The Evolutionarily Conserved Zinc Finger Motif in the Largest Subunit of Human Replication Protein A Is Required for DNA Replication and Mismatch Repair but Not for Nucleotide Excision Repair
1998; Elsevier BV; Volume: 273; Issue: 3 Linguagem: Inglês
10.1074/jbc.273.3.1453
ISSN1083-351X
AutoresYi‐Ling Lin, Mahmud K. K. Shivji, Clark Chen, Richard D. Kolodner, Richard D. Wood, Anindya Dutta,
Tópico(s)RNA Research and Splicing
ResumoThe largest subunit of the replication protein A (RPA) contains an evolutionarily conserved zinc finger motif that lies outside of the domains required for binding to single-stranded DNA or forming the RPA holocomplex. In previous studies, we showed that a point mutation in this motif (RPAm) cannot support SV40 DNA replication. We have now investigated the role of this motif in several steps of DNA replication and in two DNA repair pathways. RPAm associates with T antigen, assists the unwinding of double-stranded DNA at an origin of replication, stimulates DNA polymerases α and δ, and supports the formation of the initial short Okazaki fragments. However, the synthesis of a leading strand and later Okazaki fragments is impaired. In contrast, RPAm can function well during the incision step of nucleotide excision repair and in a full repair synthesis reaction, with either UV-damaged or cisplatin-adducted DNA. Two deletion mutants of the Rpa1 subunit (eliminating amino acids 1–278 or 222–411) were not functional in nucleotide excision repair. We report for the first time that wild type RPA is required for a mismatch repair reaction in vitro. Neither the deletion mutants nor RPAm can support this reaction. Therefore, the zinc finger of the largest subunit of RPA is required for a function that is essential for DNA replication and mismatch repair but not for nucleotide excision repair. The largest subunit of the replication protein A (RPA) contains an evolutionarily conserved zinc finger motif that lies outside of the domains required for binding to single-stranded DNA or forming the RPA holocomplex. In previous studies, we showed that a point mutation in this motif (RPAm) cannot support SV40 DNA replication. We have now investigated the role of this motif in several steps of DNA replication and in two DNA repair pathways. RPAm associates with T antigen, assists the unwinding of double-stranded DNA at an origin of replication, stimulates DNA polymerases α and δ, and supports the formation of the initial short Okazaki fragments. However, the synthesis of a leading strand and later Okazaki fragments is impaired. In contrast, RPAm can function well during the incision step of nucleotide excision repair and in a full repair synthesis reaction, with either UV-damaged or cisplatin-adducted DNA. Two deletion mutants of the Rpa1 subunit (eliminating amino acids 1–278 or 222–411) were not functional in nucleotide excision repair. We report for the first time that wild type RPA is required for a mismatch repair reaction in vitro. Neither the deletion mutants nor RPAm can support this reaction. Therefore, the zinc finger of the largest subunit of RPA is required for a function that is essential for DNA replication and mismatch repair but not for nucleotide excision repair. Human replication protein A (RPA) 1The abbreviations used are: RPA, replication protein A; RF-C, replication factor C; PCNA, proliferating cell nuclear antigen; T Ag, T antigen; DTT, dithiothreitol; ssDNA, single-stranded DNA; h, human; Ab, antibody; kb, kilobase pair(s). 1The abbreviations used are: RPA, replication protein A; RF-C, replication factor C; PCNA, proliferating cell nuclear antigen; T Ag, T antigen; DTT, dithiothreitol; ssDNA, single-stranded DNA; h, human; Ab, antibody; kb, kilobase pair(s). is a stable complex of three subunits of Rpa1 (70 kDa), Rpa2 (34 kDa), and Rpa3 (13 kDa). It was first purified from HeLa and 293 cell extracts as an essential component of SV40 DNA replication (1Wobbe C.R. Weissbach L. Borowiec J.A. Dean F.B. Murakami Y. Bullock P. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1834-1838Crossref PubMed Scopus (258) Google Scholar, 2Wold M.S. Kelly T. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2523-2527Crossref PubMed Scopus (369) Google Scholar, 3Fairman M.P. Stillman B. EMBO J. 1988; 7: 1211-1218Crossref PubMed Scopus (295) Google Scholar). Binding to single-stranded DNA with high affinity is a hallmark of human replication protein A, and purification of this protein largely takes advantage of this property (3Fairman M.P. Stillman B. EMBO J. 1988; 7: 1211-1218Crossref PubMed Scopus (295) Google Scholar, 4Henricksen L.A. Umbricht C.B. Wold M.S. J. Biol. Chem. 1994; 269: 11121-11132Abstract Full Text PDF PubMed Google Scholar). Rpa1 is the most well characterized subunit among the complex. Rpa1 alone confers the high affinity for single-stranded DNA (5Wold M.S. Weinberg D.H. Virshup D.M. Li J.J. Kelly T.J. J. Biol. Chem. 1989; 264: 2801-2809Abstract Full Text PDF PubMed Google Scholar, 6Kenny M.K. Schlegel U. Furneaux H. Hurwitz J. J. Biol. Chem. 1990; 265: 7693-7700Abstract Full Text PDF PubMed Google Scholar), yet only the whole heterotrimeric complex is active in supporting DNA replication (4Henricksen L.A. Umbricht C.B. Wold M.S. J. Biol. Chem. 1994; 269: 11121-11132Abstract Full Text PDF PubMed Google Scholar, 7Erdile L.F. Heyer W.D. Kolodner R. Kelly T.J. J. Biol. Chem. 1991; 266: 12090-12098Abstract Full Text PDF PubMed Google Scholar). Rpa1 can be subdivided into 3 domains: an N-terminal domain of protein-protein interaction, a central domain for DNA interaction, and a C-terminal domain for complex formation with Rpa2–3 (8Lin Y.L. Chen C. Keshav K.F. Winchester E. Dutta A. J. Biol. Chem. 1996; 271: 17190-17198Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 9Gomes X.V. Wold M.S. Biochemistry. 1996; 35: 10558-10568Crossref PubMed Scopus (90) Google Scholar). RPA is highly conserved throughout evolution. Homologous heterotrimeric single-stranded DNA-binding proteins have been identified in nearly all eukaryotes examined (10Heyer W.D. Rao M.R. Erdile L.F. Kelly T.J. Kolodner R.D. EMBO J. 1990; 9: 2321-2329Crossref PubMed Scopus (157) Google Scholar, 11Adachi Y. Laemmli U.K. J. Cell Biol. 1992; 119: 1-15Crossref PubMed Scopus (120) Google Scholar, 12Atrazhev A. Zhang S. Grosse F. Eur. J. Biochem. 1992; 210: 855-865Crossref PubMed Scopus (26) Google Scholar, 13Brill S.J. Stillman B. Nature. 1989; 342: 92-95Crossref PubMed Scopus (189) Google Scholar, 14Brown G.W. Melendy T.E. Ray D.S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10227-10231Crossref PubMed Scopus (37) Google Scholar, 15Georgaki A. Strack B. Podust V. Hubscher U. FEBS Lett. 1992; 308: 240-244Crossref PubMed Scopus (87) Google Scholar, 16Mitsis P.G. Kowalczykowski S.C. Lehman I.R. Biochemistry. 1993; 32: 5257-5266Crossref PubMed Scopus (56) Google Scholar, 17Nakagawa M. Tsukada S. Soma T. Shimizu Y. Miyake S. Iwamatsu A. Sugiyama H. Nucleic Acids Res. 1991; 19: 4292Crossref PubMed Scopus (13) Google Scholar). All genes reveal significant homology between species at the amino acid level. All of the known Rpa1 homologs contain a conserved putative C4-type zinc finger motif in the C-terminal third of the protein (7Erdile L.F. Heyer W.D. Kolodner R. Kelly T.J. J. Biol. Chem. 1991; 266: 12090-12098Abstract Full Text PDF PubMed Google Scholar, 11Adachi Y. Laemmli U.K. J. Cell Biol. 1992; 119: 1-15Crossref PubMed Scopus (120) Google Scholar, 18Ishiai M. Sanchez J.P. Amin A.A. Murakami Y. Hurwitz J. J. Biol. Chem. 1996; 271: 20868-20878Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 19Brown G.W. Hines J.C. Fisher P. Ray D.S. Mol. Biochem. Parasitol. 1994; 63: 135-142Crossref PubMed Scopus (27) Google Scholar). In fact, a point mutation that disrupts the putative zinc finger eliminates DNA replication, confirming the importance of this highly conserved motif (8Lin Y.L. Chen C. Keshav K.F. Winchester E. Dutta A. J. Biol. Chem. 1996; 271: 17190-17198Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 20Wold M.S. Annu. Rev. Biochem. 1997; 66: 61-91Crossref PubMed Scopus (1179) Google Scholar). RPA is essential for other cellular DNA metabolism involving single-stranded DNA intermediates. This includes DNA repair (21Coverley 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, 22He Z. Henricksen L.A. Wold M.S. Ingles C.J. Nature. 1995; 374: 566-569Crossref PubMed Scopus (374) Google Scholar) and homologous recombination (10Heyer W.D. Rao M.R. Erdile L.F. Kelly T.J. Kolodner R.D. EMBO J. 1990; 9: 2321-2329Crossref PubMed Scopus (157) Google Scholar, 23Alani E. Thresher R. Griffith J.D. Kolodner R.D. J. Mol. Biol. 1992; 227: 54-71Crossref PubMed Scopus (151) Google Scholar, 24Longhese M.P. Neecke H. Paciotti V. Lucchini G. Plevani P. 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Biol. 1991; 11: 2108-2115Crossref PubMed Google Scholar) and DNA polymerase α-primase complex to synthesize the first Okazaki fragment (29Melendy T. Stillman B. J. Biol. Chem. 1993; 268: 3389-3395Abstract Full Text PDF PubMed Google Scholar). hRPA is also important in a later elongation step where it stimulates both DNA polymerase α and DNA polymerase δ activity and cannot be substituted for byEscherichia coli SSB (30Tsurimoto T. Stillman B. EMBO J. 1989; 8: 3883-3889Crossref PubMed Scopus (189) Google Scholar). Besides the interaction with repair proteins (XPA, XPG, and XPF see below) and replication proteins, hRPA has also been implicated in interactions with transcription factors p53 (31Dutta A. Ruppert J.M. Aster J.C. Winchester E. Nature. 1993; 365: 79-82Crossref PubMed Scopus (331) Google Scholar, 32Li R. Botchan M.R. Cell. 1993; 73: 1207-1221Abstract Full Text PDF PubMed Scopus (275) Google Scholar), Gal4, VP16 (33He Z. Brinton B.T. Greenblatt J. Hassell J.A. Ingles C.J. Cell. 1993; 73: 1223-1232Abstract Full Text PDF PubMed Scopus (182) Google Scholar), RNA polymerase holoenzyme (34Maldonado E. Shiekhattar R. Sheldon M. Cho H. Drapkin R. Rickert P. Lees E. Anderson C.W. Linn S. Reinberg D. Nature. 1996; 381: 86-89Crossref PubMed Scopus (306) Google Scholar), and recombination protein RAD52 (35Smith J. Rothstein R. Mol. Cell. Biol. 1995; 15: 1632-1641Crossref PubMed Scopus (106) Google Scholar, 36Firmenich A.A. Elias-Arnanz M. Berg P. Mol. Cell. Biol. 1995; 15: 1620-1631Crossref PubMed Google Scholar, 37Park M.S. Ludwig D.L. Stigger E. Lee S.H. J. Biol. Chem. 1996; 271: 18996-19000Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). Since DNA repair reactions generally involve DNA synthesis to form a repair patch, it is perhaps not surprising that hRPA takes part in DNA repair and can modulate the repair synthesis step (38Shivji M.K. Podust V.N. Hubscher U. Wood R.D. Biochemistry. 1995; 34: 5011-5017Crossref PubMed Scopus (239) Google Scholar). However, the most important function of RPA is at an earlier stage of nucleotide excision repair (21Coverley 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, 39Coverley D. Kenny M.K. Lane D.P. Wood R.D. Nucleic Acids Res. 1992; 20: 3873-3880Crossref PubMed Scopus (137) Google Scholar), and analysis of nicking of UV-irradiated plasmid DNA during excision repair revealed that RPA is necessary for incision formation (40Shivji K.K. Kenny M.K. Wood R.D. Cell. 1992; 69: 367-374Abstract Full Text PDF PubMed Scopus (732) Google Scholar). RPA is an essential factor for dual incision of damaged DNA with purified human protein components (41Aboussekhra A. Biggerstaff M. Shivji M.K. Vilpo J.A. Moncollin V. Podust V.N. Protic M. Hubscher U. Egly J.M. Wood R.D. Cell. 1995; 80: 859-868Abstract Full Text PDF PubMed Scopus (750) Google Scholar, 42Mu D. Hsu D.S. Sancar A. J. Biol. Chem. 1996; 271: 8285-8294Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). The Rpa1 and Rpa2 subunits of hRPA interact with the nucleotide excision repair protein XPA, probably to increase the efficiency of damage recognition, and this would be expected to modulate the excision reaction (22He Z. Henricksen L.A. Wold M.S. Ingles C.J. Nature. 1995; 374: 566-569Crossref PubMed Scopus (374) Google Scholar, 43Li L. Lu X. Peterson C.A. Legerski R.J. Mol. Cell. Biol. 1995; 15: 5396-5402Crossref PubMed Scopus (227) Google Scholar, 44Matsuda T. Saijo M. Kuraoka I. Kobayashi T. Nakatsu Y. Nagai A. Enjoji T. Masutani C. Sugasawa K. Hanaoka F. Yasui A. Tanaka K. J. Biol. Chem. 1995; 270: 4152-4157Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). In addition, RPA can also modulate the efficiency of cleavage by the incision enzyme XPG and ERCC1-XPF (45Matsunaga T. Park C.H. Bessho T. Mu D. Sancar A. J. Biol. Chem. 1996; 271: 11047-11050Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). A role for hRPA in general mismatch repair in eukaryotes has not been defined. E. coli SSB protein is required in a reconstituted system of mismatch repair with purified bacterial proteins (46Lahue R.S. Au K.G. Modrich P. Science. 1989; 245: 160-164Crossref PubMed Scopus (446) Google Scholar). It therefore seems reasonable that hRPA could be involved in eukaryotic mismatch repair. To help dissect the role of RPA in DNA replication and repair, we have taken advantage of three defined mutants in the Rpa1 subunit (8Lin Y.L. Chen C. Keshav K.F. Winchester E. Dutta A. J. Biol. Chem. 1996; 271: 17190-17198Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). All of the Rpa1 mutants form a stable heterotrimeric complex and show a similar extent of binding to single-stranded DNA under physiological conditions. The present work examines their ability to function during defined steps in DNA replication, nucleotide excision repair, and mismatch repair. The experiments reveal requirements for intact domains in Rpa1 that differ depending on the DNA transaction, suggesting that specific interactions take place during each of the three DNA replication and repair processes examined. The plasmid expressing wild type and various RPA mutants were described in a previous report (8Lin Y.L. Chen C. Keshav K.F. Winchester E. Dutta A. J. Biol. Chem. 1996; 271: 17190-17198Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). The proteins were named according to the Rpa1 mutant present in the complex. The proteins were expressed and purified as described (4Henricksen L.A. Umbricht C.B. Wold M.S. J. Biol. Chem. 1994; 269: 11121-11132Abstract Full Text PDF PubMed Google Scholar). The baculovirus expressing DNA polymerase α/primase subunits were generous gifts from Drs. Teresa Wang and Ellen Fanning. DNA polymerase α/primase was expressed in Hi-5 cells with baculovirus infection and purified by immunoaffinity column chromatography with monoclonal antibody SJK-237-71 specific for the large subunit p180 (47Copeland W.C. Wang T.S. J. Biol. Chem. 1993; 268: 11028-11040Abstract Full Text PDF PubMed Google Scholar). T Ag was also purified from baculovirus-infected Hi-5 cells by immunoaffinity column chromatography with monoclonal Ab Pab 419 (48Lanford R.E. Virology. 1988; 167: 72-81Crossref PubMed Scopus (105) Google Scholar). Polymerase δ was purified from calf thymus in four steps (DEAE-cellulose, phenyl-Sepharose, S-Sepharose, and Mono-Q chromatography). 2T. Tsurimoto, personal communication. The conditions in DEAE-cellulose and phenyl-Sepharose columns were as described (49Lee M.Y. Tan C.K. Downey K.M. So A.G. Biochemistry. 1984; 23: 1906-1913Crossref PubMed Scopus (174) Google Scholar). The active fractions from phenyl-Sepharose eluate were pooled and loaded onto an S-Sepharose column (5 mg/ml bed) pre-equilibrated with buffer D (20 mm potassium phosphate, pH 7.2, 0.5 mm EDTA, 0.1 mm EGTA, 0.5 μg/ml leupeptin, 0.2 mm phenylmethylsulfonyl fluoride, 1 mm DTT, 20% glycerol). After the column was washed with 2–3 bed volumes of buffer D containing 0.1 m KCl, DNA polymerase δ activity was eluted stepwise with a KCl gradient from 0.1 to 1 m in the same buffer. Active fractions were pooled and dialyzed against buffer E (25 mm Tris-HCl, pH 8.0, 0.025 m NaCl, 1 mm EDTA, 1 mm DTT, 0.1 mm phenylmethylsulfonyl fluoride, 10% glycerol, 0.01% Nonidet P-40, 2 μg/ml leupeptin) containing 20% sucrose. The dialyzed enzyme was loaded onto a Mono-Q column pre-equilibrated with buffer E. After washing with 5 bed volumes with buffer E, polymerase δ activity was eluted with a NaCl linear gradient from 0.025 to 0.5m in the same buffer. PCNA-stimulated DNA polymerase activity eluted at 0.2 m NaCl fractions just prior to the protein peak. The resulting polymerase can be stimulated 10-fold by PCNA on a poly(dA)/oligo(dT) template and has no detectable activity on a primed M13 ssDNA template unless PCNA and RF-C were both present. Baculoviruses expressing subunits of human RF-C and calf thymus RF-C protein were the generous gift of V. Podust and E. Fanning (Vanderbilt University) (50Podust V.N. Fanning E. J. Biol. Chem. 1997; 272: 6303-6310Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Replication of a plasmid (pSV010) containing SV40 origin of DNA replication was carried out with 293 cell extracts depleted of RPA (1Wobbe C.R. Weissbach L. Borowiec J.A. Dean F.B. Murakami Y. Bullock P. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1834-1838Crossref PubMed Scopus (258) Google Scholar) and supplemented with various bacterially expressed recombinant RPA holocomplexes as described previously (4Henricksen L.A. Umbricht C.B. Wold M.S. J. Biol. Chem. 1994; 269: 11121-11132Abstract Full Text PDF PubMed Google Scholar). The products were linearized with HindIII and analyzed on a 0.8% alkaline agarose gel. The gel was fixed in 8% (w/v) trichloroacetic acid after electrophoresis and dried. The incorporation of [α-32P]dCMP was determined using the DE81 paper and scintillation counting (51Dutta A. Winchester E. Pagano M. Cell Cycle-Materials and Methods. Springer-Verlag New York Inc., NY1995: 175-185Google Scholar). The assay is essentially based on previous reports (52Bullock P.A. Seo Y.S. Hurwitz J. Mol. Cell. Biol. 1991; 11: 2350-2361Crossref PubMed Scopus (80) Google Scholar, 53Denis D. Bullock P.A. Mol. Cell. Biol. 1993; 13: 2882-2890Crossref PubMed Scopus (29) Google Scholar). Reaction mixtures (50 μl) contained 7 mm MgCl2, 0.5 mmDTT, 4 mm ATP, 40 mm creatine phosphate, 30 mm HEPES (pH 7.8), 0.2 units of creatine kinase, 0.6 μg of supercoiled SV40 origin-containing plasmid (pSV010), 0.84 μg of T Ag, 1.4 μg of recombinant RPA preparations as indicated, and RPA-depleted 293 cell lysate. Reaction mixtures were preincubated for 45 min at 37 °C in the absence of T Ag to lower T Ag-independent labeling of form II DNA and then further incubated for 15 min after the addition of T Ag. Reaction mixtures were pulse-labeled for 1 min by the addition of 3.5 μl of a solution containing [α-32P]dCTP (final concentration in the complete reaction, 1 μm, 300 cpm/fmol) dATP, dGTP, and dTTP (final concentration of 100 μm each) and CTP, GTP, and UTP (final concentration of 200 μm each). The reactions were terminated by addition of 30 μl of stop solution (60 mmEDTA, 0.3% (w/v) SDS, 2 mg/ml of Pronase) and incubated at 37 °C for another 30 min. The products were extracted with phenol/chloroform followed by ethanol precipitation, boiled for 4 min in an equal volume of formamide loading buffer, and analyzed by electrophoresis through 10% polyacrylamide gels containing 8 m urea. The gel was fixed in 10% acetic acid, 10% methanol for 15 min, dried, and analyzed by radiography. The origin unwinding reaction was carried out in 20-μl volumes containing unwinding buffer (30 mm HEPES (pH 8.0), 7 mm MgCl2, 4 mm ATP, 0.5 mm DTT, 0.1 mg/ml bovine serum albumin, 40 mm creatine phosphate, and 0.8 units of creatine phosphokinase) in the presence of 72 ng of T Ag, 0.2 μg ofSau3A-digested carrier λ DNA, 50–60 ng of radiolabeledHindIII-SphI fragment of pSV010, and the indicated amounts of recombinant RPA (54Goetz G.S. Dean F.B. Hurwitz J. Matson S.W. J. Biol. Chem. 1988; 263: 383-392Abstract Full Text PDF PubMed Google Scholar, 55Dutta A. Stillman B. EMBO J. 1992; 11: 2189-2199Crossref PubMed Scopus (223) Google Scholar). Reaction was carried out at 37 °C for 2 h and terminated by adding 10 μl of stop solution (described above) followed by continued incubation at 37 °C for 30 min. The products were extracted with phenol/chloroform followed by ethanol precipitation and then analyzed by electrophoresis through 6% acrylamide gels. The assay was carried out as described previously (56Dornreiter I. Erdile L.F. Gilbert I.U. von Winkler D. Kelly T.J. Fanning E. EMBO J. 1992; 11: 769-776Crossref PubMed Scopus (285) Google Scholar). Briefly, the wells of a 96-well microtiter plate were coated overnight with excess (1 μg) recombinant RPA proteins or E. coli single-stranded DNA-binding protein. The wells were then blocked by incubation for 2 h with 3% bovine serum albumin in phosphate-buffered saline. The indicated amounts of purified T Ag were added in 50-μl volumes of blocking solution for 2 h. After washing, bound T Ag was detected by Pab 419 monoclonal antibody (diluted 1:1000 in blocking solution), peroxidase-conjugated rabbit anti-mouse antibody (diluted 1:1000 in blocking solution), and the chromogenic substrate 2,2-azino-bis-(3-ethyl-benzothiazoline-6-sulfonic acid) (Sigma). DNA polymerase α stimulation reactions were carried out in 20-μl volumes containing 30 nm M13 ssDNA, 90 nm sequencing primer (U. S. Biochemical Corp.), 20 mm Tris acetate (pH 7.3), 5 mm magnesium acetate, 20 mm potassium acetate, 1 mm DTT, 0.1 mg/ml bovine serum albumin, 1 mmATP, 0.1 mm dATP, dGTP, TTP, and 0.025 mm dCTP (1000 cpm/pmol). DNA polymerase α was added after a 10-min preincubation period with RPA, and the reaction was continued for another 10 min in 37 °C. The incorporation of [α-32P]dCMP was determined by DE81 paper method (51Dutta A. Winchester E. Pagano M. Cell Cycle-Materials and Methods. Springer-Verlag New York Inc., NY1995: 175-185Google Scholar). DNA polymerase δ stimulation reactions were carried out in 25-μl volumes as described (57Podust V.N. Georgaki A. Strack B. Hubscher U. Nucleic Acids Res. 1992; 20: 4159-4165Crossref PubMed Scopus (73) Google Scholar). The incorporation of [α-32P]dCMP was determined by DE81 paper, and the products were analyzed on a 1% alkaline agarose gel. The gel was processed as mentioned above. The plasmids used were derivatives of pUC vectors, the 3.0-kb pBluescript KS+(Stratagene), and the 3.7-kb pHM14 (58Rydberg B. Spurr N. Karran P. J. Biol. Chem. 1990; 265: 9563-9569Abstract Full Text PDF PubMed Google Scholar). pBluescript KS+was UV-irradiated (450 J/m2). Both plasmids were treated with E. coli Nth protein, and closed circular DNA was isolated from cesium chloride and sucrose gradients (59Wood R.D. Biggerstaff M. Shivji M.K.K. Methods. 1995; 7: 163-175Crossref Scopus (70) Google Scholar). Reaction mixtures (50 μl) contained 48 μg of human CFII fraction protein depleted of RPA and PCNA (40Shivji K.K. Kenny M.K. Wood R.D. Cell. 1992; 69: 367-374Abstract Full Text PDF PubMed Scopus (732) Google Scholar), 25 ng of recombinant PCNA, 250 ng of irradiated pBluescript KS+, 250 ng of non-irradiated pHM14, 45 mm HEPES-KOH (pH 7.8), 70 mm KCl, 7.4 mm MgCl2, 0.9 mm dithiothreitol (DTT), 0.4 mm EDTA, 20 μm each of dGTP, dCTP, and TTP, 8 μm dATP, 74 kBq of [α-32P]dATP (110 TBq/mmol), 2 mm ATP, 22 mm phosphocreatine (di-Tris salt), 2.5 μg of creatine phosphokinase, 3.4% glycerol, 18 μg of bovine serum albumin and were incubated at 30 °C for 3 h. Plasmid DNA was purified from the reaction mixtures, linearized withBamHI, and loaded on a 1% agarose gel containing 0.3 μg/ml ethidium bromide. Data were analyzed by autoradiography with intensifying screens, densitometry, and liquid scintillation counting of the excised bands. Covalently closed circular DNA containing a single 1,3-intrastrand d(GpTpG)-cisplatin cross-link (Pt-GTG) was prepared as described (60Moggs J.G. Yarema K.J. Essigmann J.M. Wood R.D. J. Biol. Chem. 1996; 271: 7177-7186Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). This DNA substrate was used to analyze the dual incision process of nucleotide excision repair which leads to the excision of characteristically sized platinated oligomers 24–32 nucleotides in length. Each 150-μl reaction mixture contained 144 μg of HeLa cell CFII fraction protein, depleted of RPA and PCNA (40Shivji K.K. Kenny M.K. Wood R.D. Cell. 1992; 69: 367-374Abstract Full Text PDF PubMed Scopus (732) Google Scholar), and 4.8 μg of the indicated recombinant RPA in buffer containing 45 mmHEPES-KOH (pH 7.8), 70 mm KCl, 7.4 mmMgCl2, 0.9 mm dithiothreitol (DTT), 0.4 mm EDTA, 2 mm ATP, 22 mmphosphocreatine (di-Tris salt), 2.5 μg of creatine phosphokinase, 3.4% glycerol, and 18 μg of bovine serum albumin. After 5 min at 30 °C, either Pt-GTG DNA (750 ng) or control DNA (750 ng) without the cisplatin cross-link was added and incubation continued for 30 min at 30 °C. The DNA was purified and digested with XhoI andHindIII for 4 h at 37 °C. The reactions were stopped by adding formamide buffer containing bromphenol blue and xylene cyanol. The DNA was denatured at 95 °C for 5 min prior to loading on a 12% acrylamide gel and run until the blue dye migrated ∼30 cm from the wells. DNA was transferred by capillary action for 90 min onto a Hybond-N+ membrane soaked in 10 × Tris borate buffer. The membrane was fixed in 0.4 m NaOH for 20 min followed by a 2-min wash in 5 × SSC. The fixed membrane was incubated at 42 °C for 16 h in hybridization bottles containing 40 ml of 130 mm potassium phosphate (pH 7.0), 250 mm NaCl, 7% SDS, 10% PEG 8000, and 100 pmol of 32P-labeled 27-mer oligonucleotide which is complementary to the excised platinated oligomers (60Moggs J.G. Yarema K.J. Essigmann J.M. Wood R.D. J. Biol. Chem. 1996; 271: 7177-7186Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). The membranes were washed for 10 min in 2 × SSC buffer containing 0.1% SDS before exposure of the membrane to x-ray film. The mismatch-containing substrates used in these studies were constructed from derivatives of phagemid pBS-SK in which the polylinker between theApaI and BamHI sites had been replaced by different mutant polylinkers. Heteroduplex substrates were constructed and purified using unpublished procedures similar to those described by others (61Muster-Nassal C. Kolodner R. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 7618-7622Crossref PubMed Scopus (41) Google Scholar). The polylinker regions of the substrates used the following Sequence 1 (Sequence 1) which contains a GT mispair within overlapping XhoI and NsiI recognition sequences such that repair of the mispair can be monitored by cleavage of the product DNA with eitherXhoI or NsiI. The substrate contained a single strand break in the NsiI strand at the ScaI site (nucleotide 1106- from the mispair). Importantly, the substrates contain a unique AlwNI site 2.1 kb from the mispair. The construction of the mutant phagemids and the substrate construction methods will be described in greater detail elsewhere. The repair assay contains 50 μg of S100 extract (from 293 cells); 30 mm HEPES (pH 7.8); 7 mm MgCl2; 4 mm ATP, 200 μm CTP, GTP, and UTP; 100 μm dNTP; 40 mm creatine phosphate; 100 μg of creatine phosphokinase/ml; 15 mm sodium phosphate (pH 7.5); and 100 ng of the mismatch substrate (62Roberts J.D. Kunkel T.A. Methods Mol. Genet. 1993; 2: 295-311Google Scholar). The reaction mixture was incubated at 37 °C for 2–3 h. After the 2-h incubation, 50 μl of stop buffer (0.67% SDS; 0.025 m EDTA) and proteinase K (0.3 mg/ml final concentration) were added. After an additional 15 min incubation at 37 °C, the reaction mixture was extracted with phenol/chloroform and chloroform, and the DNA was precipitated, digested with appropriate enzymes, and run on a 1.2% agarose gel at 65 V for 2.5 h. The fractionated DNA was stained by SYBR green and visualized on a fluoroimager (Molecular Dynamics). The repair results in the restoration of an XhoI or NsiI site, and the repair efficiency observed ranged from 5 to 15%. For antibody neutralization experiments, the extract was incubated with various antibodies for 15 min at 37 °C prior to the addition of the mismatch substrate. For RPA rescue experiments, the RPA and the mismatch substrate were added simultaneously. We have previously described three human RPA complex mutants, m1–616 tRPA (two of the four cysteines in the putative zinc finger of Rpa1 (amino acid 500 and 503) are changed to serine, renamed here as RPAm), 278–616 tRPA (deletion of amino acids 1–277 of Rpa1), and Δ222–411 tRPA (deletion of amino acids 222–411 of Rpa1) (8Lin Y.L. Chen C. Keshav K.F. Winchester E. Dutta A. J. Biol. Chem. 1996; 271: 17190-17198Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). The recently solved crystal structure of the DNA binding domain of Rpa1 indicates that it is composed of two structurally homologous subdomains comprising amino acids 198–291 and 305–402 (63Bochkarev A. Pfuetzner R.A. Edwards A.M. Frappier L. Nature. 1997; 385: 176-181Crossref PubMed Scopus (471) Google Scholar, 64Pfuetzner R.A. Bochkarev A. Frappier L. Edwards A.M. J. Biol. Chem. 1997; 272: 430-434Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Salt-resistant binding of sin
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