PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of jcellbiolHomeEditorsContactInstructions for Authors
 
J Cell Biol. 1995 August 2; 130(4): 755–769.
PMCID: PMC2199964

Early events in DNA replication require cyclin E and are blocked by p21CIP1

Abstract

Using immunodepletion of cyclin E and the inhibitor protein p21WAF/CIP1, we demonstrate that the cyclin E protein, in association with Cdk2, is required for the elongation phase of replication on single-stranded substrates. Although cyclin E/Cdk2 is likely to be the major target by which p21 inhibits the initiation of sperm DNA replication, p21 can inhibit single-stranded replication through a mechanism dependent on PCNA. While the cyclin E/Cdk2 complex appears to have a role in the initiation of DNA replication, another Cdk kinase, possibly cyclin A/Cdk, may be involved in a later step controlling the switch from initiation to elongation. The provision of a large maternal pool of cyclin E protein shows that regulators of replication are constitutively present, which explains the lack of a protein synthesis requirement for replication in the early embryonic cell cycle.

Full Text

The Full Text of this article is available as a PDF (2.0M).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Adachi Y, Laemmli UK. Identification of nuclear pre-replication centers poised for DNA synthesis in Xenopus egg extracts: immunolocalization study of replication protein A. J Cell Biol. 1992 Oct;119(1):1–15. [PMC free article] [PubMed]
  • Adachi Y, Laemmli UK. Study of the cell cycle-dependent assembly of the DNA pre-replication centres in Xenopus egg extracts. EMBO J. 1994 Sep 1;13(17):4153–4164. [PubMed]
  • Amon A, Irniger S, Nasmyth K. Closing the cell cycle circle in yeast: G2 cyclin proteolysis initiated at mitosis persists until the activation of G1 cyclins in the next cycle. Cell. 1994 Jul 1;77(7):1037–1050. [PubMed]
  • Blow JJ, Nurse P. A cdc2-like protein is involved in the initiation of DNA replication in Xenopus egg extracts. Cell. 1990 Sep 7;62(5):855–862. [PubMed]
  • Blow JJ, Watson JV. Nuclei act as independent and integrated units of replication in a Xenopus cell-free DNA replication system. EMBO J. 1987 Jul;6(7):1997–2002. [PubMed]
  • Blow JJ, Laskey RA. Initiation of DNA replication in nuclei and purified DNA by a cell-free extract of Xenopus eggs. Cell. 1986 Nov 21;47(4):577–587. [PubMed]
  • Blow JJ, Laskey RA. A role for the nuclear envelope in controlling DNA replication within the cell cycle. Nature. 1988 Apr 7;332(6164):546–548. [PubMed]
  • Brizuela L, Draetta G, Beach D. p13suc1 acts in the fission yeast cell division cycle as a component of the p34cdc2 protein kinase. EMBO J. 1987 Nov;6(11):3507–3514. [PubMed]
  • Cardoso MC, Leonhardt H, Nadal-Ginard B. Reversal of terminal differentiation and control of DNA replication: cyclin A and Cdk2 specifically localize at subnuclear sites of DNA replication. Cell. 1993 Sep 24;74(6):979–992. [PubMed]
  • Chen J, Jackson PK, Kirschner MW, Dutta A. Separate domains of p21 involved in the inhibition of Cdk kinase and PCNA. Nature. 1995 Mar 23;374(6520):386–388. [PubMed]
  • Cox LS. DNA replication in cell-free extracts from Xenopus eggs is prevented by disrupting nuclear envelope function. J Cell Sci. 1992 Jan;101(Pt 1):43–53. [PubMed]
  • Draetta GF. Mammalian G1 cyclins. Curr Opin Cell Biol. 1994 Dec;6(6):842–846. [PubMed]
  • Dulić V, Lees E, Reed SI. Association of human cyclin E with a periodic G1-S phase protein kinase. Science. 1992 Sep 25;257(5078):1958–1961. [PubMed]
  • el-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R, Trent JM, Lin D, Mercer WE, Kinzler KW, Vogelstein B. WAF1, a potential mediator of p53 tumor suppression. Cell. 1993 Nov 19;75(4):817–825. [PubMed]
  • Fang F, Newport JW. Evidence that the G1-S and G2-M transitions are controlled by different cdc2 proteins in higher eukaryotes. Cell. 1991 Aug 23;66(4):731–742. [PubMed]
  • Fang F, Newport JW. Distinct roles of cdk2 and cdc2 in RP-A phosphorylation during the cell cycle. J Cell Sci. 1993 Nov;106(Pt 3):983–994. [PubMed]
  • Flores-Rozas H, Kelman Z, Dean FB, Pan ZQ, Harper JW, Elledge SJ, O'Donnell M, Hurwitz J. Cdk-interacting protein 1 directly binds with proliferating cell nuclear antigen and inhibits DNA replication catalyzed by the DNA polymerase delta holoenzyme. Proc Natl Acad Sci U S A. 1994 Aug 30;91(18):8655–8659. [PubMed]
  • Girard F, Strausfeld U, Fernandez A, Lamb NJ. Cyclin A is required for the onset of DNA replication in mammalian fibroblasts. Cell. 1991 Dec 20;67(6):1169–1179. [PubMed]
  • Gu Y, Turck CW, Morgan DO. Inhibition of CDK2 activity in vivo by an associated 20K regulatory subunit. Nature. 1993 Dec 16;366(6456):707–710. [PubMed]
  • Harper JW, Adami GR, Wei N, Keyomarsi K, Elledge SJ. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell. 1993 Nov 19;75(4):805–816. [PubMed]
  • Heichman KA, Roberts JM. Rules to replicate by. Cell. 1994 Nov 18;79(4):557–562. [PubMed]
  • Hinds PW, Mittnacht S, Dulic V, Arnold A, Reed SI, Weinberg RA. Regulation of retinoblastoma protein functions by ectopic expression of human cyclins. Cell. 1992 Sep 18;70(6):993–1006. [PubMed]
  • Howe JA, Howell M, Hunt T, Newport JW. Identification of a developmental timer regulating the stability of embryonic cyclin A and a new somatic A-type cyclin at gastrulation. Genes Dev. 1995 May 15;9(10):1164–1176. [PubMed]
  • Hunter T, Pines J. Cyclins and cancer. II: Cyclin D and CDK inhibitors come of age. Cell. 1994 Nov 18;79(4):573–582. [PubMed]
  • Hutchison CJ, Brill D, Cox R, Gilbert J, Kill I, Ford CC. DNA replication and cell cycle control in Xenopus egg extracts. J Cell Sci Suppl. 1989;12:197–212. [PubMed]
  • Hutchison CJ, Cox R, Drepaul RS, Gomperts M, Ford CC. Periodic DNA synthesis in cell-free extracts of Xenopus eggs. EMBO J. 1987 Jul;6(7):2003–2010. [PubMed]
  • King RW, Jackson PK, Kirschner MW. Mitosis in transition. Cell. 1994 Nov 18;79(4):563–571. [PubMed]
  • Knoblich JA, Sauer K, Jones L, Richardson H, Saint R, Lehner CF. Cyclin E controls S phase progression and its down-regulation during Drosophila embryogenesis is required for the arrest of cell proliferation. Cell. 1994 Apr 8;77(1):107–120. [PubMed]
  • Kobayashi H, Golsteyn R, Poon R, Stewart E, Gannon J, Minshull J, Smith R, Hunt T. Cyclins and their partners during Xenopus oocyte maturation. Cold Spring Harb Symp Quant Biol. 1991;56:437–447. [PubMed]
  • Kobayashi H, Minshull J, Ford C, Golsteyn R, Poon R, Hunt T. On the synthesis and destruction of A- and B-type cyclins during oogenesis and meiotic maturation in Xenopus laevis. J Cell Biol. 1991 Aug;114(4):755–765. [PMC free article] [PubMed]
  • Koff A, Cross F, Fisher A, Schumacher J, Leguellec K, Philippe M, Roberts JM. Human cyclin E, a new cyclin that interacts with two members of the CDC2 gene family. Cell. 1991 Sep 20;66(6):1217–1228. [PubMed]
  • Koff A, Giordano A, Desai D, Yamashita K, Harper JW, Elledge S, Nishimoto T, Morgan DO, Franza BR, Roberts JM. Formation and activation of a cyclin E-cdk2 complex during the G1 phase of the human cell cycle. Science. 1992 Sep 18;257(5077):1689–1694. [PubMed]
  • Koff A, Ohtsuki M, Polyak K, Roberts JM, Massagué J. Negative regulation of G1 in mammalian cells: inhibition of cyclin E-dependent kinase by TGF-beta. Science. 1993 Apr 23;260(5107):536–539. [PubMed]
  • Lees E, Faha B, Dulic V, Reed SI, Harlow E. Cyclin E/cdk2 and cyclin A/cdk2 kinases associate with p107 and E2F in a temporally distinct manner. Genes Dev. 1992 Oct;6(10):1874–1885. [PubMed]
  • Leibovici M, Gusse M, Bravo R, Méchali M. Characterization and developmental expression of Xenopus proliferating cell nuclear antigen (PCNA). Dev Biol. 1990 Sep;141(1):183–192. [PubMed]
  • Leno GH, Downes CS, Laskey RA. The nuclear membrane prevents replication of human G2 nuclei but not G1 nuclei in Xenopus egg extract. Cell. 1992 Apr 3;69(1):151–158. [PubMed]
  • Lew DJ, Dulić V, Reed SI. Isolation of three novel human cyclins by rescue of G1 cyclin (Cln) function in yeast. Cell. 1991 Sep 20;66(6):1197–1206. [PubMed]
  • Lohka MJ, Masui Y. Roles of cytosol and cytoplasmic particles in nuclear envelope assembly and sperm pronuclear formation in cell-free preparations from amphibian eggs. J Cell Biol. 1984 Apr;98(4):1222–1230. [PMC free article] [PubMed]
  • Lohka MJ, Maller JL. Induction of nuclear envelope breakdown, chromosome condensation, and spindle formation in cell-free extracts. J Cell Biol. 1985 Aug;101(2):518–523. [PMC free article] [PubMed]
  • Mills AD, Blow JJ, White JG, Amos WB, Wilcock D, Laskey RA. Replication occurs at discrete foci spaced throughout nuclei replicating in vitro. J Cell Sci. 1989 Nov;94(Pt 3):471–477. [PubMed]
  • Minden JS, Agard DA, Sedat JW, Alberts BM. Direct cell lineage analysis in Drosophila melanogaster by time-lapse, three-dimensional optical microscopy of living embryos. J Cell Biol. 1989 Aug;109(2):505–516. [PMC free article] [PubMed]
  • Minshull J, Golsteyn R, Hill CS, Hunt T. The A- and B-type cyclin associated cdc2 kinases in Xenopus turn on and off at different times in the cell cycle. EMBO J. 1990 Sep;9(9):2865–2875. [PubMed]
  • Murray AW, Kirschner MW. Cyclin synthesis drives the early embryonic cell cycle. Nature. 1989 May 25;339(6222):275–280. [PubMed]
  • Nakayasu H, Berezney R. Mapping replicational sites in the eucaryotic cell nucleus. J Cell Biol. 1989 Jan;108(1):1–11. [PMC free article] [PubMed]
  • Newmeyer DD, Forbes DJ. An N-ethylmaleimide-sensitive cytosolic factor necessary for nuclear protein import: requirement in signal-mediated binding to the nuclear pore. J Cell Biol. 1990 Mar;110(3):547–557. [PMC free article] [PubMed]
  • Newport J, Spann T. Disassembly of the nucleus in mitotic extracts: membrane vesicularization, lamin disassembly, and chromosome condensation are independent processes. Cell. 1987 Jan 30;48(2):219–230. [PubMed]
  • Newport JW, Kirschner MW. Regulation of the cell cycle during early Xenopus development. Cell. 1984 Jul;37(3):731–742. [PubMed]
  • Ohtsubo M, Roberts JM. Cyclin-dependent regulation of G1 in mammalian fibroblasts. Science. 1993 Mar 26;259(5103):1908–1912. [PubMed]
  • Ohtsubo M, Theodoras AM, Schumacher J, Roberts JM, Pagano M. Human cyclin E, a nuclear protein essential for the G1-to-S phase transition. Mol Cell Biol. 1995 May;15(5):2612–2624. [PMC free article] [PubMed]
  • Pagano M, Pepperkok R, Lukas J, Baldin V, Ansorge W, Bartek J, Draetta G. Regulation of the cell cycle by the cdk2 protein kinase in cultured human fibroblasts. J Cell Biol. 1993 Apr;121(1):101–111. [PMC free article] [PubMed]
  • Pagano M, Pepperkok R, Verde F, Ansorge W, Draetta G. Cyclin A is required at two points in the human cell cycle. EMBO J. 1992 Mar;11(3):961–971. [PubMed]
  • Pines J. Cyclins and cyclin-dependent kinases: take your partners. Trends Biochem Sci. 1993 Jun;18(6):195–197. [PubMed]
  • Pines J, Hunter T. Isolation of a human cyclin cDNA: evidence for cyclin mRNA and protein regulation in the cell cycle and for interaction with p34cdc2. Cell. 1989 Sep 8;58(5):833–846. [PubMed]
  • Polyak K, Kato JY, Solomon MJ, Sherr CJ, Massague J, Roberts JM, Koff A. p27Kip1, a cyclin-Cdk inhibitor, links transforming growth factor-beta and contact inhibition to cell cycle arrest. Genes Dev. 1994 Jan;8(1):9–22. [PubMed]
  • Polyak K, Lee MH, Erdjument-Bromage H, Koff A, Roberts JM, Tempst P, Massagué J. Cloning of p27Kip1, a cyclin-dependent kinase inhibitor and a potential mediator of extracellular antimitogenic signals. Cell. 1994 Jul 15;78(1):59–66. [PubMed]
  • Raska I, Koberna K, Jarník M, Petrasovicová V, Bednár J, Raska K, Jr, Bravo R. Ultrastructural immunolocalization of cyclin/PCNA in synchronized 3T3 cells. Exp Cell Res. 1989 Sep;184(1):81–89. [PubMed]
  • Resnitzky D, Gossen M, Bujard H, Reed SI. Acceleration of the G1/S phase transition by expression of cyclins D1 and E with an inducible system. Mol Cell Biol. 1994 Mar;14(3):1669–1679. [PMC free article] [PubMed]
  • Sawin KE, Mitchison TJ. Mitotic spindle assembly by two different pathways in vitro. J Cell Biol. 1991 Mar;112(5):925–940. [PMC free article] [PubMed]
  • Sheehan MA, Mills AD, Sleeman AM, Laskey RA, Blow JJ. Steps in the assembly of replication-competent nuclei in a cell-free system from Xenopus eggs. J Cell Biol. 1988 Jan;106(1):1–12. [PMC free article] [PubMed]
  • Sherr CJ. G1 phase progression: cycling on cue. Cell. 1994 Nov 18;79(4):551–555. [PubMed]
  • Ito T, Smith CL, Cantor CR. Sequence-specific DNA purification by triplex affinity capture. Proc Natl Acad Sci U S A. 1992 Jan 15;89(2):495–498. [PubMed]
  • Simanis V, Lane DP. An immunoaffinity purification procedure for SV40 large T antigen. Virology. 1985 Jul 15;144(1):88–100. [PubMed]
  • Sobczak-Thepot J, Harper F, Florentin Y, Zindy F, Brechot C, Puvion E. Localization of cyclin A at the sites of cellular DNA replication. Exp Cell Res. 1993 May;206(1):43–48. [PubMed]
  • Solomon MJ, Glotzer M, Lee TH, Philippe M, Kirschner MW. Cyclin activation of p34cdc2. Cell. 1990 Nov 30;63(5):1013–1024. [PubMed]
  • Jacks T, Remington L, Williams BO, Schmitt EM, Halachmi S, Bronson RT, Weinberg RA. Tumor spectrum analysis in p53-mutant mice. Curr Biol. 1994 Jan 1;4(1):1–7. [PubMed]
  • Toyoshima H, Hunter T. p27, a novel inhibitor of G1 cyclin-Cdk protein kinase activity, is related to p21. Cell. 1994 Jul 15;78(1):67–74. [PubMed]
  • van den Heuvel S, Harlow E. Distinct roles for cyclin-dependent kinases in cell cycle control. Science. 1993 Dec 24;262(5142):2050–2054. [PubMed]
  • Waga S, Bauer G, Stillman B. Reconstitution of complete SV40 DNA replication with purified replication factors. J Biol Chem. 1994 Apr 8;269(14):10923–10934. [PubMed]
  • Waga S, Hannon GJ, Beach D, Stillman B. The p21 inhibitor of cyclin-dependent kinases controls DNA replication by interaction with PCNA. Nature. 1994 Jun 16;369(6481):574–578. [PubMed]
  • Xiong Y, Hannon GJ, Zhang H, Casso D, Kobayashi R, Beach D. p21 is a universal inhibitor of cyclin kinases. Nature. 1993 Dec 16;366(6456):701–704. [PubMed]
  • Yan H, Newport J. An analysis of the regulation of DNA synthesis by cdk2, Cip1, and licensing factor. J Cell Biol. 1995 Apr;129(1):1–15. [PMC free article] [PubMed]
  • Zhang H, Hannon GJ, Beach D. p21-containing cyclin kinases exist in both active and inactive states. Genes Dev. 1994 Aug 1;8(15):1750–1758. [PubMed]
  • Zuber M, Yasui W, Tan EM, Ryoji M. Quantitation and subcellular localization of proliferating cell nuclear antigen (PCNA/cyclin) in oocytes and eggs of Xenopus laevis. Exp Cell Res. 1989 Jun;182(2):384–393. [PubMed]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press