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• W2090410902 abstract "Replication protein A (RPA) is involved in multiple stages of DNA mismatch repair (MMR); however, the modulation of its functions between different stages is unknown. We show here that phosphorylation likely modulates RPA functions during MMR. Unphosphorylated RPA initially binds to nicked heteroduplex DNA to facilitate assembly of the MMR initiation complex. The unphosphorylated protein preferentially stimulates mismatch-provoked excision, possibly by cooperatively binding to the resultant single-stranded DNA gap. The DNA-bound RPA begins to be phosphorylated after extensive excision, resulting in severalfold reduction in the DNA binding affinity of RPA. Thus, during the phase of repair DNA synthesis, the phosphorylated RPA readily disassociates from DNA, making the DNA template available for DNA polymerase δ-catalyzed resynthesis. These observations support a model of how phosphorylation alters the DNA binding affinity of RPA to fulfill its differential requirement at the various stages of MMR. Replication protein A (RPA) is involved in multiple stages of DNA mismatch repair (MMR); however, the modulation of its functions between different stages is unknown. We show here that phosphorylation likely modulates RPA functions during MMR. Unphosphorylated RPA initially binds to nicked heteroduplex DNA to facilitate assembly of the MMR initiation complex. The unphosphorylated protein preferentially stimulates mismatch-provoked excision, possibly by cooperatively binding to the resultant single-stranded DNA gap. The DNA-bound RPA begins to be phosphorylated after extensive excision, resulting in severalfold reduction in the DNA binding affinity of RPA. Thus, during the phase of repair DNA synthesis, the phosphorylated RPA readily disassociates from DNA, making the DNA template available for DNA polymerase δ-catalyzed resynthesis. These observations support a model of how phosphorylation alters the DNA binding affinity of RPA to fulfill its differential requirement at the various stages of MMR. Defects in DNA mismatch repair (MMR) 4The abbreviations used are: MMR, mismatch repair; RPA, replication protein A; PCNA, proliferating cellular nuclear antigen; nt, nucleotides; DNA-PK, DNA-dependent protein kinase; ssDNA, single-stranded DNA; PI, phosphatidylinositol; pol, polymerase; WT, wild-type; RFC, replicative factor C. lead to a hypermutable phenotype and a predisposition to cancer (1Kolodner R.D. Marsischky G.T. Curr. Opin. Genet. Dev. 1999; 9: 89-96Crossref PubMed Scopus (730) Google Scholar, 2Li G.M. Front. Biosci. 2003; 8: d997-d1017Crossref PubMed Scopus (68) Google Scholar, 3Modrich P. Lahue R. Annu. Rev. Biochem. 1996; 65: 101-133Crossref PubMed Scopus (1337) Google Scholar), demonstrating the importance in maintaining genome stability. The genome maintenance functions of MMR include repair of DNA replication errors (1Kolodner R.D. Marsischky G.T. Curr. Opin. Genet. Dev. 1999; 9: 89-96Crossref PubMed Scopus (730) Google Scholar, 3Modrich P. Lahue R. Annu. Rev. Biochem. 1996; 65: 101-133Crossref PubMed Scopus (1337) Google Scholar, 4Kunkel T.A. Erie D.A. Annu. Rev. Biochem. 2005; 74: 681-710Crossref PubMed Scopus (1023) Google Scholar), suppression of DNA recombination between two divergent sequences (5Harfe B.D. Jinks-Robertson S. Annu. Rev. Genet. 2000; 34: 359-399Crossref PubMed Scopus (505) Google Scholar, 6Myung K. Datta A. Chen C. Kolodner R.D. Nat. Genet. 2001; 27: 113-116Crossref PubMed Scopus (268) Google Scholar, 7Schofield M.J. Hsieh P. Annu. Rev. Microbiol. 2003; 57: 579-608Crossref PubMed Scopus (399) Google Scholar), and participation in DNA damage signaling to trigger cell cycle arrests and/or apoptosis (8Li G.M. Oncol. Res. 1999; 11: 393-400PubMed Google Scholar, 9Fishel R. Cancer Res. 2001; 61: 7369-7374PubMed Google Scholar, 10Stojic L. Brun R. Jiricny J. DNA Repair. 2004; 3: 1091-1101Crossref PubMed Scopus (333) Google Scholar). Among these functions, the correction of biosynthetic errors is best characterized. The molecular mechanism by which MMR corrects biosynthetic errors is conserved from bacteria to human cells. Using the methyl-directed MMR system in Escherichia coli as a model, essential components required for eukaryotic MMR have been identified to include MutSα (a heterodimer of MSH2-MSH6), MutSβ (a heterodimer of MSH2-MSH3), MutLα (a heterodimer of MLH1-PMS2 in mammalian or MLH1-PMS1 in yeast), proliferating cellular nuclear antigen (PCNA), replication protein A (RPA), EXO1, HMGB1, replication factor C (RFC), DNA polymerase δ, and DNA ligase I (Refs. 4Kunkel T.A. Erie D.A. Annu. Rev. Biochem. 2005; 74: 681-710Crossref PubMed Scopus (1023) Google Scholar and 11Zhang 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 (299) Google Scholar, and references therein). Recently, the human MMR reaction has been reconstituted using purified proteins (11Zhang 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 (299) Google Scholar, 12Constantin N. Dzantiev L. Kadyrov F.A. Modrich P. J. Biol. Chem. 2005; 280: 39752-39761Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). For 5′ nick-directed MMR, formation of a complex between mismatched DNA and MMR proteins MutSα or MutSβ, MutLα, EXO1, RPA, and HMGB1 initiates mismatch-provoked excision at the strand break (11Zhang 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 (299) Google Scholar). The excision reaction is terminated immediately after the removal of the mismatch in a manner dependent on MutLα and RPA (11Zhang 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 (299) Google Scholar). The resulting ssDNA gap is filled by DNA polymerase δ, and the repair is completed by DNA ligase I (11Zhang 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 (299) Google Scholar). For 3′ nick-directed MMR, the reaction additionally requires RFC and PCNA and is strongly enhanced by the addition of EXO1 (12Constantin N. Dzantiev L. Kadyrov F.A. Modrich P. J. Biol. Chem. 2005; 280: 39752-39761Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). RPA, an ubiquitous MMR component, has been shown to play important roles in both the excision and resynthesis reactions of MMR (11Zhang 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 (299) Google Scholar, 13Lin 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 (129) Google Scholar, 14Dzantiev 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 (200) Google Scholar, 15Genschel J. Modrich P. Mol. Cell. 2003; 12: 1077-1086Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 16Ramilo 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 (65) Google Scholar). RPA stimulates the processivity of EXO1 (11Zhang 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 (299) Google Scholar, 14Dzantiev 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 (200) Google Scholar, 15Genschel J. Modrich P. Mol. Cell. 2003; 12: 1077-1086Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar), protects the ssDNA gap generated during excision from attacks by nucleases, and facilitates the termination of MMR excision and repair DNA synthesis (11Zhang 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 (299) Google Scholar, 14Dzantiev 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 (200) Google Scholar, 15Genschel J. Modrich P. Mol. Cell. 2003; 12: 1077-1086Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 16Ramilo 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 (65) Google Scholar). RPA is additionally involved in other DNA repair pathways and DNA damage response (17Binz S.K. Sheehan A.M. Wold M.S. DNA Repair. 2004; 3: 1015-1024Crossref PubMed Scopus (239) Google Scholar, 18Sancar A. Lindsey-Boltz L.A. Unsal-Kacmaz K. Linn S. Annu. Rev. Biochem. 2004; 73: 39-85Crossref PubMed Scopus (2557) Google Scholar, 19Coverley D. Kenny M.K. Munn M. Rupp W.D. Lane D.P. Wood R.D. Nature. 1991; 349: 538-541Crossref PubMed Scopus (198) Google Scholar, 20Dianov G.L. Jensen B.R. Kenny M.K. Bohr V.A. Biochemistry. 1999; 38: 11021-11025Crossref PubMed Scopus (48) Google Scholar, 21Otterlei M. Warbrick E. Nagelhus T.A. Haug T. Slupphaug G. Akbari M. Aas P.A. Steinsbekk K. Bakke O. Krokan H.E. EMBO J. 1999; 18: 3834-3844Crossref PubMed Scopus (297) Google Scholar, 22Zou Y. Liu Y. Wu X. Shell S.M. J. Cell. Physiol. 2006; 208: 267-273Crossref PubMed Scopus (257) Google Scholar). During cellular responses to DNA damage, RPA, a heterotrimer composed of subunits of 70 (RPA1), 34 (RPA2), and 14 kDa (RPA3) (23Wold M.S. Annu. Rev. Biochem. 1997; 66: 61-92Crossref PubMed Scopus (1188) Google Scholar), can be hyper-phosphorylated by members of the phosphatidylinositol (PI) 3-kinase family (24Zernik-Kobak M. Vasunia K. Connelly M. Anderson C.W. Dixon K. J. Biol. Chem. 1997; 272: 23896-23904Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), which includes DNA-dependent protein kinase (DNA-PK), ATM, and ATR (17Binz S.K. Sheehan A.M. Wold M.S. DNA Repair. 2004; 3: 1015-1024Crossref PubMed Scopus (239) Google Scholar, 25Gately D.P. Hittle J.C. Chan G.K. Yen T.J. Mol. Biol. Cell. 1998; 9: 2361-2374Crossref PubMed Scopus (164) Google Scholar, 26Niu H. Erdjument-Bromage H. Pan Z.Q. Lee S.H. Tempst P. Hurwitz J. J. Biol. Chem. 1997; 272: 12634-12641Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 27Blackwell L.J. Borowiec J.A. Masrangelo I.A. Mol. Cell. Biol. 1996; 16: 4798-4807Crossref PubMed Scopus (114) Google Scholar). Phosphorylation occurs primarily within the N-terminal 33 residues of RPA2 (28Lee S.H. Kim D.K. J. Biol. Chem. 1995; 270: 12801-12807Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 29Henricksen L.A. Carter T. Dutta A. Wold M.S. Nucleic Acids Res. 1996; 24: 3107-3112Crossref PubMed Scopus (56) Google Scholar). Although RPA phosphorylation is not essential for nucleotide excision repair (30Ariza R.R. Keyse S.M. Moggs J.G. Wood R.D. Nucleic Acids Res. 1996; 24: 433-440Crossref PubMed Google Scholar, 31Pan Z.Q. Park C.H. Amin A.A. Hurwitz J. Sancar A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4636-4640Crossref PubMed Scopus (63) Google Scholar), its role and relevance in MMR remains unknown. In this study, we monitored the time-dependent association of several key MMR proteins in HeLa nuclear extracts with a biotin-streptavitin-bound mismatched DNA substrate, and demonstrate here that RPA, MSH2 (a subunit of human MutS heterodimers), MLH1 (a subunit of human MutL heterodimers), PCNA, and DNA polymerase δ bind to the heteroduplex in a sequential manner. Surprisingly, RPA binds to the DNA substrate at a time earlier than the known MMR initiation factors MutSα, MutLα, and PCNA and remains bound throughout the repair reaction. Additionally, we show that the functions of RPA in MMR are regulated by phosphorylation. Unphosphorylated RPA possesses a high DNA binding affinity and preferentially stimulates mismatch-provoked excision; phosphorylated RPA preferentially facilitates DNA resynthesis via reducing its DNA binding ability, allowing its displacement by DNA polymerases to make DNA template available for nucleotide polymerization. Preparations of Nuclear Extracts and Proteins—HeLa S3 cells were purchased from the National Cell Culture Center (Minneapolis, MN), and nuclear extracts were prepared as described (32Parsons R. Li G.M. Longley M.J. Fang W.H. Papadopoulos N. Jen J. de la Chapelle A. Kinzler K.W. Vogelstein B. Modrich P. Cell. 1993; 75: 1227-1236Abstract Full Text PDF PubMed Scopus (966) Google Scholar). Three different forms of human RPA were used in this study, the wild-type RPA and its hyperphosphorylated (RPA2D) and non-phosphorylated (RPA2A) isoforms (33Binz S.K. Lao Y. Lowry D.F. Wold M.S. J. Biol. Chem. 2003; 278: 35584-35591Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). These RPA isoforms were expressed in E. coli and purified essentially as described (34Henricksen L.A. Umbricht C.B. Wold M.S. J. Biol. Chem. 1994; 269: 11121-11132Abstract Full Text PDF PubMed Google Scholar). Recombinant MutSα, MutLα, PCNA, EXO1, RFC, and pol δ were expressed and purified as described (11Zhang 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 (299) Google Scholar). DNA-PK was purified from HeLa nuclear extracts essentially as described (35Chan D.W. Mody C.H. Ting N.S. Lees-Miller S.P. Biochem. Cell Biol. 1996; 74: 67-73Crossref PubMed Scopus (83) Google Scholar). DNA Substrate Preparation and MMR Assays—A 6.4-kb circular substrate containing either a G-T mismatch and a strand break 128 bp 5′ to the mismatch or a 171-nt gap was constructed as described (36Guo 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). The circular DNA substrates were digested with BspHI to produce linear duplexes with 5′ overhang sequences of 5′-CATG, which served as a template for 3′ biotin labeling (see Fig. 1) in the presence of Klenow DNA polymerase, dCTP, and biotin-dATP as described (37Gabrielsen O.S. Huet J. Methods Enzymol. 1993; 218: 508-525Crossref PubMed Scopus (43) Google Scholar). An otherwise identical linear homoduplex was similarly prepared. The biotinylated DNA substrates were incubated with streptavidin-Sepharose at 4 °C for 2 h. The resulting complex was used for MMR assay essentially as described (32Parsons R. Li G.M. Longley M.J. Fang W.H. Papadopoulos N. Jen J. de la Chapelle A. Kinzler K.W. Vogelstein B. Modrich P. Cell. 1993; 75: 1227-1236Abstract Full Text PDF PubMed Scopus (966) Google Scholar) with minor modifications. Briefly, the repair assay was performed in a 45-μl reaction containing 140 fmol of biotin-streptavidin-attached DNA heteroduplex and 250 μg of HeLa nuclear extracts. The repair reactions were incubated at 37 °C for an indicated time, on a rotating rack, and the DNA samples were recovered and digested with BspDI and HindIII to score for repair on agarose gel. To analyze mismatch-provoked excision intermediates, reactions were assembled identically to the MMR reaction but in the absence of dNTPs. After digestion with SspI, DNA excision products were fractionated through a denaturing 6% polyacrylamide gel. DNA samples were subjected to Southern blot analysis using a 32P-labeled probe (5′-ATTGTTCTGGATATTACC-3′) as described (38McCulloch S.D. Gu L. Li G.M. J. Biol. Chem. 2003; 278: 50803-50809Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Protein Pulldown Assay—To pull down proteins participating in MMR, 250 μg of HeLa nuclear extracts were incubated with 500 or 140 fmol of biotinylated heteroduplex or homoduplex DNA substrates attached to streptavidin at 37 °C for an indicated time under the repair conditions (32Parsons R. Li G.M. Longley M.J. Fang W.H. Papadopoulos N. Jen J. de la Chapelle A. Kinzler K.W. Vogelstein B. Modrich P. Cell. 1993; 75: 1227-1236Abstract Full Text PDF PubMed Scopus (966) Google Scholar). Reactions were terminated by the addition of 800 μl of a low salt washing buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% Nonidet P-40, 0.5% Triton X-100, and protease inhibitors (0.3 mg/ml benzamidine hydrochloride, 0.5 μg/ml of pepstatin A, 0.5 μg/ml of leupeptin, 0.5 μg/ml of antipain)). The beads were recovered by centrifugation and washed once each with a high salt washing buffer (same as the low salt washing buffer but with 500 mm NaCl) and the low salt washing buffer to remove nonspecifically bound proteins. Proteins that remained on the beads were eluted with the SDS gel loading buffer and separated by 10% SDS-polyacrylamide gels, followed by Western blot analysis. Mismatch-excision and DNA Gap-filling Assays—Excision assays were performed in 20-μl reactions containing 24 fmol of the 5′ G-T heteroduplex (see Fig. 1A, substrate I), 5 fmol of EXO1, 400 fmol of MutSα, 260 fmol of MutLα, 190 fmol of RFC, 290 fmol of homotrimer of PCNA, and 800 fmol of the indicated forms of RPA. The reactions were incubated at 37 °C for 10 min as described previously (11Zhang 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 (299) Google Scholar). DNA samples were recovered by phenol extraction and ethanol precipitation. Excision was scored by NheI digestion. DNA gap-filling assay was performed by incubating purified proteins (pol δ and one of RPA isoforms) with a circular DNA duplex containing a 171-nt gap (see Fig. 1A, substrate III). After incubation for the indicated times, DNA samples were digested with SspI, fractionated through a 6% polyacrylamide gel, and subjected to Southern blot analysis using a 32P-labeled probe (5′-AAAATTTAACGCGAATTTT-3′) as described (38McCulloch S.D. Gu L. Li G.M. J. Biol. Chem. 2003; 278: 50803-50809Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Stopped-flow Assays—The 21-mer (5′-GCTGAAGCAGAAGGCTTGCAA-3′) and its 5′-hexachlorofluorescein-labeled analog were synthesized by Gene Link (Hawthorne, NY) and purified as described (39Bao K.K. Skalka A.M. Wong I. J. Biol. Chem. 2002; 277: 12099-12108Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar). Concentrations were determined spectrophotometrically using extinction coefficients calculated according to Cantor et al. (40Cantor C.R. Warshaw M.M. Shapiro H. Biopolymers. 1970; 9: 1059-1077Crossref PubMed Scopus (880) Google Scholar). Real-time fluorescence changes were measured using a KinTek SF 2001 stopped-flow spectrophotometer (KinTek Instruments, State College, PA) with upgraded Hamamatsu light source/monochromator fitted with a 150-watt xenon arc lamp. Reactions containing the indicated RPA and oligonucleotides were maintained at a constant 37 °C with a Neslab RTE-111 refrigerated bath. Intrinsic protein fluorescence was excited at λex = 290 nm while monitoring total emission at wavelengths >325 nm using an Oriel 51960 filter. Hexachlorofluorescein fluorescence was excited at λex = 535 nm while monitoring total emission at wavelengths >560 nm using a Corion LG-560-F filter. Time courses were fitted by nonlinear least-square regression to a single of exponential function, y = A1 (1 − e−ktt) + C. Dynamic Protein-DNA Association during the MMR Process—A biotinylated linear DNA heteroduplex containing a G-T mismatch and a nicked 128 base pairs 5′ to the mismatch immobilized on streptavidin beads (Fig. 1A, substrate II) was constructed to use in pulldown assays during MMR. In HeLa nuclear extracts, the linear heteroduplex was efficiently repaired (Fig. 2A) with appearance of repair products after 6 min incubation at 37 °C. Mismatch-provoked excision assay under the condition of limited DNA synthesis (38McCulloch S.D. Gu L. Li G.M. J. Biol. Chem. 2003; 278: 50803-50809Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar) showed little excision before and at 1 min (Fig. 2B), as almost all DNA molecules remained as nicked substrate (second band from top) and a small amount of directly ligated side product (top band). Onset of bands corresponding to smaller excision products indicated the initiation of mismatch-provoked excision at 2 min. By 4 min, extensive excision was observed. However, as little repair products were detected at 6 min in the presence of dNTPs (Fig. 2A), DNA resynthesis past the mismatch required at least 6 min for the majority of molecules. To correlate the repair progress with the repair proteins assembled on the heteroduplex substrate in real time, the resin-bound substrate was used to pull down proteins involved in MMR (Fig. 2F). To ensure recovery of sufficient proteins for analysis, 500 fmol of the streptavidin-bound heteroduplex (3.5-fold more compared with the repair reaction) was incubated with 250 μg of HeLa nuclear extract under the repair conditions. Proteins pulled down at various times were analyzed by Western blotting to profile proteins bound during the course of the MMR reaction (Fig. 2C). As expected, MMR initiation components, MSH2, MLH1, and PCNA, were detected 0.5 min after incubation at 37 °C, the amounts of these proteins apparently increased, particularly after the mismatch had been removed (at 6 min). However, this increase in the level of MSH2 and MLH1 may not necessarily reflect their requirement for the late steps of the reaction. Instead, it is likely due to their association with unrepaired heteroduplexes, where these proteins could spread along the nick-ligated and ends-blocked molecules after loading to the mismatch. Due to the poor specificity of the commercial antibodies, EXO1 was not directly detectable; however, the appearance of pol δ binding at ∼2 min (Fig. 2C) implied that excision must have occurred at or prior to 2 min, consistent with the excision profile shown in Fig. 2B. When antibodies against the 70-(RPA1) and 34-kDa (RPA2) subunits of RPA were used to monitor the status of RPA during MMR, two surprising phenomena were observed: (i) RPA bound to the DNA substrate earlier than MSH2, MLH1, and PCNA (Fig. 2C), suggesting involvement of RPA in the initiation step of MMR; (ii) the RPA2 antibody detected two species (a fast-migrating and a slow-migrating) of RPA2, with the fast migrating species appearing at the earlier steps of the reaction and the slow migrating one covering the later steps of the reaction (Fig. 2C). The two RPA2 species are reminiscent of phosphorylated (slow-migrating) and unphosphorylated (fast-migrating) RPA2 observed during cellular response to DNA damage in published reports (41Carty M.P. Zernik-Kobak M. McGrath S. Dixon K. EMBO J. 1994; 13: 2114-2123Crossref PubMed Scopus (169) Google Scholar, 42Liu V.F. Weaver D.T. Mol. Cell. Biol. 1993; 13: 7222-7231Crossref PubMed Scopus (188) Google Scholar). This was confirmed in experiments supplemented with 4 μm wortmannin, a potent inhibitor of PI 3-kinases, where the fast migrating species was no longer detectable (data not shown), suggesting strongly that the slow migrating RPA2 was likely formed from the fast migrating species via phosphorylation by PI 3-kinases (43Brush G.S. Anderson C.W. Kelly T.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12520-12524Crossref PubMed Scopus (156) Google Scholar, 44Brush G.S. Morrow D.M. Hieter P. Kelly T.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15075-15080Crossref PubMed Scopus (163) Google Scholar, 45Dutta A. Stillman B. EMBO J. 1992; 11: 2189-2199Crossref PubMed Scopus (224) Google Scholar). Comparison with the excision profile (Fig. 2B) revealed that unphosphorylated RPA, appearing from 0 to 6 min (Fig. 2C), was associated with excision initiation and complete removal of the mismatch (from 0 to 6 min, see Fig. 2B), whereas phosphorylated RPA appeared just before the binding of pol δ to the DNA substrate and accumulated throughout the resynthesis phase of the reaction (Fig. 2C). These results, therefore, suggest that unphosphorylated RPA is required for the excision step, whereas phosphorylated RPA is necessary for the resynthesis step of MMR. In control experiments performed with an otherwise identical, nicked DNA substrate without the mismatch, little MSH2 and MLH1 binding to the homoduplex substrate was detected initially as expected (MLH1 was detected only at a much later reaction time, Fig. 2D). The binding profile of other protein components to the nicked homoduplex also appeared similar. However, RPA2 phosphorylation in the homoduplex reaction appeared much faster as complete phosphorylation was observed at 4 min as compared with the 10 min required for the heteroduplex reaction (Fig. 2, C and D). These results are repeatable and likely reflect the known differences in the length of the ssDNA gap generated in these two reactions (see “Discussion” for details). Significantly, in either case, RPA2 phosphorylation consistently coincided with or slightly preceded pol δ binding, suggesting again that RPA2 phosphorylation may play a critical role in triggering DNA synthesis, even in the homoduplex reaction where a significant amount of DNA synthesis unrelated to MMR has been reported previously (46Holmes Jr., J. Clark S. Modrich P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5837-5841Crossref PubMed Scopus (336) Google Scholar, 47Thomas D.C. Roberts J.D. Kunkel T.A. J. Biol. Chem. 1991; 266: 3744-3751Abstract Full Text PDF PubMed Google Scholar). Additionally, although RPA (RPA2) was readily pulled down by both the nicked hetero- and homo-duplexes, identical constructs lacking the nick were poor substrates for RPA binding (Fig. 2E). The requirement for a nick in the substrate implies that RPA likely binds to the DNA substrate at or via the strand break. Unphosphorylated RPA Promotes the Excision Reaction, but Phosphorylated RPA Facilitates the Resynthesis in MMR—The human MMR system has recently been reconstituted using purified proteins, and it can be subdivided into the excision and resynthesis reactions (11Zhang 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 (299) Google Scholar). To determine the role of RPA phosphorylation in MMR, two RPA isoforms that functionally mimic phosphorylated and unphosphorylated RPA (48Vassin V.M. Wold M.S. Borowiec J.A. Mol. Cell. Biol. 2004; 24: 1930-1943Crossref PubMed Scopus (140) Google Scholar) were used in the reconstituted excision and resynthesis reactions. These RPA isoforms were obtained by substituting the phosphorylation sites of wild-type RPA2 with aspartate (RPA2D) and alanine (RPA2A), respectively (33Binz S.K. Lao Y. Lowry D.F. Wold M.S. J. Biol. Chem. 2003; 278: 35584-35591Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar), and the individual RPA trimers containing RPA2D and RPA2A were referred to as RPA2D or RPA2A here, respectively. The mobility of RPA2A and RPA2D in SDS-PAGE correlated well with that of unphosphorylated and hyperphosphorylated RPA2, respectively (Fig. 1B). Fig. 3A shows the excision assay in the reconstituted system in the presence of these different RPA isoforms. Relative to the reaction with the phosphorylated isoform, RPA2D, 2.2- and 1.7-fold higher excision activities were observed in reactions using unphosphorylated wild-type RPA (RPAWT) and the unphosphorylated isoform, RPA2A. These results suggest a preferential role for the unphosphorylated form of RPA in stimulating the mismatch-provoked excision phase of the reaction. To explore the role of RPA phosphorylation in the resynthesis step of MMR, gap-filling activity was assayed in a defined system containing purified pol δ and either phosphorylated or unphosphorylated RPA using a circular DNA substrate with a 171-nt single-stranded gap (Fig. 1A, III) to mimic the MMR excision product. Resynthesis products were detected by Southern blot analysis (Fig. 3B) and the percentage of resynthesis products are plotted as a function of time in Fig. 3C. As previously observed, pol δ by itself can efficiently fill the 171-nt gap (11Zhang 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 (299) Google Scholar). The addition of RPAWT to the reaction resulted in a severe, greater than 4-fold reduction in the rate of gap-filling (Fig. 3C). Gap filling activity was also similarly inhibited in reactions supplemented with RPA2A. However, a greater than 2-fold recovery of gap filling activity was observed when RPA2D was substituted for RPAWT or RPA2A, which partially mitigated but did not completely abolish the overall inhibitory effect of adding RPA (Fig. 3, B and C). These results suggest a preferential role for phosphorylated RPA during DNA resynthesis. To demonstrate that the faster gap filling observed with RPA2D was due to RPA phosphorylation, gap filling assay was conducted using RPAWT in the presence or absence of purified DNA-PK, which has been shown to be capable of phosphorylating RPA in vitro (17Binz S.K. Sheehan A.M. Wold M.S. DNA Repair. 2004; 3: 1015-1024Crossref PubMed Scopus (239) Google Scholar, 26Niu H. Erdjument-Bromage H. Pan Z.Q. Lee S.H. Tempst P. Hurwitz J. J. Biol. Chem. 1997; 272: 12634-12641Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Consistent with previous observations, we found that DNA-PK purified from HeLa cells (see Fig. 1B) could efficiently phosphorylate recombinant RPA (data not shown), although we do not know whether or not RPA was hyperphosphorylated in this manner. As shown in Fig. 3D, addition of DNA-PK to the reaction containing pol δ and RPAWT stimulated the gap filling activity by more than 1.7-fold, and the stimulation by DNA-PK was abolished in the presence of wortmannin, a potent inhibitor of" @default.
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• W2090410902 date "2006-08-01" @default.
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• W2090410902 title "Regulation of Replication Protein A Functions in DNA Mismatch Repair by Phosphorylation" @default.
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