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Changes in the subcellular localization of replication initiation proteins and cell cycle proteins during G1- to S-phase transition in mammalian cells

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Abstract

DNA replication in eukaryotic cells is restricted to the S-phase of the cell cycle. In a cell-free replication model system, using SV40 origin-containing DNA, extracts from G1 cells are inefficient in supporting DNA replication. We have undertaken a detailed analysis of the subcellular localization of replication proteins and cell cycle regulators to determine when these proteins are present in the nucleus and therefore available for DNA replication. Cyclin A and cdk2 have been implicated in regulating DNA replication, and may be responsible for activating components of the DNA replication mitiation complex on entry into S-phase. G1 cell extracts used for in vitro replication contain the replication proteins RPA (the eukaryotic single-stranded DNA binding protein) and DNA polymerase α as well as cdk2, but lack cyclin A. On localizing these components in G1 cells we find that both RPA and DNA polymerase α are present as nuclear proteins, while cdk2 is primarily cytoplasmic and there is no detectable cyclin A. An apparent change in the distribution of these proteins occurs as the cell enters S-phase. Cyclin A becomes abundant and both cyclin A and cdk2 become localized to the nucleus in S-phase. In contrast, the RPA-34 and RPA-70 subunits of RPA, which are already nuclear, undergo a transition from the uniform nuclear distribution observed during G1, and now display a distinct punctate nuclear pattern. The initiation of DNA replication therefore most likely occurs by modification and activation of these replication initiation proteins rather than by their recruitment to the nuclear compartment.

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References

  • Adachi Y, Laemmli UK (1992) Identification of nuclear pre-replication centers poised for DNA synthesis in Xenopus egg extracts: immunolocalization study of replication protein A. J Cell Biol 119:1–15

    Google Scholar 

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

    Google Scholar 

  • Beach D, Durkacz B, Nurse P (1982) Functionally homologous cell cycle control genes in budding and fission yeast. Nature 300:706–709

    Google Scholar 

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

    Google Scholar 

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

    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

    Google Scholar 

  • Din S-U, 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

    Google Scholar 

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

    Google Scholar 

  • Dornreiter I, Copeland WC, Wang TS-F (1993) Initiation of Simian Virus 40 DNA replication requires the interaction of a specific domain of human DNA polymerase α with large T antigen. Mol Cell Biol 13:809–820

    Google Scholar 

  • Dulic V, Lees E, Reed SI (1992) Association of human cyclin E with a periodic G1-S phase protein kinase. Science 257:1958–1961

    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

    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

    Google Scholar 

  • Elledge SJ, Spottswood MR (1991) A new human p34 protein kinase, cdk2, identified by complementation of a CDC28 mutation is Saccharomyces cerevisiae, is a homolog of Xenopus Eg1. EMBO J 10:2653–2659

    Google Scholar 

  • Elledge SJ, Richman R, Hall FL, Williams RT, Lodgsson N, Harper JH (1992) Cdk2 encodes a 33 kDa cyclin A-associated protein kinase and is expressed before cdc2 in the cell-cycle. Proc Natl Acad Sci USA 89:2907–2911

    Google Scholar 

  • Endo T, Nadal-Ginard B (1989) SV40 T antigen induces reentry of terminally differentiated myotubes into the cell cycle. In: Kedes LH, Stockdale FE (eds) Cell and molecular biology of muscle development. (UCLA Symp Mol Cell Biol) Alan R. Liss, NY, pp 95–104

    Google Scholar 

  • Erdile LF, Wold MS, Kelly TJ (1990) The primary structure of the 32-kDa subunit of human replication protein A. J Biol Chem 265:3177–3182

    Google Scholar 

  • Erdile LF, Heyer W-D, Kolodner R, Kelly TJ (1991) Characterization of a cDNA encoding the 70-kDa single-stranded DNA-binding subunit of human replication protein A and the role of the protein in DNA replication. J Biol Chem 266:12090–12098

    Google Scholar 

  • Fairman MP, Stillman B (1988) Cellular factors required for multiple stages of SV40 DNA replication in vitro. EMBO J 7: 1211–1218

    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

    Google Scholar 

  • Fanning E, Knippers R (1992) Structure and function of simian virus 40 large tumor antigen. Annu Rev Biochem 61:55–85

    Google Scholar 

  • Fotedar R, Roberts JM (1989) Multistep pathway for replication-dependent nucleosome assembly. Proc Natl Acad Sci USA 86:6459–6463

    Google Scholar 

  • Fotedar R, Roberts JM (1991) Cell cycle dependent association of p34cdc2 with replicating DNA. Cold Spring Harbor Symp Quant Biol LVI:325–333

    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

    Google Scholar 

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

    Google Scholar 

  • Guilly MN, Dannon F, Brouet JC, Bornens M, Courvalin JC (1987) Autoantibodies to nuclear lamin B in a patient with thrombopenia. Eur J Cell Biol 43:266–272

    Google Scholar 

  • Henricksen LA, Umbricht CB, Wold MS (1994) Recombinant replication protein A: expression, complex formation, and functional characterization. J Biol Chem 269:11121–11132

    Google Scholar 

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

    Google Scholar 

  • Kelly TJ (1988) SV40 DNA replication. J Biol Chem 263:17889–17892

    Google Scholar 

  • Kenny MK, Lee S-H, Hurwitz J (1989) Multiple functions of human single-stranded-DNA binding protein in simian virus 40 DNA replication: single-strand stabilization and stimulation of DNA polymerases α and δ. Proc Natl Acad Sci USA 86:9757–9761

    Google Scholar 

  • Kenny MK, Schlegel U, Furneaux H, Hurwitz J (1990) The role of human single-stranded DNA binding protein and its individual subunits in simian virus 40 DNA replication. J Biol Chem 265:7693–7700

    Google Scholar 

  • Kill IR, Bridger JM, Campbell KHS, Maldonado-Codina G, Hutchison CJ (1991) The timing of the formation and usage of replicase clusters in S-phase nuclei of human diploid fibroblasts. J Cell Sci 100:869–876

    Google Scholar 

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

    Google Scholar 

  • Lanford RE (1988) Expression of Simian virus 40 T antigen in insect cells using a baculovirus expression vector. Virology 167:72–81

    Google Scholar 

  • Li JJ, Kelly TJ (1984) Simian virus 40 DNA replication in vitro. Proc Natl Acad Sci USA 81:6973–6977

    Google Scholar 

  • Maridor G, Gallant P, Golsteyn R, Nigg EA (1993) Nuclear localization of vertebrate cyclin A correlates with its ability to form complexes with cdk catalytic subunits. J Cell Sci 106:535–544

    Google Scholar 

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

    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

    Google Scholar 

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

    Google Scholar 

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

    Google Scholar 

  • Murakami Y, Hurwitz J (1993) Functional interactions between SV40 T antigen and other replication proteins at the replication fork. J Biol Chem 268:11008–11017

    Google Scholar 

  • Ninomiya-Tsuji J, Nomoto S, Yasuda H, Reed SI, Matsumoto K (1991) Cloning of a human cDNA encoding a cdc2-related kinase by complementation of a budding yeast cdc28 mutation. Proc Natl Acad Sci USA 88:9006–9010

    Google Scholar 

  • Nishikori M, Hansen H, Jhanwar S, Fried J, Sordillo P, Koziner B, Lloyd K, Clarkson B (1984) Establishment of a near-tetraploid B-cell lymphoma line with duplication of the 8;14 translocation. Cancer Genet Cytogenet 12:39–50

    Google Scholar 

  • Pagano M, Draetta G, Jansen-Durr P (1992a) Association of cdk2 kinase with the transcription factor E2F during S phase. Science 254:1144–1147

    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

    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

    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

    Google Scholar 

  • Pines J, Hunter T (1991) Human cyclins A and B1 are differentially located in the cell and undergo cell cycle-dependent nuclear transport. J Cell Biol 115:1–17

    Google Scholar 

  • Prelich G, Tan C-K, Kostura M, Mathews MB, So AG, Downey KM, Stillman B (1987) Functional identity of proliferating cell nuclear antigen and a DNA polymerase-δ auxiliary protein. Nature 326:517–520

    Google Scholar 

  • Reed SI (1992) The role of p34 kinases in the G1 to S-phase transition. Annu Rev Cell Biol 8:529–561

    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

    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

    Google Scholar 

  • Simanis V, Lane DP (1985) An immunoaffinity purification procedure for SV40 large T antigen. Virology 144:88–100

    Google Scholar 

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

    Google Scholar 

  • Stigger E, Dean FB, Hurwitz J, Lee S-H (1994) Reconstitution of functional human single-stranded DNA-binding protein from individual subunits expressed by recombinant baculoviruses. Proc Natl Acad Sci USA 91:579–583

    Google Scholar 

  • Stillman B (1986) Chromatin assembly during SV40 DNA replication in vitro. Cell 45:555–565

    Google Scholar 

  • Stillman B, Gluzman Y (1985) Replication and supercoiling of simian virus 40 DNA in cell extracts from human cells. Mol Cell Biol 5:2051–2060

    Google Scholar 

  • Tsai L-H, Harlow E, Meyerson M (1991) Isolation of the human cdk2 gene that encodes the cyclin A- and adenovirus E1A-associated p33 kinase. Nature 353:174–177

    Google Scholar 

  • Tsai L-H, Lees E, Faha B, Harlow E, Riabowol K (1993) The cdk2 kinase is required for the G1-to-S transition in mammalian cells. Oncogene 8:1593–1602

    Google Scholar 

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

    Google Scholar 

  • Umbricht CB, Erdile LF, Jabs EW, Kelly TJ (1993) Cloning, over-expression, and genomic mapping of the 14-kDa subunit of human replication protein A. J Biol Chem 268:6131–6138

    Google Scholar 

  • Wobbe CR, Dean FB, 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

    Google Scholar 

  • Wobbe CR, Weissbach L, Borowiec JA, Dean FB, Murakami Y, Bullock P, Hurwitz J (1987) Replication of simian virus 40 origin-containing DNA in vitro with purified proteins. Proc Natl Acad Sci USA 84:1834–1838

    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

    Google Scholar 

  • Wold MS, Kelly TJ (1988) Purification and characterization of replication protein A, a cellular protein required for in vitro replication of simian virus 40 DNA. Proc Natl Acad Sci USA 85:2523–2527

    Google Scholar 

  • Wold MS, Weinberg DH, Virshup DM, Li JJ, Kelly TJ (1989) Identification of cellular proteins required for simian virus 40 DNA replication in vitro. J Biol Chem 264:2801–2809

    Google Scholar 

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

    Google Scholar 

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

    Françoise Brénot-Bosc, Sunita Gupta, Robert L. Margolis & Rati Fotedar

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  1. Françoise Brénot-Bosc
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  2. Sunita Gupta
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  3. Robert L. Margolis
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  4. Rati Fotedar
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Brénot-Bosc, F., Gupta, S., Margolis, R.L. et al. 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 (1995). https://doi.org/10.1007/BF00355316

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