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Association of cyclin A and cdk2 with SV40 DNA in replication initiation complexes is cell cycle dependent

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Abstract

The cell cycle is driven by the sequential activation of a family of cyclin-dependent kinases (CDK) in association with cyclins. In mammalian cells the timing of activation of cyclin A-associated kinase activity coincides with the onset of DNA synthesis in S-phase. Using in vitro replication of SV40 origin-containing DNA as a model system, we have analyzed the proteins associated with DNA during initiation of DNA replication in S-phase cell extracts. This analysis reveals that, in addition to replication initiation proteins, cyclin A and cdk2 are also specifically associated with DNA. The association of cyclin A and cdk2 with DNA during initiation is cell cycle regulated and occurs specifically in the presence of SV40 origin-containing plasmid and SV40 T antigen (the viral replication initiator protein). The interactions among proteins involved in initiation play an important role in DNA replication. We therefore investigated the ability of cyclin A and cdk2 to associate with replication initiation proteins. Under replication initiation conditions, cyclin A and cdk2 from S-phase extracts specifically associate with SV40 T antigen. Further, the interaction of cyclin A-cdk2 with SV40 T antigen is mediated via cyclin A, and purified recombinant cyclin A associates directly with SV40 T antigen. Taken together, our results suggest that cyclin A and cdk2 are components of the SV40 replication initiation complex, and that protein-protein interactions between cyclin A-cdk2 and T antigen may facilitate the association of cyclin A-cdk2 with the complex.

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References

  • Adamczewski JP, Gannon JV, Hunt T (1993) Simian virus 40 large T antigen associates with cyclin A and p33cdk2. J Virol 67:6551–6557

    PubMed  CAS  Google Scholar 

  • Atherton-Fessler S, Parker LL, Geahlen RL, Piwnica-Worms H (1993) Mechanisms of p34cdc2 regulation. Mol Cell Biol 13:1675–1685

    PubMed  CAS  Google Scholar 

  • Blow JJ, Nurse P (1990) A cdc2-like protein is involved in the initiation of DNA replication inXenopus egg extracts. Cell 62: 855–862

    Article  PubMed  CAS  Google Scholar 

  • Borowiec JA, Dean FB, Bullock PA, Hurwitz J (1990) Binding and unwinding—how T antigen engages the SV40 origin of DNA replication. Cell 60: 181–184

    Article  PubMed  CAS  Google Scholar 

  • Brenot-Bosc F, Gupta S, Margolis RL, Fotedar R (1995) Changes in the subcellular localization of replication initiation proteins and cell cycle proteins during G1- to S-phase transition in mammalian cells. Chromosoma 103: 517–527

    PubMed  CAS  Google Scholar 

  • Cardoso MC, Leonhardt H, Nadal-Ginard B (1993) Reversal of terminal differentiation and control of DNA replication: cyclin A and Cdk2 specifically localize at subnuclear sites of DNA replication. Cell 74: 979–992

    Article  PubMed  CAS  Google Scholar 

  • Chen J, Jackson PK, Kirschner MW, Dutta A (1995) Separate domains of p21 involved in the inhibition of Cdk kinase and PCNA. Nature 374: 386–388

    Article  PubMed  CAS  Google Scholar 

  • Chevalier S, Tassan JP, Cox R, Philippe M, Ford C (1995) Both cdc2 and cdk2 promote S phase initiation inXenopus egg extracts. J Cell Sci 108: 1831–1841

    PubMed  CAS  Google Scholar 

  • Collins KL, Kelly TJ (1991) Effects of T antigen and replication protein A on the initiation of DNA synthesis by DNA polymerase alpha-primase. Mol Cell Biol 11: 2108–2115

    PubMed  CAS  Google Scholar 

  • D'Urso G, Marraccino RL, Marshak DR, Roberts JM (1990) Cell cycle control of DNA replication by a homologue from human cells of the p34cdc2 protein kinase. Science 250: 786–791

    Article  PubMed  Google Scholar 

  • Din S, Brill SJ, Fairman MP, Stillman B (1990) Cell-cycle-regulated phosphorylation of DNA replication factor A from human and yeast cells. Genes Dev 4: 968–977

    PubMed  CAS  Google Scholar 

  • Dornreiter I, Erdile LF, Gilbert IU, von Winkler D, Kelly TJ, Fanning E (1992) Interaction of DNA polymerase alpha-primase with cellular replication protein A and SV40 T antigen. EMBO J 11: 769–776

    PubMed  CAS  Google Scholar 

  • Dornreiter I, Copeland WC, Wang TS (1993) Initiation of simian virus 40 DNA replication requires the interaction of a specific domain of human DNA polymerase alpha with larger T antigen. Mol Cell Biol 13: 809–820

    PubMed  CAS  Google Scholar 

  • Dutta A, Stillman B (1992) cdc2 family kinases phosphorylate a human cell DNA replication factor, RPA, and activate DNA replication. EMBO J 11: 2189–2199

    PubMed  CAS  Google Scholar 

  • Dyson N, Buchkovich K, Whyte P, Harlow E (1989) The cellular 107K protein that binds to adenovirus E1A also associates with the large T antigens of SV40 and JC virus. Cell 58: 249–255

    Article  PubMed  CAS  Google Scholar 

  • Ewen ME, Ludlow JW, Marsilio E, DeCaprio JA, Millikan RC, Cheng SH, Paucha E, Livingston DM (1989) An N-terminal transformation-governing sequence of SV40 large T antigen contributes to the binding of both p110Rb and a second cellular protein, p120. Cell 58: 257–267

    Article  PubMed  CAS  Google Scholar 

  • Ewen ME, Faha B, Harlow E, Livingston DM (1992) Interaction of p107 with cyclin A independent of complex formation with viral oncoproteins. Science 255: 85–87

    Article  PubMed  CAS  Google Scholar 

  • Faha B, Ewen ME, Tsai LH, Livingston DM, Harlow E (1992) Interaction between human cyclin A and adenovirus E1 A-associated p107 protein. Science 255: 87–90

    Article  PubMed  CAS  Google Scholar 

  • Fang F, Newport JW (1991) Evidence that the G1-S and G2-M transitions are controlled by different cdc2 proteins in higher eukaryotes. Cell 66: 731–742

    Article  PubMed  CAS  Google Scholar 

  • Fotedar A, Cannella D, Fitzgerald P, Rousselle T, Gupta S, Doree M, Fotedar R (1996) Role for cyclin A dependent kinase in DNA replication in human S phase cell extracts. J Biol Chem 271 (in press)

  • Fotedar R, Fotedar A (1995) Cell cycle control of DNA replications. In: Meijer L, Guidet S, Tung HYL (eds) Progress in Cell Cycle Research. Plenum Press, New York, pp 73–89

    Google Scholar 

  • Fotedar R, Roberts JM (1991) Association of p34cdc2 with replicating DNA. Cold Spring Harbor Symp Quant Biol 56: 325–333

    PubMed  CAS  Google Scholar 

  • Fotedar R, Roberts JM (1992) Cell cycle regulated phosphorylation of RPA-32 occurs within the replication initiation complex. EMBO J 11: 2177–2187

    PubMed  CAS  Google Scholar 

  • Fotedar R, Fitzgerald P, Rousselle T, Cannella D, Doree M, Messier H, Fotedar A (1996) p21 contains independent binding sites for cyclin and cdk2: Both sites are required to inhibit cdk2 kinase activity. Oncogene 12: 2155–2164

    PubMed  CAS  Google Scholar 

  • Gannon JV, Lane DP (1987) p53 and DNA polymerase alpha compete for binding to SV40 T antigen. Nature 329: 456–458

    Article  PubMed  CAS  Google Scholar 

  • Girard F, Strausfeld U, Fernandez A, Lamb NJ (1991) Cyclin A is required for the onset of DNA replication in mammalian fibroblasts. Cell 67: 1169–1179

    Article  PubMed  CAS  Google Scholar 

  • Hoss A, Moarefi I, Scheidtmann KH, Cisek LJ, Corden JL, Dornreiter I, Arthur AK, Fanning E (1990) Altered phosphorylation pattern of simian virus 40 T antigen expressed in insect cells by using a baculovirus vector. J Virol 64: 4799–4807

    PubMed  CAS  Google Scholar 

  • Hsi KL, Copeland WC, Wang TS (1990) Human DNA polymerase alpha catalytic polypeptide binds ConA and RCA and contains a specific labile site in the N-terminus. Nucleic Acids Res. 18: 6231–6237

    PubMed  CAS  Google Scholar 

  • Hurwitz J, Dean FB, Kwong AD, Lee SH (1990) The in vitro replication of DNA containing the SV40 origin. J Biol Chem 265: 18043–18046

    PubMed  CAS  Google Scholar 

  • Jackson PK, Chevalier S, Philippe M, Kirschner MW (1995) Early events in DNA replication require cyclin E and are blocked by p21CIP1. J Cell Biol 130: 755–769

    Article  PubMed  CAS  Google Scholar 

  • Koff A, Giordano A, Desai D, Yamashita K, Harper JW, Elledge S, Nishimoto T, Morgan DO, Franza BR, Roberts JM (1992) Formation and activation of a cyclin E-cdk2 complex during the G1 phase of the human cell cycle. Science 257: 1689–1694

    Article  PubMed  CAS  Google Scholar 

  • Lee MG, Nurse P (1987) Complementation used to clone a human homologue of the fission yeast cell cycle control gene cdc2. Nature 327: 31–35

    Article  PubMed  CAS  Google Scholar 

  • Maller JL (1990)Xenopus, oocytes and the biochemistry of cell division. Biochemistry 29: 3157–3166

    Article  PubMed  CAS  Google Scholar 

  • McVey D, Brizuela L, Mohr I, Marshak DR, Gluzman Y, Beach D (1989) Phosphorylation of large tumour antigen by cdc2 stimulates SV40 DNA replication. Nature 341: 503–507

    Article  PubMed  CAS  Google Scholar 

  • McVey D, Ray S, Gluzman Y, Berger L, Wildeman AG, Marshak DR, Tegtmeyer P (1993) cdc2 phosphorylation of threonini 124 activates the origin-unwinding functions of simian virus 40 T antigen. J Virol 67: 5206–5215

    PubMed  CAS  Google Scholar 

  • Melendy T, Stillman B (1993) An interaction between replication protein A and SV40 T antigen appears essential for primosome assembly during SV40 DNA replication. J Biol Chem 268: 3389–3395

    PubMed  CAS  Google Scholar 

  • Meyerson M, Enders GH, Wu CL, Su LK, Gorka C, Nelson C, Harlow E, Tsai LH (1992) A family of human cdc2-related protein kinases. EMBO J, 11: 2909–2917

    PubMed  CAS  Google Scholar 

  • Morris GF, Mathews MB (1989) Regulation of proliferating cell nuclear antigen during the cell cycle. J Biol Chem 264: 13856–13864

    PubMed  CAS  Google Scholar 

  • Muzi-Falconi M, Brown GW, Kelly TJ (1996) Controlling initiation during the cell cycle. Curr Biol 6: 229–233

    Article  PubMed  CAS  Google Scholar 

  • Nasheuer HP, Moore A, Wahl AF, Wang TS (1991) Cell cycle-dependent phosphorylation of human DNA polymerase alpha. J Biol Chem 266: 7893–7903

    PubMed  CAS  Google Scholar 

  • Pagano M, Draetta G, Jansen-Durr P (1992a) Association of cdk2 kinase with the transcription factor E2F during S phase. Science 255: 1144–1147

    Article  PubMed  CAS  Google Scholar 

  • Pagano M, Pepperkok R, Verde F, Ansorge W, Draetta G (1992b) Cyclin A is required at two points in the human cell cycle. EMBO J 11: 961–971

    PubMed  CAS  Google Scholar 

  • Pagano M, Pepperkok R, Lukas J, Baldin V, Ansorge W, Bartek J, Draetta G (1993) Regulation of the cell cycle by the cdk2 protein kinase in cultured human fibroblasts. J Cell Biol 121: 101–111

    Article  PubMed  CAS  Google Scholar 

  • Pines J (1993) Cyclins and cyclin-dependent kinases: take your partners. Trends Biochem Sci 18: 195–197

    Article  PubMed  CAS  Google Scholar 

  • Pines J, Hunter T (1990) Human cyclin A is adenovirus E1A-associated protein p60 and behaves differently from cyclin B. Nature 346: 760–763

    Article  PubMed  CAS  Google Scholar 

  • Prosperi E, Scovassi AI, Stivala LA, Bianchi L (1994) Proliferating cell nuclear antigen bound to DNA synthesis sites: phosphorylation and association with cyclin D1 and cyclin A. Exp Cell Res 215: 257–262

    Article  PubMed  CAS  Google Scholar 

  • Roberts JM, D'Urso G (1988) An origin unwinding activity regulates initiation of DNA replication during mammalian cell cycle. Science 241: 1486–1489

    Article  PubMed  CAS  Google Scholar 

  • Rosenblatt J, Gu Y, Morgan DO (1992) Human cyclin-dependent kinase 2 is activated during the S and G2 phases of the cell cycle and associates with cyclin A. Proc Natl Acad Sci USA 89: 2824–2828

    Article  PubMed  CAS  Google Scholar 

  • Sherr CJ (1993) Mammalian G1 cyclins. Cell 73: 1059–1065

    Article  PubMed  CAS  Google Scholar 

  • Smale ST, Tjian R (1986) T-antigen-DNA polymerase alpha complex implicated in simian virus 40 DNA replication. Mol Cell Biol 6: 4077–4087

    PubMed  CAS  Google Scholar 

  • Sobczak-Thepot J, Harper F, Florentin Y, Zindy F, Brechot C, Puvion E (1993) Localization of cyclin A at the sites of cellular DNA replication. Exp Cell Res 206: 43–48

    Article  PubMed  CAS  Google Scholar 

  • Stillman B (1994) Initiation of chromosomal DNA replication in eukaryotes. Lessons from lambda. J Biol Chem 269: 7047–7050

    PubMed  CAS  Google Scholar 

  • Strausfeld UP, Howell M, Rempel R, Maller JL, Hunt T, Blow JJ (1994) Cip1 blocks the initiation of DNA replication inXenopus extracts by inhibition of cyclin-dependent kinases. Curr Biol 4: 876–883

    Article  PubMed  CAS  Google Scholar 

  • Tsurimoto T, Fairman MP; Stillman B (1989) Simian virus 40 DNA replication in vitro: identification of multiple stages of initiation. Mol Cell Biol 9: 3839–3849

    PubMed  CAS  Google Scholar 

  • van den Heuvel S, Harlow E (1993) Distinct roles for cyclin-dependent kinases in cell cycle control. Science 262: 2050–2054

    Article  PubMed  Google Scholar 

  • Vogelstein B, Pardoll DM, Coffey DS (1980) Supercoiled loops and eucaryotic DNA replication. Cell 22: 79–85

    Article  PubMed  CAS  Google Scholar 

  • Waga S, Bauer G, Stillman B (1994a) Reconstitution of complete SV40 DNA replication with purified replication factors. J Biol Chem 269: 10923–10934

    PubMed  CAS  Google Scholar 

  • Waga S, Hannon GJ, Beach D, Stillman B (1994b) The p21 inhibitor of cyclin-dependent kinases controls DNA replication by interaction with PCNA. Nature 369: 574–578

    Article  PubMed  CAS  Google Scholar 

  • Wessel R, Schweizer J, Stahl H (1992) Simian virus 40 T-antigen DNA helicase is a hexamer which forms a binary complex during bidirectional unwinding from the viral origin of DNA replication. J Virol 66: 804–815

    PubMed  CAS  Google Scholar 

  • Wobbe CR, Dean F, Weissbach L, Hurwitz J (1985) In vitro replication of duplex circular DNA containing the simian virus 40 DNA origin site. Proc Natl Acad Sci USA 82: 5710–5714

    Article  PubMed  CAS  Google Scholar 

  • Wold MS, Li JJ, Kelly TJ (1987) Initiation of simian virus 40 DNA replication in vitro: large-tumor-antigen- and origin-dependent unwinding of the template. Proc Natl Acad Sci USA 84: 3643–3647

    Article  PubMed  CAS  Google Scholar 

  • Zeng XR, Hao H, Jiang Y, Lee MY (1994) Regulation of human DNA polymerase delta during the cell cycle. J Biol Chem 269: 24027–24033

    PubMed  CAS  Google Scholar 

  • Zindy F, Lamas E, Chenivesse X, Sobczak J, Wang J, Fesquet D, Henglein B, Brechot C (1992) Cyclin A is required in S phase in normal epithelial cells. Biochem Biophys Res Commun 182: 1144–1154

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Institut de Biologie Structurale J.-P. Ebel, 41 Avenue des Martyrs, F-38027, Grenoble, Cedex 1, France

    Dominique Cannella & Rati Fotedar

  2. Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 98104, Seattle, WA, USA

    James M. Roberts

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  1. Dominique Cannella
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  2. James M. Roberts
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Correspondence to Rati Fotedar.

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Edited by: J. Huberman

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Cannella, D., Roberts, J.M. & Fotedar, R. Association of cyclin A and cdk2 with SV40 DNA in replication initiation complexes is cell cycle dependent. Chromosoma 105, 349–359 (1997). https://doi.org/10.1007/BF02529750

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  • DOI: https://doi.org/10.1007/BF02529750

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