Identification of replication factor C from Saccharomyces cerevisiae: a component of the leading-strand DNA replication complex.
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A number of proteins have been isolated from human cells on the basis of their ability to support DNA replication in vitro of the simian virus 40 (SV40) origin of DNA replication. One such protein, replication factor C (RFC), functions with the proliferating cell nuclear antigen (PCNA), replication protein A (RPA), and DNA polymerase delta to synthesize the leading strand at a replication fork. To determine whether these proteins perform similar roles during replication of DNA from origins in cellular chromosomes, we have begun to characterize functionally homologous proteins from the yeast Saccharomyces cerevisiae. RFC from S. cerevisiae was purified by its ability to stimulate yeast DNA polymerase delta on a primed single-stranded DNA template in the presence of yeast PCNA and RPA. Like its human-cell counterpart, RFC from S. cerevisiae (scRFC) has an associated DNA-activated ATPase activity as well as a primer-template, structure-specific DNA binding activity. By analogy with the phage T4 and SV40 DNA replication in vitro systems, the yeast RFC, PCNA, RPA, and DNA polymerase delta activities function together as a leading-strand DNA replication complex. Now that RFC from S. cerevisiae has been purified, all seven cellular factors previously shown to be required for SV40 DNA replication in vitro have been identified in S. cerevisiae.Keywords:
Replication factor C
DNA polymerase delta
Origin recognition complex
DNA polymerase II
Replication protein A
DNA clamp
Primer (cosmetics)
Pre-replication complex
Ter protein
Replication factor C
Origin recognition complex
Pre-replication complex
Licensing factor
Ter protein
DNA polymerase delta
Replication protein A
Minichromosome maintenance
S phase
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A number of proteins have been isolated from human cells on the basis of their ability to support DNA replication in vitro of the simian virus 40 (SV40) origin of DNA replication. One such protein, replication factor C (RFC), functions with the proliferating cell nuclear antigen (PCNA), replication protein A (RPA), and DNA polymerase delta to synthesize the leading strand at a replication fork. To determine whether these proteins perform similar roles during replication of DNA from origins in cellular chromosomes, we have begun to characterize functionally homologous proteins from the yeast Saccharomyces cerevisiae. RFC from S. cerevisiae was purified by its ability to stimulate yeast DNA polymerase delta on a primed single-stranded DNA template in the presence of yeast PCNA and RPA. Like its human-cell counterpart, RFC from S. cerevisiae (scRFC) has an associated DNA-activated ATPase activity as well as a primer-template, structure-specific DNA binding activity. By analogy with the phage T4 and SV40 DNA replication in vitro systems, the yeast RFC, PCNA, RPA, and DNA polymerase delta activities function together as a leading-strand DNA replication complex. Now that RFC from S. cerevisiae has been purified, all seven cellular factors previously shown to be required for SV40 DNA replication in vitro have been identified in S. cerevisiae.
Replication factor C
DNA polymerase delta
Origin recognition complex
DNA polymerase II
Replication protein A
DNA clamp
Primer (cosmetics)
Pre-replication complex
Ter protein
Cite
Citations (55)
Human replication protein A (RPA) is a three subunit protein complex involved in DNA replication, repair, and recombination. We investigated the role of the 34-kDa subunit (p34) of RPA in DNA replication by generating a series of p34 mutants. While deletion of the N-terminal domain of p34 prevented its phosphorylation by both cyclin-dependent kinase (Cdk) and DNA-dependent kinase, a double point mutant that lacks the major phosphorylation sites for Cdk could be phosphorylated by DNA-dependent kinase. In simian virus 40 (SV40) DNA replication, RPA containing either of these mutants functioned as efficiently as wild-type RPA. However, mutant RPA containing C-terminally deleted p34 was only marginally active. This indicates that the C-terminal region, but not the phosphorylation domain of p34, is necessary for RPA function in DNA replication. Furthermore, RPA containing the C-terminally deleted p34 mutant could stimulate DNA polymerase α, and bind to single-stranded DNAs but was limited in its ability to unwind DNA or interact with SV40 large T antigen (T Ag). These results suggest that RPA p34 interacts with SV40 T Ag during the initiation of SV40 DNA replication and may be necessary for DNA unwinding. Human replication protein A (RPA) is a three subunit protein complex involved in DNA replication, repair, and recombination. We investigated the role of the 34-kDa subunit (p34) of RPA in DNA replication by generating a series of p34 mutants. While deletion of the N-terminal domain of p34 prevented its phosphorylation by both cyclin-dependent kinase (Cdk) and DNA-dependent kinase, a double point mutant that lacks the major phosphorylation sites for Cdk could be phosphorylated by DNA-dependent kinase. In simian virus 40 (SV40) DNA replication, RPA containing either of these mutants functioned as efficiently as wild-type RPA. However, mutant RPA containing C-terminally deleted p34 was only marginally active. This indicates that the C-terminal region, but not the phosphorylation domain of p34, is necessary for RPA function in DNA replication. Furthermore, RPA containing the C-terminally deleted p34 mutant could stimulate DNA polymerase α, and bind to single-stranded DNAs but was limited in its ability to unwind DNA or interact with SV40 large T antigen (T Ag). These results suggest that RPA p34 interacts with SV40 T Ag during the initiation of SV40 DNA replication and may be necessary for DNA unwinding. INTRODUCTIONThe in vitro simian virus 40 (SV40)1( 1The abbreviations used are: SV40simian virus 40pol α and δDNA polymerase α and δ, respectivelytopotopoisomerasePCRpolymerase chain reactionRPAreplication protein ASSBsingle stranded DNA-binding proteinssDNAsingle-stranded DNAT AgSV40 large tumor antigenDTTdithiothreitolTBETris borate-EDTA bufferPBSphosphate-buffered salineELISAenzyme-linked immunosorbent assayCdkcyclin-dependent kinasekbkilobase pair(s)ABTS2,2-azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid.) DNA replication system has been used extensively as a model to understand eukaryotic DNA replication because it uses the host replication machinery for its own DNA replication together with the virally encoded SV40 large T antigen (T Ag). The development of cell-free SV40 DNA replication (Li and Kelly, 1984Li J.J. Kelly T.J. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 6973-6977Crossref PubMed Scopus (351) Google Scholar; Wobbe et al., 1985Wobbe C.R. Dean F.B. Weissbach L. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 5710-5714Crossref PubMed Scopus (226) Google Scholar; Stillman and Gluzman, 1985Stillman B. Gluzman Y. Mol. Cell. Biol. 1985; 5: 2051-2060Crossref PubMed Scopus (252) Google Scholar) has led to the identification of a number of human replication factors involved in SV40 DNA replication in vitro (Challberg and Kelly, 1989Challberg M.D. Kelly T.J. Annu. Rev. Biochem. 1989; 58: 671-717Crossref PubMed Google Scholar; Stillman, 1989Stillman B. Annu. Rev. Cell Biol. 1989; 5: 197-245Crossref PubMed Scopus (282) Google Scholar; Hurwitz et al., 1990Hurwitz J. Dean F.B. Kwong A.D. Lee S.-H. J. Biol. Chem. 1990; 265: 18043-18046Abstract Full Text PDF PubMed Google Scholar) including human replication protein A (RPA, also called human single-stranded DNA-binding protein or HSSB) (Wobbe et al., 1987Wobbe 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 (255) Google Scholar; Fairman and Stillman, 1988Fairman M.P. Stillman B. EMBO J. 1988; 7: 1211-1218Crossref PubMed Scopus (291) Google Scholar; Wold and Kelly, 1988Wold M.S. Kelly T.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2523-2527Crossref PubMed Scopus (365) Google Scholar). Human RPA comprises three subunits of 70, 34, and 11 kDa (p70, p34, and p11, respectively), which are tightly associated with each other (Fairman and Stillman, 1988Fairman M.P. Stillman B. EMBO J. 1988; 7: 1211-1218Crossref PubMed Scopus (291) Google Scholar) and are conserved among other species (Brill and Stillman, 1989Brill S.J. Stillman B. Nature. 1989; 342: 92-95Crossref PubMed Scopus (186) Google Scholar, Brill and Stillman, 1991Brill S.J. Stillman B. Genes & Dev. 1991; 5: 1589-1600Crossref PubMed Scopus (188) Google Scholar; Mitsis et al., 1993Mitsis P.G. Kowalczykowski S.C. Lehman I.R. Biochemistry. 1993; 32: 5257-5266Crossref PubMed Scopus (54) Google Scholar; Brown et al., 1993Brown G.W. Melendy T. Ray D.S. Mol. Biochem. Parasitol. 1993; 59: 323-325Crossref PubMed Scopus (8) Google Scholar). RPA subunits are assembled in an ordered process; p34 forms a stable complex with p11 to which p70 binds (Stigger et al., 1994Stigger E. Dean F. Hurwitz J. Lee S.-H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 579-583Crossref PubMed Scopus (58) Google Scholar; Henricksen et al., 1994Henricksen L.A. Umbricht C.B. Wold M.S. J. Biol. Chem. 1994; 269: 11121-11132Abstract Full Text PDF PubMed Google Scholar).The cDNAs encoding the human RPA subunits are about 30% homologous to their yeast counterparts (Erdile et al., 1990Erdile L.F. Wold M.S. Kelly T.J. J. Biol. Chem. 1990; 265: 3177-3182Abstract Full Text PDF PubMed Google Scholar, Erdile et al., 1991Erdile L.F. Heyer W.-D. Kolodner R. Kelly T.J. J. Biol. Chem. 1991; 266: 12090-12098Abstract Full Text PDF PubMed Google Scholar; Heyer et al., 1990Heyer W.-D. Rao M.R.S. Erdile L.F. Kelly T.J. Kolodner R. EMBO J. 1990; 9: 2321-2329Crossref PubMed Scopus (156) Google Scholar; Brill and Stillman, 1991Brill S.J. Stillman B. Genes & Dev. 1991; 5: 1589-1600Crossref PubMed Scopus (188) Google Scholar; Umbricht et al., 1993Umbricht C.B. Erdile L.F. Jabs E.W. Kelly T.J. J. Biol. Chem. 1993; 268: 6131-6138Abstract Full Text PDF PubMed Google Scholar). However, yeast RPA substitutes poorly for human RPA in the in vitro SV40 replication system (Brill and Stillman, 1989Brill S.J. Stillman B. Nature. 1989; 342: 92-95Crossref PubMed Scopus (186) Google Scholar), indicating that highly specific protein-protein interactions occur between RPA and other replication protein(s). SV40 T Ag, RPA, and the DNA polymerase α-primase complex (pol α-primase) functionally interact in vitro to form a primosome complex that is required for both primer synthesis (Collins and Kelly, 1991Collins K.L. Kelly T.J. Mol. Cell. Biol. 1991; 11: 2108-2115Crossref PubMed Google Scholar; Melendy and Stillman, 1993Melendy T. Stillman B. J. Biol. Chem. 1993; 268: 3389-3395Abstract Full Text PDF PubMed Google Scholar) and DNA synthesis at the replication fork (Murakami and Hurwitz, 1993Murakami Y. Hurwitz J. J. Biol. Chem. 1993; 268: 11008-11017Abstract Full Text PDF PubMed Google Scholar). Physical interactions between the various components of the primosome have been shown by enzyme-linked immunosorbent assay (ELISA) and immunoblotting (Dornreiter et al., 1992Dornreiter I. Erdile L.F. Gilbert I.U. von Winkler D. Kelly T.J. Fanning E. EMBO J. 1992; 11: 769-776Crossref PubMed Scopus (284) Google Scholar; Collins et al., 1993Collins K.L. Russo A.A.R. Tseng B.Y. Kelly T.J. EMBO J. 1993; 12: 4555-4566Crossref PubMed Scopus (94) Google Scholar). RPA functions at the initiation stage of SV40 DNA replication by promoting both T Ag-dependent presynthetic unwinding of SV40 DNA (Dean et al., 1987Dean F.B. Bullock P. Murakami Y. Wobbe C.R. Weissbach L. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 16-20Crossref PubMed Scopus (213) Google Scholar; Wold et al., 1987Wold M.S. Li J.J. Kelly T.J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 3643-3647Crossref PubMed Scopus (159) Google Scholar; Borowiec et al., 1990Borowiec J.A. Dean F.B. Bullock P. Hurwitz J. Cell. 1990; 60: 181-184Abstract Full Text PDF PubMed Scopus (285) Google Scholar) and pol α-primase activity (Matsumoto et al., 1990Matsumoto T. Eki T. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9712-9716Crossref PubMed Scopus (93) Google Scholar). During the elongation stage, RPA stimulates the activities of pol α-primase, pol δ, and pol ε (Kenny et al., 1989Kenny M.K. Lee S.-H. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9757-9761Crossref PubMed Scopus (164) Google Scholar; Lee et al., 1991Lee S.-H. Kwong A.D. Pan Z.-Q. Hurwitz J. J. Biol. Chem. 1991; 266: 594-602Abstract Full Text PDF PubMed Google Scholar). While RPAs from Escherichia coli (SSB), adenovirus (DNA-binding protein (Ad-DBP)), and herpesvirus (ICP8) can substitute for human RPA to stimulate T Ag-catalyzed unwinding of SV40 DNA and pol δ activity in the SV40 system, they cannot replace human RPA to stimulate pol α-primase activity (Kenny et al., 1989Kenny M.K. Lee S.-H. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9757-9761Crossref PubMed Scopus (164) Google Scholar). This suggests that the interaction between RPA and pol α-primase may contribute to the species specificity of SV40 replication. The roles of the individual RPA subunits, particularly p34 and p11, in DNA metabolism are not known. In yeast, all three RPA genes, rpa1, rpa2, and rpa3 (the RPA p70, p34, and p11 homologs, respectively), are essential for cell viability (Brill and Stillman, 1991Brill S.J. Stillman B. Genes & Dev. 1991; 5: 1589-1600Crossref PubMed Scopus (188) Google Scholar). Although in yeast, RPA is not required for cell cycle entry, disruption of the rpa genes results in an abnormal phenotype that is consistent with an S-phase defect (Brill and Stillman, 1991Brill S.J. Stillman B. Genes & Dev. 1991; 5: 1589-1600Crossref PubMed Scopus (188) Google Scholar).RPA p34 is phosphorylated on serine residues at the G1/S transition and then dephosphorylated during mitosis (Din et al., 1990Din S.-U. Brill S.J. Fairman M.P. Stillman B. Genes & Dev. 1990; 4: 968-977Crossref PubMed Scopus (244) Google Scholar; Dutta et al., 1991Dutta A. Din S. Brill S.J. Stillman B. Cold Spring Harbor Symp. Quant. Biol. 1991; 56: 315-324Crossref PubMed Scopus (21) Google Scholar; Dutta and Stillman, 1992Dutta A. Stillman B. EMBO J. 1992; 11: 2189-2199Crossref PubMed Scopus (221) Google Scholar). One of the kinases responsible for p34 phosphorylation is a cyclin-dependent kinase (Cdk) (Elledge et al., 1992Elledge S.J. Richman R. Hall F.L. Williams R.T. Lodgson N. Harper J.W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2907-2911Crossref PubMed Scopus (168) Google Scholar; Dutta and Stillman, 1992Dutta A. Stillman B. EMBO J. 1992; 11: 2189-2199Crossref PubMed Scopus (221) Google Scholar; Pan et al., 1993Pan Z.-Q. Amin A. Hurwitz J. J. Biol. Chem. 1993; 268: 20443-20451Abstract Full Text PDF PubMed Google Scholar; Pan and Hurwitz, 1993Pan Z.-Q. Hurwitz J. J. Biol. Chem. 1993; 268: 20433-20442Abstract Full Text PDF PubMed Google Scholar). Several reports implicate this kinase in DNA replication. First, in SV40 DNA unwinding experiments with T Ag and HeLa cell cytosolic extracts, G1-phase cell extracts have a much lower DNA unwinding activity than S-phase or G2-phase extracts. However, G1-phase cell extracts could be activated by the addition of Cdk (Roberts and D'Urso, 1988Roberts J.M. D'Urso G. Science. 1988; 241: 1486-1489Crossref PubMed Scopus (32) Google Scholar; D'Urso et al., 1990D'Urso G. Marraccina R.L. Marshak D.R. Roberts J.M. Science. 1990; 250: 786-791Crossref PubMed Scopus (200) Google Scholar). Second, depletion of Cdk from Xenopus egg extracts results in the inability of those extracts to support DNA replication (Fang and Newport, 1991Fang F. Newport J.W. Cell. 1991; 66: 731-742Abstract Full Text PDF PubMed Scopus (375) Google Scholar; Blow and Nurse, 1990Blow J.J. Nurse P. Cell. 1990; 62: 855-862Abstract Full Text PDF PubMed Scopus (140) Google Scholar). To date, there is no direct evidence that the activation of DNA replication is due to RPA p34 phosphorylation by Cdk.Recent evidence from Cdk-depleted S-phase extracts suggests that RPA can associate with both single-stranded DNA and SV40 origin-containing DNA, and then become phosphorylated even in the absence of S-phase kinases (Fotedar and Roberts, 1992Fotedar R. Roberts J.M. EMBO J. 1992; 11: 2177-2187Crossref PubMed Scopus (119) Google Scholar). It may be that the cell cycle-dependent phosphorylation of RPA p34 results from the coordinated action of Cdk and DNA-dependent kinase both of which function at the G1/S transition. RPA p34 is also phosphorylated following DNA damage caused by UV (Carty et al., 1994Carty M.P. Zernik-Kobak M. McGrath S. Dixon K. EMBO J. 1994; 13: 2114-2123Crossref PubMed Scopus (168) Google Scholar) or ionizing radiation (Liu and Weaver, 1993Liu V.F. Weaver D.T. Mol. Cell. Biol. 1993; 13: 7222-7231Crossref PubMed Scopus (186) Google Scholar). However, the role of RPA p34 phosphorylation in DNA metabolism remains unknown.In this report, we address the role of RPA p34 in DNA replication by comparing the function of wild-type RPA with that of a series of mutants. Deletion of the N-terminal region of RPA p34 abolished its phosphorylation by both Cdk and DNA-dependent kinase but did not affect the mutant's ability to support SV40 DNA replication in vitro. A double point mutant that lacks Cdk phosphorylation sites also retains its activity in the SV40 replication system. However, mutant RPA lacking the C-terminal region of p34 only weakly supported DNA replication and unwinding, and interacted poorly with SV40 T Ag. We discuss the implications of these results on the role of RPA p34 in DNA replication.EXPERIMENTAL PROCEDURESCell Extracts, Proteins, Antibodies, and DNAHeLa cell cytosolic extracts and their ammonium sulfate fractions were prepared as described previously (Wobbe et al., 1985Wobbe C.R. Dean F.B. Weissbach L. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 5710-5714Crossref PubMed Scopus (226) Google Scholar, Wobbe et al., 1987Wobbe 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 (255) Google Scholar), as was SV40 T Ag (Lee et al., 1991Lee S.-H. Kwong A.D. Pan Z.-Q. Hurwitz J. J. Biol. Chem. 1991; 266: 594-602Abstract Full Text PDF PubMed Google Scholar). Pol α-primase and topoisomerase I (topo I) were isolated from HeLa cells using the procedure described by Ishimi et al., 1988Ishimi Y. Claude A. Bullock P. Hurwitz J. J. Biol. Chem. 1988; 263: 19723-19733Abstract Full Text PDF PubMed Google Scholar. Monoclonal antibodies against RPA p70 and p34 were generated as described earlier (Kenny et al., 1990Kenny M.K. Schlegel U. Furneaux H. Hurwitz J. J. Biol. Chem. 1990; 265: 7693-7700Abstract Full Text PDF PubMed Google Scholar), and DNA primers for PCR, oligo(dT)50, and oligo(dT)17 were synthesized by the Molecular Resource Center at St Jude Children's Research Hospital, Memphis, TN.Recombinant BaculovirusesRecombinant baculovirus constructs encoding wild-type p70, p34, and p11 have been described previously (Stigger et al., 1994Stigger E. Dean F. Hurwitz J. Lee S.-H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 579-583Crossref PubMed Scopus (58) Google Scholar). The double point mutant, in which alanine is substituted for serine at amino acids 23 and 29 in p34 (p34(S/A:23,29)), was generated by polymerase chain reaction (PCR) using the full-length cDNA and the following sets of primers: 5′-CGC GGA TCC ATG TGG AAC-3′ and 5′-GCG CTC CAA AGC CCC CCG GGG ACC GCG-3′ (representing the N-terminal sequences and amino acids 23-29), and 5′-CGC GGT CCC CGG GGG GCT TTG GAG CGC-3′ and 5′-CGC GGA TCC TCT CAG GTA CCC AGT T-3′ (representing amino acids 23-29 and the C-terminal sequences). PCRs (30 cycles) were carried out at 94°C for 1 min, 42°C for 1 min, and 72°C for 2 min. The PCR products were then gel isolated and restricted with XmaI, which cuts once between amino acids 23 and 29. The products were then ligated and directly used for a second PCR reaction with primers representing N-terminal and C-terminal sequences. p34 lacking amino acids 2-30 (p34Δ2-30) was prepared by PCR using a set of primers (5′-AGG ATC CAT GGC ACC TTC TCA AGC CGA A-3′ and 5′-CGC GGA TCC TCT CAG GTA CCC AGT T-3′) to generate the mutant under the same conditions as described above. The PCR products, after gel isolation, were cloned into the BamHI site of pVL941. p34 lacking 33 amino acids at the C terminus (p34Δ33C) was prepared similarly to p34Δ2-30 except that the following primers were used: 5′-CGC GGA TCC ATG TGG AAC-3′ and 5′-AGG ATC CTT ACA TGT GTT TCA GCT GGT T-3′.Baculovirus Infection, Metabolic Labeling, and ImmunoprecipitationInsect (Sf9) cell culture and preparation of recombinant baculoviruses have been described previously (Stigger et al., 1994Stigger E. Dean F. Hurwitz J. Lee S.-H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 579-583Crossref PubMed Scopus (58) Google Scholar). Sf9 cells (2.0 × 106) were plated on a 60-mm dish and infected with each recombinant baculovirus at a multiplicity of infection of 30 for 40 h at 27°C. The cells were then labeled for 4 h with Tran35S-labeled methionine at 200 μCi/ml (1000 Ci/mmol) in 1.5 ml of methionine-free medium/5% dialyzed fetal calf serum and washed with phosphate-buffered saline (PBS), prior to being lysed for 1 h on ice in 0.5 ml of EBC buffer (50 mM Tris-HCl at pH 8.0, 120 mM NaCl, 0.5% Nonidet P-40, 1 mM DTT, 1 mM EDTA, 0.1 mM NaF, 10 mM β-glycerophosphate, 0.1 mM sodium orthovanadate, 0.1 mM phenylmethylsulfonyl fluoride, 0.1 mg/ml leupeptin, and 0.2 mg/ml antipain). Cleared cell lysates (50 μl) were subsequently incubated overnight with 5 μg of purified monoclonal antibody in the presence of bovine serum albumin (200 μg/ml) at 4°C with rocking. Protein G-Sepharose was then added, and the lysates were incubated for 1 h at 4°C. Finally, the immunoprecipitates were collected by centrifugation, washed five times with EBC buffer, and then analyzed by SDS-PAGE.Protein IsolationWild-type RPA and RPA mutants were isolated from insect cells coinfected with recombinant baculoviruses encoding p70, p11, and either wild-type or mutant p34 (p34(S/A:23,29), p34Δ33C, or p34Δ2-30) as described earlier (Stigger et al., 1994Stigger E. Dean F. Hurwitz J. Lee S.-H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 579-583Crossref PubMed Scopus (58) Google Scholar).SV40 DNA Replication in VitroThe reactions were carried out as described by Wobbe et al., 1985Wobbe C.R. Dean F.B. Weissbach L. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 5710-5714Crossref PubMed Scopus (226) Google Scholar. In brief, the reaction mixtures (40 μl) included 40 mM creatine phosphate-di-Tris salt (pH 7.7), 1 μg of creatine kinase, 7 mM MgCl2, 0.5 mM DTT, 4 mM ATP, 200 μM UTP, GTP, and CTP, 100 μM dATP, dGTP, and dCTP, 25 μM [3H]dTTP (300 cpm/pmol), 0.6 μg of SV40 T Ag, 0.23 μg of pSV01ΔEP, and the indicated amounts of replication proteins or extracts. The reactions ran for 1 h at 37°C, after which the acid-insoluble radioactivity was measured.Replication products were analyzed using [α-32P]dCTP (30,000 cpm/pmol) instead of [3H]dTTP in the reactions just described. After incubation, the reactions were stopped by the addition of 80 μl of a solution containing 20 mM EDTA, 1% sodium dodecyl sulfate, and E. coli tRNA (0.5 mg/ml). DNA was isolated and electrophoretically separated in a 1.2% alkaline-agarose gel (40 mM NaOH and 1 mM EDTA) for 12-14 h at 2 V/cm. The gel was subsequently dried and exposed to x-ray film.In Vitro Phosphorylation of RPAReaction mixtures (30 μl) contained 40 mM creatine phosphate-di-Tris salt (pH 7.7), 1 μg creatine kinase, 7 mM MgCl2, 0.5 mM DTT, 5 μg of bovine serum albumin, 4 mM ATP, 200 μM UTP, GTP, and CTP, 100 μM dATP, dGTP, dTTP, and dCTP, 1 mM Na3VO4, 10 mM NaF, and 0.6 μg of SV40 T Ag. Where indicated, 100 μg of ammonium sulfate fraction 35-65% (AS 35-65) of HeLa cell cytosolic extract, 1.0 μg of RPA, or 0.3 μg of SV40 origin-containing DNA was included in the reactions which were incubated at 37°C for 1 h. Proteins were then separated by 11% SDS-PAGE, transferred to nitrocellulose, immunoblotted with an anti-p34 polyclonal antibody (rabbit), and visualized by 125I-protein A autoradiography (Stigger et al., 1994Stigger E. Dean F. Hurwitz J. Lee S.-H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 579-583Crossref PubMed Scopus (58) Google Scholar).DNA Polymerase α Assay and SV40 DNA Unwinding AssaysDNA pol α activity and the unwinding of SV40 origin-containing DNA (pSV01ΔEP) were assayed as described previously (Lee et al., 1989Lee S.-H. Eki T. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7361-7365Crossref PubMed Scopus (112) Google Scholar; Stigger et al., 1994Stigger E. Dean F. Hurwitz J. Lee S.-H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 579-583Crossref PubMed Scopus (58) Google Scholar).Interaction between RPA and SV40 T AgProtein interaction was determined by the modified ELISA described by Dornreiter et al., 1992Dornreiter I. Erdile L.F. Gilbert I.U. von Winkler D. Kelly T.J. Fanning E. EMBO J. 1992; 11: 769-776Crossref PubMed Scopus (284) Google Scholar. The 96-well plates were coated with 1.0 μg of either wild-type or mutant RPA overnight at 4°C. After washing with PBS, wells were blocked with 3% bovine serum albumin in PBS for 1 h at 37°C. The indicated amounts of SV40 T Ag were added, and the wells were incubated for another hour at room temperature before being washed extensively with PBS. RPA-bound SV40 T Ag was measured by incubating the wells with a horseradish peroxidase-conjugated SV40 T Ag monoclonal antibody (Pab 419) for 1 h at 37°C. Conjugation of the antibody to horseradish peroxidase (Zymed Actizyme-Peroxidase kit) was done according to the manufacturer's specifications. After extensive washing with PBS, the chromogenic substrate ABTS acid and hydrogen peroxide were added, and the colorimetric reaction was monitored at 410 nm.ssDNA Binding AssayThe assay was performed according to method used by Kim et al., 1992Kim C. Snyder R.O. Wold M.S. Mol. Cell. Biol. 1992; 12: 3050-3059Crossref PubMed Scopus (239) Google Scholar with the following modifications. The reaction mixture (20 μl) contained 50 mM Hepes-KOH (pH 7.5), 150 mM NaCl, 1 mM MgCl2, 0.5 mM DTT, 10% glycerol, 50 fmol of 5′-32P-labeled oligo(dT)50 (2200 cpm/fmol), plus the indicated amount of RPA, and was incubated for 15 min at 25°C. The complex was electrophoretically separated on a 5% polyacrylamide gel in 0.5 × TBE (89 mM Tris borate, 2 mM EDTA) at 15 V/cm. The gel was then dried and exposed to x-ray film. To quantitate the data, the protein-DNA complex bands were excised and analyzed by liquid scintillation counting.RESULTSRPA p34 MutantsTo study the function of RPA p34 in DNA replication, we created two p34 deletion mutants and a p34 mutant that lacks the putative Cdk recognition sites (by serine to alanine substitutions at amino acids 23 and 29). We first examined whether mutant p34 can form a complex with other RPA subunits. After we had coinfected Sf9 cells with recombinant baculoviruses encoding p34 (wild-type or mutant), p11, and p70, RPA complexes were immunoprecipitated from cell lysates using an anti-p34 monoclonal antibody or an anti-p70 antibody (Fig. 1). Both the N-terminal and the C-terminal deletion mutants (p34Δ2-30 and p34Δ33C, respectively) were able to form heterotrimeric complexes with p70 and p11. Chromatographic behaviors and yields of these deletion mutants were similar to those of wild-type RPA (data not shown). p34Δ33C was immunoprecipitated by the anti-p70 antibody but not by the anti-p34 monoclonal antibody, suggesting that the C terminus of p34 (amino acids 238-270) contains the p34 monoclonal antibody recognition site.The N-terminal Region of RPA p34 Is Required for Its Phosphorylation by Cdk and DNA-dependent KinaseAt least two different kinases (cyclin-dependent kinase and DNA-dependent kinase) can phosphorylate RPA p34 in vitro (Elledge et al., 1992Elledge S.J. Richman R. Hall F.L. Williams R.T. Lodgson N. Harper J.W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2907-2911Crossref PubMed Scopus (168) Google Scholar; Dutta and Stillman, 1992Dutta A. Stillman B. EMBO J. 1992; 11: 2189-2199Crossref PubMed Scopus (221) Google Scholar; Fotedar and Roberts, 1992Fotedar R. Roberts J.M. EMBO J. 1992; 11: 2177-2187Crossref PubMed Scopus (119) Google Scholar; Pan and Hurwitz, 1993Pan Z.-Q. Hurwitz J. J. Biol. Chem. 1993; 268: 20433-20442Abstract Full Text PDF PubMed Google Scholar). Therefore, we examined the phosphorylation of both wild-type and mutant RPA p34 under replication conditions. The 35-65% ammonium sulfate fraction (AS 35-65) of HeLa cell cytosolic extracts was used in this assay because it contains all of the required host replication factors except RPA (Wobbe et al., 1987Wobbe 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 (255) Google Scholar). The amount of RPA present in AS 35-65 was negligible compared to the exogenous RPA (∼1.0 μg) in this experiment (Fig. 2, lane 1). In the absence of SV40 origin-containing DNA, less than 50% of wild-type RPA was phosphorylated and appeared as a slow migrating band on the denaturing gel (Fig. 2, lane 3). This slow migrating band, which disappears on phosphatase treatment (data not shown), probably represents RPA that was phosphorylated by cyclin A-Cdk2 (or another cyclin-dependent kinase), because, under the same conditions, p34 mutants (p34(S/A:23,29) and p34Δ2-30) lacking the major Cdk consensus sites were not phosphorylated. p34(S/A:23,29) can be phosphorylated by purified Cdk2-cyclin A in vitro, indicating that additional Cdk target sites exist.( 2S.-H. Lee, unpublished data.) 2 In the presence of SV40 DNA, both wild-type RPA and RPA containing p34(S/A:23,29) were efficiently phosphorylated by DNA-dependent kinase (indicated by the highly retarded bands in lanes 4 and 7 of Fig. 2). This suggests that the cyclin-dependent kinase phosphorylation sites are different from those targeted by DNA-dependent kinase. C-terminally deleted RPA p34 (p34Δ33C) displayed a phosphorylation pattern similar to that of wild-type RPA with the exception that this mutant was phosphorylated less efficiently in the presence than in the absence of SV40 DNA (Fig. 2, lanes 11-13). These data indicate that the C-terminal region of p34 is not required for RPA p34 phosphorylation but may be necessary for efficient replication. However, the p34Δ2-30 mutant was not phosphorylated under either conditions, as measured by both immunoblotting (Fig. 2, lanes 8-10) and assaying for kinase activity (data not shown), indicating that the N-terminal region of p34 is necessary for its phosphorylation by both kinases. This region contains 5 serine residues (amino acids 4, 8, 11, 12, and 13), in addition to the major consensus sites for Cdk (amino acids 23 and 29).Figure 2:In vitro phosphorylation of wild-type and mutant RPA p34. Reaction conditions were described in the experimental procedures. Where indicated, 1.0 μg of either wild-type or mutant RPA, 100 μg of an ammonium sulfate fraction (35-65%) of HeLa cell cytosolic extract, or 0.3 μg of SV40 origin-containing DNA were included. After the reactions, RPA p34 was visualized using an anti-p34 polyclonal antibody (rabbit).View Large Image Figure ViewerDownload Hi-res image Download (PPT)The C-terminal but Not the N-terminal Region of RPA p34 Is Required for Efficient SV40 DNA ReplicationIn vivo, RPA p34 is phosphorylated at the G1/S boundary and remains phosphorylated during S-phase, suggesting that p34 phosphorylation may be important to DNA replication. Since p34Δ2-30 is not phosphorylated by kinases present in HeLa cell cytosolic extracts, this mutant should help determine whether RPA p34 phosphorylation is directly involved in the replication process.We, therefore, examined whether our RPA p34 mutants supported SV40 DNA replication in vitro. RPA containing p34Δ2-30 or p34(S/A:23,29) functioned as efficient as wild-type RPA, when added to the AS 35-65 fraction, in our SV40 DNA replication system (Fig. 3, panel A), whereas p34Δ33C poorly supported DNA replication. We also examined the function of these mutants in the SV40 monopolymerase system which contains pol α-primase, topo I, and SV40 T Ag (Fig. 3, panel B). Similarly to SV40 replication with crude extracts (AS 35-65), p34Δ33C supported only about 20% of the activity supported by wild-type RPA or the other two mutants. This result indicates that RPA p34 requires its C-terminal domain, but not phosphorylation, to function effectively in an SV40 DNA replication system.Figure 3:Comparison between wild-type and mutant RPA function in SV40 DNA replication in vitro. A, SV40 DNA replication in vitro with crude extracts. Replication reactions comprised SV40 origin-containing DNA (pSV01ΔEP), SV40 T Ag, the 35-65% ammonium sulfate fraction (AS 35-65) of HeLa cell cytosolic extract (100 μg), [3H]dTTP, and the indicated amounts of eith
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Replication factor A (RF-A) is a heterotrimeric single-stranded-DNA-binding protein which is conserved in all eukaryotes. Since the availability of conditional mutants is an essential step to define functions and interactions of RF-A in vivo, we have produced and characterized mutations in the RFA1 gene, encoding the p70 subunit of the complex in Saccharomyces cerevisiae. This analysis provides the first in vivo evidence that RF-A function is critical not only for DNA replication but also for efficient DNA repair and recombination. Moreover, genetic evidence indicate that p70 interacts both with the DNA polymerase alpha-primase complex and with DNA polymerase delta.
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Replication of plasmid DNA molecules containing the simian virus 40 (SV40) origin of DNA replication has been reconstituted with seven highly purified cellular proteins plus the SV40 large tumor (T) antigen. Initiation of DNA synthesis is absolutely dependent upon T antigen, replication protein A, and the DNA polymerase alpha-primase complex and is stimulated by the catalytic subunit of protein phosphatase 2A. Efficient elongation of nascent chains additionally requires proliferating cell nuclear antigen, replication factor C, DNA topoisomerase I, and DNA polymerase delta. Electron microscopic studies indicate that DNA replication begins at the viral origin and proceeds via intermediates containing two forks that move in opposite directions. These findings indicate that the reconstituted replication reaction has many of the characteristics expected of authentic viral DNA replication.
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A number of proteins have been isolated from human cells on the basis of their ability to support DNA replication in vitro of the simian virus 40 (SV40) origin of DNA replication. One such protein, replication factor C (RFC), functions with the proliferating cell nuclear antigen (PCNA), replication protein A (RPA), and DNA polymerase delta to synthesize the leading strand at a replication fork. To determine whether these proteins perform similar roles during replication of DNA from origins in cellular chromosomes, we have begun to characterize functionally homologous proteins from the yeast Saccharomyces cerevisiae. RFC from S. cerevisiae was purified by its ability to stimulate yeast DNA polymerase delta on a primed single-stranded DNA template in the presence of yeast PCNA and RPA. Like its human-cell counterpart, RFC from S. cerevisiae (scRFC) has an associated DNA-activated ATPase activity as well as a primer-template, structure-specific DNA binding activity. By analogy with the phage T4 and SV40 DNA replication in vitro systems, the yeast RFC, PCNA, RPA, and DNA polymerase delta activities function together as a leading-strand DNA replication complex. Now that RFC from S. cerevisiae has been purified, all seven cellular factors previously shown to be required for SV40 DNA replication in vitro have been identified in S. cerevisiae.
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A number of proteins are involved in DNA replication, which is essential for the inheritance of genetic information. These proteins assemble to form a huge complex, called replisome, and accomplish each function through highly regulated manner. Electron microscopic single particle analysis is one of the most powerful methods to study such complex system, which is difficult to study by X‐ray crystallography. DNA replication in archaea and eukaryotes is executed by family B DNA polymerases, which exhibit full activity when complexed with the DNA clamp, proliferating cell nuclear antigen (PCNA). PCNA has a trimeric ring structure that encircles the DNA, and increases the processivity of the bound DNA polymerase by tethering it to the DNA. It is known now, that PCNA also interacts with multiple partners to control DNA replication, DNA repair, and cell cycle progression, and works not only as the platform, but also as the conductor for the recruitment and release of these factors. However, the molecular architectures as well as the mechanism of the regulation of these replication factors are not known in detail. We have been focusing our interest on the mechanism of synthesis and maturation of Okazaki fragments during lagging strand DNA replication in which three replication factors, i.e. DNA polymerase, Flap endonuclease, DNA ligase, are playing essential roles (Fig. 1A‐C). As all of these 3 enzymes are known to interact with PCNA trimer, a switching mechanism between these factors has been proposed, called PCNA tool belt model, which is considered to increase the efficiency of these sequential reactions (Fig 1D). Recent biochemical study, on the other hand, suggests a sequential switching mechanism of these factors. Little is known regarding the switching mechanism, due to the lack of the structural data of these complexes. We have investigated the three‐dimensional structure of the core components of the replisome, such as DNA polymerase B(PolB)‐PCNA‐DNA, and DNA ligase‐PCNA‐DNA ternary complexes, by single particle analysis (2‐ 3). Besides the authentic interaction through a PCNA‐interacting protein box (PIP‐box), we could find a novel contact between both polymerase‐PCNA and ligase‐PCNA. Mutant analysis showed that these contacts are involved in the regulation of the replication factors, such as the switching between the polymerizing and editing modes of the PolB. Our results, showing that both factors interacting with two subunits of the PCNA trimer ring, were inconsistent with the standard tool belt model. However, the third PCNA subunit was still free in both complexes, thus we analyzed the complex structures with two replication factors bound to a PCNA ring, in order to investigate the switching mechanism between them in more detail.
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Replication factor A (RF-A) is a heterotrimeric single-stranded-DNA-binding protein which is conserved in all eukaryotes. Since the availability of conditional mutants is an essential step to define functions and interactions of RF-A in vivo, we have produced and characterized mutations in the RFA1 gene, encoding the p70 subunit of the complex in Saccharomyces cerevisiae. This analysis provides the first in vivo evidence that RF-A function is critical not only for DNA replication but also for efficient DNA repair and recombination. Moreover, genetic evidence indicate that p70 interacts both with the DNA polymerase alpha-primase complex and with DNA polymerase delta.
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The single-stranded DNA-binding protein, Replication Protein A (RPA), is a heterotrimeric complex with subunits of 70, 32 and 14 kDa involved in DNA metabolism. RPA may be a target for cellular regulation; the 32 kDa subunit (RPA32) is phosphorylated by several cellular kinases including the DNA-dependent protein kinase (DNA-PK). We have purified a mutant hRPA complex lacking amino acids 1–33 of RPA32 (rhRPA•32Δ1–33). This mutant bound ssDNA and supported DNA replication; however, (rhRPA•32Δ1–33) was not phosphorylated under replication conditions or directly by DNA-PK. Proteolytic mapping revealed that all the sites phosphorylated by DNA-PK are contained on residues 1–33 of RPA32. When wild-type RPA was treated with DNA-PK and the mixture added to SV40 replication assays, DNA replication was supported. In contrast, when rhRPA•32Δ1–33 was treated with DNA-PK, DNA replication was strongly inhibited. Because untreated (rhRPA•32Δ1–33) is fully functional, this suggests that the N-terminus of RPA is needed to overcome inhibitory effects of DNA-PK on other components of the DNA replication system. Thus, phosphorylation of RPA may modulate DNA replication indirectly, through interactions with other proteins whose activity is modulated by phosphorylation.
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INTRODUCTION DNA replication requires the concerted action of many enzymes, as well as other protein and non-protein cofactors. The DNA, in preparation for DNA synthesis, has to become single-strand to serve as a template for the replicative DNA polymerases (pols). It is this form of the DNA that is especially prone to damage of any kind. Nature has provided a set of proteins that support the replicative pols in performing processive, accurate, and rapid DNA synthesis. Furthermore, such proteins also prevent damage to the transient single-strand (ss) DNA. These proteins are called DNA replication accessory proteins. The three best known are the proliferating cell nuclear antigen (PCNA), replication factor C (RF-C), and replication protein A (RP-A). In this chapter, we focus on these three protein classes and compare them to their selected counterparts in eukaryotic viruses. Additional replication proteins that also assist the proper function of pols, such as the 3′ → 5′ exonuclease, DNA primase, RNase H, 5′ → 3′ exonuclease, DNA helicases, DNA ligases, and DNA topoisomerases, are covered in various other chapters. Early Discovery of Replication Accessory Proteins in Prokaryotes by Genetics and Defined In Vitro Replication Systems Fifteen years ago it was realized that bacteriophages of Escherichia coli provide a window to understand the cellular events of DNA replication (Kornberg and Baker 1992). By using ssDNA from φX174, G4, and M13 as model replicons, the requirements for a ssDNA-binding protein (SSB) and a DNA synthesis complex were identified. The latter includes the pol III holoenzyme, which...
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