (293) Google Scholar). In addition, the non-histone protein HMGB1 has been found to be required for mismatch repair in partially fractionated mammalian nuclear extracts (14Yuan F. Gu L. Guo S. Wang C. Li G.M. J. Biol. Chem. 2004; 279: 20935-20940Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). This small DNA binding protein has been postulated to functionally complement RPA in this 5′-directed system (12Zhang 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 (293) Google Scholar). replication protein A bovine serum albumin proliferative cell nuclear antigen mouse embryo fibroblast. replication protein A bovine serum albumin proliferative cell nuclear antigen mouse embryo fibroblast. Supplementation of MutSα, MutLα, RPA, and Exo1 with the replication clamp PCNA proliferative cell nuclear antigen (PCNA) and the clamp loader replication factor C (RFC) yields a system that supports mismatch-provoked excision directed by a 3′ or 5′ strand break (11Dzantiev 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 (198) Google Scholar). The basis of the bidirectional excision capability of this system was clarified with the demonstration that MutLα is a latent endonuclease that is activated in a mismatch-, MutSα-, RFC-, and ATP-dependent fashion (15Kadyrov F.A. Dzantiev L. Constantin N. Modrich P. Cell. 2006; 126: 297-308Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar). Incision by activated MutLα is restricted to the discontinuous strand of a nicked heteroduplex and tends to occur on the distal side of the mismatch. Thus, for a nicked heteroduplex in which the strand break resides 3′ to the mismatch, activated MutLα introduces an additional strand break 5′ to the mispair. This 5′ strand discontinuity provides the loading site for the 5′ to 3′ excision system described above, which removes the mismatch. MutSα has been shown to activate the 5′ to 3′ hydrolytic function of Exo1 on heteroduplex DNA, rendering the exonuclease highly processive, an effect attributed to physical interaction of the two activities (10Genschel J. Modrich P. Mol. Cell. 2003; 12: 1077-1086Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). However, there are differences in the literature with respect to the roles of RPA and MutLα in the control of this processive activity that leads to termination of 5′-directed excision (10Genschel J. Modrich P. Mol. Cell. 2003; 12: 1077-1086Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 12Zhang 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 (293) Google Scholar). One study has ascribed the primary excision termination function to RPA, which was shown to reduce the processive hydrolytic tracts of the MutSα·Exo1 complex from ≈2,000 to ≈250 nucleotides and by binding to gaps, to restrict Exo1 access to 5′ termini in excision intermediates and products (10Genschel J. Modrich P. Mol. Cell. 2003; 12: 1077-1086Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Analysis of excision intermediates as a function of nick-mismatch separation distance led to the conclusion that MutSα is able to reload Exo1 at an RPA-filled gap provided that a mismatch remains in the molecule; however, excision is attenuated upon mismatch removal because MutSα is no longer able to assist in this regard. In this mechanism MutLα functions in excision termination by suppressing nonspecific hydrolysis of the mismatch-free product, an effect attributed to its function as a general negative regulator of Exo1 (10Genschel J. Modrich P. Mol. Cell. 2003; 12: 1077-1086Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 16Nielsen F.C. Jäger A.C. Lützen A. Bundgaard J.R. Rasmussen L.J. Oncogene. 2004; 23: 1457-1468Crossref PubMed Scopus (65) Google Scholar). By contrast, a second study has attributed the primary termination function in this 5′-directed excision system to MutLα, with RPA playing a secondary role (12Zhang 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 (293) Google Scholar). In this mechanism RPA has several proposed functions. It is postulated to bind to the 5′ strand break where MutSα recruits HMGB1. RPA and HGB1 then partially melt the helix in the vicinity of the strand break leading to recruitment of Exo1, which initiates processive 5′ to 3′ hydrolysis. Binding of RPA to the ensuing gap results in displacement of MutSα and HMGB1 from the DNA and promotes physical interaction of the exonuclease with MutLα. This results in Exo1 inactivation and dissociation of the MutLα·Exo1 complex from the substrate. As in the other mechanism described above, Exo1 is reloaded if a mismatch remains within the DNA. To distinguish between these models, we have reassessed the roles of MutLα, RPA, and HMGB1 in mammalian mismatch repair. We demonstrate that 5′-directed excision terminates normally in extracts of A2780/AD cells, which support 5′-directed mismatch repair despite deficiency of MLH1 and PMS2 (17Drummond J.T. Anthoney A. Brown R. Modrich P. J. Biol. Chem. 1996; 271: 19645-19648Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar). Reanalysis of 5′-directed mismatch-provoked excision in the purified system described above has confirmed a direct role for RPA in terminating processive excision by MutSα-activated Exo1 but is inconsistent with direct participation of MutLα in this process. These experiments also indicate that while HMGB1 can attenuate Exo1 excision, this effect is distinct from that mediated by RPA. We also show that in contrast to results obtained with partially fractionated HeLa extracts (14Yuan F. Gu L. Guo S. Wang C. Li G.M. J. Biol. Chem. 2004; 279: 20935-20940Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), extracts derived from HMGB1−/− mouse embryo fibroblast cells are fully proficient in mismatch repair. DISCUSSIONSeveral drug-resistant human cell lines, in which the MLH1 loci are silenced (30Strathdee G. MacKean M.J. Illand M. Brown R. Oncogene. 1999; 18: 2335-2341Crossref PubMed Scopus (309) Google Scholar),3 have been shown to be defective in 3′- but not 5′-directed mismatch rectification (17Drummond J.T. Anthoney A. Brown R. Modrich P. J. Biol. Chem. 1996; 271: 19645-19648Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar). We have shown here that extracts derived from one of these cell lines, A2780/AD, supports mismatch-dependent 5′-directed excision, proving that the 5′-directed rectification that was previously documented with this line is mismatch-provoked and hence corresponds to bona fide mismatch repair. As observed previously with respect to repair (17Drummond J.T. Anthoney A. Brown R. Modrich P. J. Biol. Chem. 1996; 271: 19645-19648Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar), we have also found that MutLα supplementation modestly enhances 5′-directed excision in these extracts, suggesting the potential existence of MutLα-dependent and -independent modes of 5′-directed excision in mammalian cells. Interestingly, previous studies have documented two distinct modes of 5′-directed excision in HeLa nuclear extracts with respect to PCNA involvement. While essentially all 3′-directed excision events that occur in HeLa extracts are PCNA-dependent, both PCNA-dependent and -independent modes of 5′-directed excision have been documented in this system (supplemental data in Refs. 10Genschel J. Modrich P. Mol. Cell. 2003; 12: 1077-1086Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 31Guo 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 (45) Google Scholar).We have also found that 5′-directed excision tracts that occur A2780/AD extracts terminate within the same region as do those that occur in extracts derived from MutLα-proficient HeLa cells (Figs. 2 and supplemental S1). Thus, the postulated requirement for MutLα in the termination of mismatch-provoked excision (12Zhang 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 (293) Google Scholar) can be ruled out for the extract system. Analysis of MutLα effects on the termination of hydrolysis by MutSα-activated Exo1 in a purified system is also consistent with this view. We have demonstrated previously in the presence of MutLα (10Genschel J. Modrich P. Mol. Cell. 2003; 12: 1077-1086Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar) and confirm here in the absence or presence of MutLα, the dramatic effects of RPA on mismatch-provoked excision by MutSα-activated Exo1. Surprisingly, RPA has both positive and negative effector roles in this reaction. The single-strand-binding protein not only stimulates substrate turnover but also functions to stabilize reaction products after mismatch removal (compare right panels of Fig. 5 with the center panels of Fig. 3). Furthermore, the reaction products produced in the presence of RPA and MutLα are similar to those produced in the presence of RPA alone, although it is clear that MutLα does enhance their lifetime (compare right and center panels of Fig. 3). This substantiates the previous conclusion (10Genschel J. Modrich P. Mol. Cell. 2003; 12: 1077-1086Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar) that RPA functions as the primary termination activity in this system, with MutLα functioning after the fact to stabilize primary hydrolytic products against further Exo1 degradation.We have also confirmed here the previous finding that RPA functions as a negative regulator of the MutSα·Exo1 complex, reducing its processivity from about 2,000 to about 200 nucleotides (Figs. 6 and supplemental S2). By contrast, MutLα is without significant effect on the processive behavior of activated Exo1. As described previously (10Genschel J. Modrich P. Mol. Cell. 2003; 12: 1077-1086Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar), we attribute the activating effect of RPA on mismatch-provoked excision to its ability to regulate processive behavior of the MutSa·Exo1 complex. In particular, we suggest that RPA-mediated displacement of MutSα and/or Exo1 excision intermediates and products permits the system to turn over.We have also confirmed the ability of the nonhistone protein HMGB1 to stimulate excision by MutSα-activated Exo1 and have evaluated its effects on the processive behavior of this complex. In both cases HMGB1 behaved in a manner distinct from RPA. In amounts comparable to that present in 100 μg of nuclear extract (≈120 ng as judged by quantitative Western blot, data not shown), HMGB1 significantly stimulated mismatch-provoked excision (compare center and right panels of Fig. 5), but less effectively than RPA. As in the case of RPA, this effect may be due to the ability of HMGB1 to interfere with progression of the processive hydrolysis, forcing turnover of the hydrolytic complex. However, as can be seen in Fig. 6, HMGB1 was less effective than RPA with respect to its ability to disrupt processive excision.As noted above, the most compelling evidence for an HMGB1 requirement in mammalian mismatch repair has derived from analysis of partially fractionated HeLa cell extracts, a system in which repair was shown to be strongly dependent on HMGB1 addition (14Yuan F. Gu L. Guo S. Wang C. Li G.M. J. Biol. Chem. 2004; 279: 20935-20940Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Inasmuch as HMGB1-proficient and -null cell lines are available (18Calogero S. Grassi F. Aguzzi A. Voigtländer T. Ferrier P. Ferrari S. Bianchi M.E. Nat. Genet. 1999; 22: 276-280Crossref PubMed Scopus (439) Google Scholar, 19Lange S.S. Mitchell D.L. Vasquez K.M. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 10320-10325Crossref PubMed Scopus (157) Google Scholar), we have evaluated the mismatch repair requirement for this protein in whole cell extracts. In contrast to results obtained with partially fractionated extracts, we have found that unfractionated HMGB1-deficient extracts support robust mismatch repair that can be directed by either a 3′ or 5′ strand break. There are several possible explanations for these different findings. For example, mammalian cell extracts may possess a second activity, which provides a mismatch repair function that is redundant with respect to HMGB1; in this case, the fractionation method employed by Yuan et al. (14Yuan F. Gu L. Guo S. Wang C. Li G.M. J. Biol. Chem. 2004; 279: 20935-20940Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar) may have resolved these two activities. An alternate possibility is that the fractionation method employed by Yuan et al. enriched for an inhibitor of the reaction, the action of which is reversed by HMGB1. Distinction between these and other possibilities must await further studies, but we note that as in the case of the unfractionated extracts used here, several purified systems have been described that support highly efficient mismatch repair in the absence of HMGB1 (12Zhang 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 (293) Google Scholar, 13Constantin N. Dzantiev L. Kadyrov F.A. Modrich P. J. Biol. Chem. 2005; 280: 39752-39761Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar).Comparison of results obtained with the extract and purified systems described here has revealed significant differences in termination zones in the two cases. Excision occurring in extracts is characterized with a single primary termination zone centered about 130–140 nucleotides beyond the mispair. By contrast, two major termination zones occur in the purified system, located about 40–100 nucleotides or 220–350 nucleotides beyond the mismatch, with the former appearing to be a precursor to the latter. As described previously, we regard the reconstituted mismatch repair systems that have been described to date as minimal systems (4Modrich P. J. Biol. Chem. 2006; 281: 30305-30309Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar) and attribute the sort of difference noted above to as yet unidentified extract components that significantly modulate various steps of the reaction. Several drug-resistant human cell lines, in which the MLH1 loci are silenced (30Strathdee G. MacKean M.J. Illand M. Brown R. Oncogene. 1999; 18: 2335-2341Crossref PubMed Scopus (309) Google Scholar),3 have been shown to be defective in 3′- but not 5′-directed mismatch rectification (17Drummond J.T. Anthoney A. Brown R. Modrich P. J. Biol. Chem. 1996; 271: 19645-19648Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar). We have shown here that extracts derived from one of these cell lines, A2780/AD, supports mismatch-dependent 5′-directed excision, proving that the 5′-directed rectification that was previously documented with this line is mismatch-provoked and hence corresponds to bona fide mismatch repair. As observed previously with respect to repair (17Drummond J.T. Anthoney A. Brown R. Modrich P. J. Biol. Chem. 1996; 271: 19645-19648Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar), we have also found that MutLα supplementation modestly enhances 5′-directed excision in these extracts, suggesting the potential existence of MutLα-dependent and -independent modes of 5′-directed excision in mammalian cells. Interestingly, previous studies have documented two distinct modes of 5′-directed excision in HeLa nuclear extracts with respect to PCNA involvement. While essentially all 3′-directed excision events that occur in HeLa extracts are PCNA-dependent, both PCNA-dependent and -independent modes of 5′-directed excision have been documented in this system (supplemental data in Refs. 10Genschel J. Modrich P. Mol. Cell. 2003; 12: 1077-1086Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 31Guo 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 (45) Google Scholar). We have also found that 5′-directed excision tracts that occur A2780/AD extracts terminate within the same region as do those that occur in extracts derived from MutLα-proficient HeLa cells (Figs. 2 and supplemental S1). Thus, the postulated requirement for MutLα in the termination of mismatch-provoked excision (12Zhang 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 (293) Google Scholar) can be ruled out for the extract system. Analysis of MutLα effects on the termination of hydrolysis by MutSα-activated Exo1 in a purified system is also consistent with this view. We have demonstrated previously in the presence of MutLα (10Genschel J. Modrich P. Mol. Cell. 2003; 12: 1077-1086Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar) and confirm here in the absence or presence of MutLα, the dramatic effects of RPA on mismatch-provoked excision by MutSα-activated Exo1. Surprisingly, RPA has both positive and negative effector roles in this reaction. The single-strand-binding protein not only stimulates substrate turnover but also functions to stabilize reaction products after mismatch removal (compare right panels of Fig. 5 with the center panels of Fig. 3). Furthermore, the reaction products produced in the presence of RPA and MutLα are similar to those produced in the presence of RPA alone, although it is clear that MutLα does enhance their lifetime (compare right and center panels of Fig. 3). This substantiates the previous conclusion (10Genschel J. Modrich P. Mol. Cell. 2003; 12: 1077-1086Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar) that RPA functions as the primary termination activity in this system, with MutLα functioning after the fact to stabilize primary hydrolytic products against further Exo1 degradation. We have also confirmed here the previous finding that RPA functions as a negative regulator of the MutSα·Exo1 complex, reducing its processivity from about 2,000 to about 200 nucleotides (Figs. 6 and supplemental S2). By contrast, MutLα is without significant effect on the processive behavior of activated Exo1. As described previously (10Genschel J. Modrich P. Mol. Cell. 2003; 12: 1077-1086Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar), we attribute the activating effect of RPA on mismatch-provoked excision to its ability to regulate processive behavior of the MutSa·Exo1 complex. In particular, we suggest that RPA-mediated displacement of MutSα and/or Exo1 excision intermediates and products permits the system to turn over. We have also confirmed the ability of the nonhistone protein HMGB1 to stimulate excision by MutSα-activated Exo1 and have evaluated its effects on the processive behavior of this complex. In both cases HMGB1 behaved in a manner distinct from RPA. In amounts comparable to that present in 100 μg of nuclear extract (≈120 ng as judged by quantitative Western blot, data not shown), HMGB1 significantly stimulated mismatch-provoked excision (compare center and right panels of Fig. 5), but less effectively than RPA. As in the case of RPA, this effect may be due to the ability of HMGB1 to interfere with progression of the processive hydrolysis, forcing turnover of the hydrolytic complex. However, as can be seen in Fig. 6, HMGB1 was less effective than RPA with respect to its ability to disrupt processive excision. As noted above, the most compelling evidence for an HMGB1 requirement in mammalian mismatch repair has derived from analysis of partially fractionated HeLa cell extracts, a system in which repair was shown to be strongly dependent on HMGB1 addition (14Yuan F. Gu L. Guo S. Wang C. Li G.M. J. Biol. Chem. 2004; 279: 20935-20940Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Inasmuch as HMGB1-proficient and -null cell lines are available (18Calogero S. Grassi F. Aguzzi A. Voigtländer T. Ferrier P. Ferrari S. Bianchi M.E. Nat. Genet. 1999; 22: 276-280Crossref PubMed Scopus (439) Google Scholar, 19Lange S.S. Mitchell D.L. Vasquez K.M. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 10320-10325Crossref PubMed Scopus (157) Google Scholar), we have evaluated the mismatch repair requirement for this protein in whole cell extracts. In contrast to results obtained with partially fractionated extracts, we have found that unfractionated HMGB1-deficient extracts support robust mismatch repair that can be directed by either a 3′ or 5′ strand break. There are several possible explanations for these different findings. For example, mammalian cell extracts may possess a second activity, which provides a mismatch repair function that is redundant with respect to HMGB1; in this case, the fractionation method employed by Yuan et al. (14Yuan F. Gu L. Guo S. Wang C. Li G.M. J. Biol. Chem. 2004; 279: 20935-20940Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar) may have resolved these two activities. An alternate possibility is that the fractionation method employed by Yuan et al. enriched for an inhibitor of the reaction, the action of which is reversed by HMGB1. Distinction between these and other possibilities must await further studies, but we note that as in the case of the unfractionated extracts used here, several purified systems have been described that support highly efficient mismatch repair in the absence of HMGB1 (12Zhang 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 (293) Google Scholar, 13Constantin N. Dzantiev L. Kadyrov F.A. Modrich P. J. Biol. Chem. 2005; 280: 39752-39761Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). Comparison of results obtained with the extract and purified systems described here has revealed significant differences in termination zones in the two cases. Excision occurring in extracts is characterized with a single primary termination zone centered about 130–140 nucleotides beyond the mispair. By contrast, two major termination zones occur in the purified system, located about 40–100 nucleotides or 220–350 nucleotides beyond the mismatch, with the former appearing to be a precursor to the latter. As described previously, we regard the reconstituted mismatch repair systems that have been described to date as minimal systems (4Modrich P. J. Biol. Chem. 2006; 281: 30305-30309Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar) and attribute the sort of difference noted above to as yet unidentified extract components that significantly modulate various steps of the reaction. We thank K. Vasquez and M. Bianchi for the generous gift of HMGB1+/+ and HMGB1−/− mouse embryonic fibroblast cell lines. Supplementary Material Download .pdf (.1 MB) Help with pdf files Download .pdf (.1 MB) Help with pdf files
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