{"doi":"10.1074/jbc.270.21.12801","title":"The Role of the 34-kDa Subunit of Human Replication Protein A in Simian Virus 40 DNA Replication in Vitro","abstract":null,"journal":"Journal of Biological Chemistry","year":1995,"id":590861,"datarank":4.194601872210994,"base_score":4.356708826689592,"endowment":4.356708826689592,"self_citation_contribution":0.6535063240034389,"citation_network_contribution":3.541095548207555,"self_endowment_contribution":0.6535063240034389,"citer_contribution":3.541095548207555,"corpus_percentile":null,"corpus_rank":null,"citation_count":77,"citer_count":67,"citers_with_citation_signal":64,"citers_with_endowment":64,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":null,"is_data_producer":false,"deposit_databanks":null,"is_oa":false,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":null,"fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1511696,"name":"Dong Kyoo Kim","orcid":null,"position":1,"is_corresponding":false},{"id":191539,"name":"Suk-Hee Lee","orcid":null,"position":0,"is_corresponding":false}],"reference_count":0,"raw_metadata":{"has_enrichment":true,"resolved":true,"title":"The Role of the 34-kDa Subunit of Human Replication Protein A in Simian Virus 40 DNA Replication in Vitro","abstract":"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 with and Stillman, 1988Fairman M.P. Stillman B. EMBO J. 1988; 7: 1211-1218Crossref PubMed Scopus (291) Google Scholar) and are and Stillman, Stillman B. 1989; PubMed Scopus Google and Stillman, Stillman B. 5: PubMed Scopus Google Scholar; et al., PubMed Scopus Google Scholar; et al., Mol. Biochem. PubMed Scopus Google Scholar). RPA subunits are in p34 a complex with to which et al., Dean Hurwitz J. Lee S.-H. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar; et al., Wold M.S. J. Biol. Chem. Full Text PDF PubMed Google the human RPA subunits are to et al., Wold M.S. Kelly T.J. J. Biol. Chem. 1990; 265: Full Text PDF PubMed Google et al., Kelly T.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar; et al., Kelly T.J. EMBO J. 1990; PubMed Scopus Google Scholar; and Stillman, Stillman B. 5: PubMed Scopus Google Scholar; et al., Kelly T.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). However, RPA for human RPA in the in vitro SV40 replication system and Stillman, Stillman B. 1989; PubMed Scopus Google that RPA and replication SV40 T and the DNA polymerase complex interact in vitro to a complex that is for both and Kelly, Kelly T.J. Mol. Cell. Biol. PubMed Google Scholar; and Stillman, Stillman B. J. Biol. Chem. Full Text PDF PubMed Google Scholar) and DNA the replication and Y. Hurwitz J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). the of the been by immunosorbent and et al., Kelly T.J. EMBO J. PubMed Scopus Google Scholar; et al., Kelly T.J. EMBO J. PubMed Scopus Google Scholar). RPA the initiation of SV40 DNA replication by both T of SV40 DNA et al., F.B. Bullock P. Murakami Y. Wobbe C.R. Weissbach L. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: PubMed Scopus Google Scholar; Wold et al., M.S. J.J. Kelly T.J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: PubMed Scopus Google Scholar; Borowiec et al., J.A. Dean F.B. Bullock P. Hurwitz J. Cell. 1990; Full Text PDF PubMed Scopus Google Scholar) and et al., Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1990; PubMed Scopus Google Scholar). the RPA the of δ, and et al., Lee S.-H. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1989; PubMed Scopus Google Scholar; Lee et al., S.-H. Kwong A.D. Hurwitz J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). While protein and for human RPA to stimulate T of SV40 DNA and in the SV40 human RPA to stimulate et al., Lee S.-H. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1989; PubMed Scopus Google Scholar). This that the RPA and may to the of SV40 replication. The of the RPA p34 and p11, in DNA are not In three RPA and RPA p34, and respectively), are for and Stillman, Stillman B. 5: PubMed Scopus Google Scholar). in RPA is not for of the results in that is with and Stillman, Stillman B. 5: PubMed Scopus Google p34 is phosphorylated the and during et al., Fairman M.P. Stillman B. 1990; PubMed Scopus Google Scholar; et al., A. S. Stillman B. Biol. PubMed Scopus Google Scholar; and Stillman, A. Stillman B. EMBO J. PubMed Scopus Google Scholar). of the for p34 phosphorylation is a cyclin-dependent kinase (Cdk) et al., Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar; and Stillman, A. Stillman B. EMBO J. PubMed Scopus Google Scholar; et al., A. Hurwitz J. J. Biol. Chem. Full Text PDF PubMed Google Scholar; and Hurwitz J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). kinase in DNA replication. in SV40 DNA with T Ag and a DNA or However, could be by the of Cdk and 1988; PubMed Scopus Google Scholar; et al., 1990; PubMed Scopus Google Scholar). of Cdk results in the of to DNA replication and Cell. Full Text PDF PubMed Scopus Google Scholar; and J.J. P. Cell. 1990; Full Text PDF PubMed Scopus Google Scholar). is that the of DNA replication is to RPA p34 phosphorylation by that RPA with both single-stranded DNA and SV40 and phosphorylated in the of and EMBO J. PubMed Scopus Google Scholar). may be that the phosphorylation of RPA p34 results the of Cdk and DNA-dependent kinase both of which function the RPA p34 is also phosphorylated DNA by et al., M.P. S. EMBO J. PubMed Scopus Google Scholar) or and Mol. Cell. Biol. PubMed Scopus Google Scholar). However, the role of RPA p34 phosphorylation in DNA the role of RPA p34 in DNA replication by the function of wild-type RPA with that of a series of mutants. of the N-terminal of RPA p34 its phosphorylation by both Cdk and DNA-dependent kinase but not the ability to SV40 DNA replication in A double point mutant that lacks Cdk phosphorylation sites also its in the SV40 replication However, mutant RPA the C-terminal of p34 only DNA replication and and with SV40 T We the of these results the role of RPA p34 in DNA and and as (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 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 as was SV40 T Ag et al., S.-H. Kwong A.D. Hurwitz J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). and the by et al., Y. A. Bullock P. Hurwitz J. J. Biol. Chem. 1988; Full Text PDF PubMed Google RPA and p34 as et al., U. Hurwitz J. J. Biol. Chem. 1990; 265: Full Text PDF PubMed Google and DNA for and by the wild-type p34, and been et al., Dean Hurwitz J. Lee S.-H. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The double point in which is for and in p34 was by polymerase chain the and the of and the N-terminal and and and and the C-terminal for for and for The and with which and The and used for a with N-terminal and C-terminal p34 was by a of and to the mutant the as The the of p34 the was to that the and and and of been et al., Dean Hurwitz J. Lee S.-H. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). a and with a of of for 40 The for with in of and with to for in of and with of in the of with was and the for the by with and by RPA and RPA mutants with p11, and either wild-type or mutant p34 or as et al., Dean Hurwitz J. Lee S.-H. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google DNA in as 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 In the 40 of kinase, and and of SV40 T of and the of replication or The for which the was of in the the by the of of a containing and DNA was and in a and for The was and to of 40 kinase, of and and and of SV40 T of of of or of SV40 DNA was in the which for by to with and by A et al., Dean Hurwitz J. Lee S.-H. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google α and SV40 DNA α and the of SV40 DNA as et al., S.-H. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1989; PubMed Scopus Google Scholar; et al., Dean Hurwitz J. Lee S.-H. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google RPA and SV40 T was by the by et al., Kelly T.J. EMBO J. PubMed Scopus Google The with of either wild-type or mutant RPA with with in for The of SV40 T Ag and the for extensively with SV40 T Ag was by the with a SV40 T Ag for of the to was to the with the and and the was was to used by et al., Wold M.S. Mol. Cell. Biol. PubMed Scopus Google with the The of the of and was for The complex was a in The was and to the the complex and by p34 the function of RPA p34 in DNA replication, p34 deletion mutants and a p34 mutant that lacks the Cdk sites to and We mutant p34 a complex with RPA with p34 or p11, and RPA or the N-terminal and the C-terminal deletion mutants and to with and and of these deletion mutants to of wild-type RPA not was by the but not by the that the of p34 the p34 N-terminal of RPA p34 for by Cdk and DNA-dependent kinase and DNA-dependent RPA p34 in vitro et al., Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar; and Stillman, A. Stillman B. EMBO J. PubMed Scopus Google Scholar; and EMBO J. PubMed Scopus Google Scholar; and Hurwitz J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). the phosphorylation of both wild-type and mutant RPA p34 replication The of was used in because it of the host replication factors 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 of RPA in was to the RPA in In the of SV40 of wild-type RPA was phosphorylated and as a the This which not RPA that was phosphorylated by cyclin-dependent the p34 mutants and the major Cdk sites not be phosphorylated by A in that Cdk sites In the of SV40 both wild-type RPA and RPA containing efficiently phosphorylated by DNA-dependent kinase by the in and of This that the cyclin-dependent kinase phosphorylation sites are by DNA-dependent kinase. C-terminally deleted RPA p34 a phosphorylation to that of wild-type RPA with the that mutant was phosphorylated efficiently in the in the of SV40 DNA These that the C-terminal of p34 is not for RPA p34 phosphorylation but may be necessary for replication. However, the mutant was not phosphorylated either as by both and for kinase not that the N-terminal of p34 is necessary for its phosphorylation by both This and in to the major sites for Cdk and vitro phosphorylation of wild-type and mutant RPA in the of either wild-type or mutant of of or of SV40 DNA the RPA p34 was C-terminal but the N-terminal of RPA p34 for SV40 DNA RPA p34 is phosphorylated the and phosphorylated during that p34 phosphorylation may be to DNA replication. is not phosphorylated by in mutant RPA p34 phosphorylation is involved in the replication RPA p34 mutants SV40 DNA replication in RPA containing or functioned as as wild-type to the in SV40 DNA replication system DNA replication. We also the function of these mutants in the SV40 system which and SV40 T Ag to SV40 replication with only of the by wild-type RPA or the mutants. This indicates that RPA p34 its C-terminal but not to function in SV40 DNA replication wild-type and mutant RPA function in SV40 DNA replication in SV40 DNA replication in vitro with SV40 DNA SV40 T the of and the of either wild-type or mutant RPA. for and the for SV40 DNA replication in vitro with The replication of SV40 T complex and respectively), and the of wild-type or mutant as as of for and for p34 in the but in the of DNA which of DNA replication are by by the of SV40 DNA replication in containing with wild-type RPA or RPA containing The replication and the in A the and indicates the of DNA DNA was in the containing in containing wild-type However, the in both of that the mutant DNA the of initiation of SV40 DNA in the of wild-type or mutant RPA. containing SV40 T and of either wild-type or mutant and in and SV40 T and the in RPA. the and by was by and the single-stranded and single-stranded of replication including either wild-type RPA or RPA containing for to the of chain initiation the of DNA in mutant to that in wild-type the of was both wild-type and mutant RPA function in chain during SV40 DNA replication in in the of for and the by for DNA chain the as in the for of either wild-type or mutant RPA was in and to in and with the that SV40 T Ag was the in and and the single-stranded and single-stranded of in of RPA a role for RPA p34 in DNA replication, the of RPA containing with that of wild-type RPA in the of α the of SV40 SV40 T Ag and to the subunit of RPA was to be by α to stimulate the of et al., Wold M.S. Kelly T.J. J. Biol. Chem. 1990; 265: Full Text PDF PubMed Google Scholar). However, in the of the p34 and p11, the of may be the of wild-type RPA as as RPA containing α these both wild-type and mutant RPA α by that the replication of RPA containing is not to its ability to stimulate of wild-type and mutant RPA DNA α The of either wild-type or mutant RPA and was to the which of human α, and of for the of RPA and human RPA in SV40 et al., Lee S.-H. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1989; PubMed Scopus Google that the role of RPA in DNA is not to single-stranded the of wild-type and mutant in that as the of the that to that with wild-type RPA. However, DNA was in the containing This could SV40 DNA replication with the mutant is the ability of wild-type and mutant RPA to unwind SV40 The was in the of of SV40 T and and and and and of either wild-type or mutant RPA as the of the indicates the of and the of DNA and DNA ability to bind to and to interact with SV40 T RPA that are to be involved in DNA unwinding. A of wild-type or mutant RPA was to and with of SV40 T et al., Kelly T.J. EMBO J. PubMed Scopus Google wild-type RPA with SV40 T Ag in RPA containing lacks p34 phosphorylation with SV40 T Ag to a as wild-type RPA. RPA containing not interact with SV40 T The of in with T Ag and RPA containing of mutant that of is not to protein of wild-type and mutant RPA with SV40 T or mutant with of SV40 T Ag for SV40 T Ag to RPA was with a SV40 T Ag as wild-type RPA and mutant RPA containing for a with as a et al., Wold M.S. Mol. Cell. Biol. PubMed Scopus Google Scholar; and Borowiec J.A. Mol. Cell. Biol. PubMed Scopus Google as and Borowiec J.A. Mol. Cell. Biol. PubMed Scopus Google that wild-type RPA the but a RPA both wild-type and mutant RPA Furthermore, the of the wild-type complex as a the mutant as and We results as the in of not of wild-type and mutant RPA. of either wild-type or mutant RPA with of for The DNA by has been in the of DNA replication and three subunit is the a role for However, function for either the p34 or the subunit has been the role of the RPA p34 a of mutants. deletion mutants which lacks the phosphorylation sites by A and DNA-dependent and which lacks the by that SV40 DNA replication in deletion of the N-terminal of p34 its phosphorylation by both Cdk and DNA-dependent kinase, a double point mutant of p34 which lacks the major Cdk phosphorylation was efficiently phosphorylated by DNA-dependent kinase. This that the phosphorylation of by DNA-dependent kinase is of its phosphorylation by The and in the N-terminal of p34 may be the for DNA-dependent kinase the mutant could not be phosphorylated by either kinase. the of the N-terminal may be necessary for the kinase to RPA p34, to phosphorylation mutants to be to has been that RPA phosphorylation by Cdk is involved in the of that SV40 DNA replication et al., 1990; PubMed Scopus Google Scholar; and Stillman, A. Stillman B. EMBO J. PubMed Scopus Google Scholar). suggest that RPA p34 by either Cdk or DNA-dependent kinase, is not for SV40 DNA replication in be that SV40 is a model and that RPA p34 phosphorylation may be involved in as DNA kinase RPA p34 only RPA with and EMBO J. PubMed Scopus Google Scholar; et al., A. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar; that RPA p34 and A to the replication during et al., B. Cell. Full Text PDF PubMed Scopus Google Scholar). These results suggest in RPA p34 phosphorylation may only RPA has been to the replication it may be involved in the of DNA Furthermore, and of human been to of RPA p34 and Mol. Cell. Biol. PubMed Scopus Google Scholar; et al., M.P. S. EMBO J. PubMed Scopus Google Scholar) and to the ability of RPA to in vitro DNA replication et al., M.P. S. EMBO J. PubMed Scopus Google Scholar). This that RPA phosphorylation may be involved in the of DNA replication by DNA of the of RPA p34 the of SV40 DNA replication in the of that of p34 is necessary for RPA function in DNA replication. suggest that the C-terminal of RPA p34 is involved in the RPA and SV40 T to during the initiation of DNA replication. The of mutant RPA was to that of wild-type that the RPA and SV40 T Ag is for of SV40 be that the of RPA containing was that of wild-type RPA of RPA. be necessary to the role of RPA p34 in DNA both in vitro SV40 DNA replication and the of RPA α However, has the of SV40 DNA et al., U. Hurwitz J. J. Biol. Chem. 1990; 265: Full Text PDF PubMed Google Scholar). In mutant RPA that lacks the C-terminal of RPA SV40 DNA replication because it to interact with SV40 T The stimulate α These results could be as to the to p34 and the which is necessary for α the the domain is and interact with the p34 SV40 DNA mutant RPA containing C-terminally deleted p34 DNA unwinding. This be by the that the deleted both the and the necessary for SV40 DNA unwinding. These sites the deleted 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 with and Stillman, 1988Fairman M.P. Stillman B. EMBO J. 1988; 7: 1211-1218Crossref PubMed Scopus (291) Google Scholar) and are and Stillman, Stillman B. 1989; PubMed Scopus Google and Stillman, Stillman B. 5: PubMed Scopus Google Scholar; et al., PubMed Scopus Google Scholar; et al., Mol. Biochem. PubMed Scopus Google Scholar). RPA subunits are in p34 a complex with to which et al., Dean Hurwitz J. Lee S.-H. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar; et al., Wold M.S. J. Biol. Chem. Full Text PDF PubMed Google the human RPA subunits are to et al., Wold M.S. Kelly T.J. J. Biol. Chem. 1990; 265: Full Text PDF PubMed Google et al., Kelly T.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar; et al., Kelly T.J. EMBO J. 1990; PubMed Scopus Google Scholar; and Stillman, Stillman B. 5: PubMed Scopus Google Scholar; et al., Kelly T.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). However, RPA for human RPA in the in vitro SV40 replication system and Stillman, Stillman B. 1989; PubMed Scopus Google that RPA and replication SV40 T and the DNA polymerase complex interact in vitro to a complex that is for both and Kelly, Kelly T.J. Mol. Cell. Biol. PubMed Google Scholar; and Stillman, Stillman B. J. Biol. Chem. Full Text PDF PubMed Google Scholar) and DNA the replication and Y. Hurwitz J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). the of the been by immunosorbent and et al., Kelly T.J. EMBO J. PubMed Scopus Google Scholar; et al., Kelly T.J. EMBO J. PubMed Scopus Google Scholar). RPA the initiation of SV40 DNA replication by both T of SV40 DNA et al., F.B. Bullock P. Murakami Y. Wobbe C.R. Weissbach L. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: PubMed Scopus Google Scholar; Wold et al., M.S. J.J. Kelly T.J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: PubMed Scopus Google Scholar; Borowiec et al., J.A. Dean F.B. Bullock P. Hurwitz J. Cell. 1990; Full Text PDF PubMed Scopus Google Scholar) and et al., Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1990; PubMed Scopus Google Scholar). the RPA the of δ, and et al., Lee S.-H. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1989; PubMed Scopus Google Scholar; Lee et al., S.-H. Kwong A.D. Hurwitz J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). While protein and for human RPA to stimulate T of SV40 DNA and in the SV40 human RPA to stimulate et al., Lee S.-H. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1989; PubMed Scopus Google Scholar). This that the RPA and may to the of SV40 replication. The of the RPA p34 and p11, in DNA are not In three RPA and RPA p34, and respectively), are for and Stillman, Stillman B. 5: PubMed Scopus Google Scholar). in RPA is not for of the results in that is with and Stillman, Stillman B. 5: PubMed Scopus Google p34 is phosphorylated the and during et al., Fairman M.P. Stillman B. 1990; PubMed Scopus Google Scholar; et al., A. S. Stillman B. Biol. PubMed Scopus Google Scholar; and Stillman, A. Stillman B. EMBO J. PubMed Scopus Google Scholar). of the for p34 phosphorylation is a cyclin-dependent kinase (Cdk) et al., Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar; and Stillman, A. Stillman B. EMBO J. PubMed Scopus Google Scholar; et al., A. Hurwitz J. J. Biol. Chem. Full Text PDF PubMed Google Scholar; and Hurwitz J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). kinase in DNA replication. in SV40 DNA with T Ag and a DNA or However, could be by the of Cdk and 1988; PubMed Scopus Google Scholar; et al., 1990; PubMed Scopus Google Scholar). of Cdk results in the of to DNA replication and Cell. Full Text PDF PubMed Scopus Google Scholar; and J.J. P. Cell. 1990; Full Text PDF PubMed Scopus Google Scholar). is that the of DNA replication is to RPA p34 phosphorylation by that RPA with both single-stranded DNA and SV40 and phosphorylated in the of and EMBO J. PubMed Scopus Google Scholar). may be that the phosphorylation of RPA p34 results the of Cdk and DNA-dependent kinase both of which function the RPA p34 is also phosphorylated DNA by et al., M.P. S. EMBO J. PubMed Scopus Google Scholar) or and Mol. Cell. Biol. PubMed Scopus Google Scholar). 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