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. 1994 Sep 1;13(17):4153–4164. doi: 10.1002/j.1460-2075.1994.tb06733.x

Study of the cell cycle-dependent assembly of the DNA pre-replication centres in Xenopus egg extracts.

Y Adachi 1, U K Laemmli 1
PMCID: PMC395338  PMID: 8076611

Abstract

RPA is a cellular, three-subunit, single-stranded (ss) DNA binding protein, which assists T-antigen in the assembly of the pre-priming complex in the SV40 replication system. By immunodepletion and complementation, we have identified RPA as an essential factor for cellular DNA replication in Xenopus extracts. RPA assembles post-mitotically on the decondensing chromosomes into numerous subnuclear pre-replication centres (preRCs) which serve, upon formation of the nuclear membrane, as RCs for the initiation of DNA synthesis. By a variety of experiments including the use of isolated components, we demonstrate that an inactive cdc2-cyclin B kinase complex is essential to allow post-mitotic assembly of the preRCs. In contrast, the active cdk2-cyclin A kinase does not impede or facilitate the assembly of preRCs. Digestion analysis using the single-strand-specific P1 nuclease as well as competition experiments with ssDNA, reveal that replication-associated unwinding of the DNA, assisted by RPA, requires the formation of the nuclear membrane. The p21 cdk-interacting protein Cip1 appears to inhibit DNA replication prior to the unwinding DNA step, but after assembly of preRC and nuclear reconstruction.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adachi Y., Laemmli U. K. 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. doi: 10.1083/jcb.119.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Adachi Y., Luke M., Laemmli U. K. Chromosome assembly in vitro: topoisomerase II is required for condensation. Cell. 1991 Jan 11;64(1):137–148. doi: 10.1016/0092-8674(91)90215-k. [DOI] [PubMed] [Google Scholar]
  3. Alberts B. M., Amodio F. J., Jenkins M., Gutmann E. D., Ferris F. L. Studies with DNA-cellulose chromatography. I. DNA-binding proteins from Escherichia coli. Cold Spring Harb Symp Quant Biol. 1968;33:289–305. doi: 10.1101/sqb.1968.033.01.033. [DOI] [PubMed] [Google Scholar]
  4. Almouzni G., Wolffe A. P. Nuclear assembly, structure, and function: the use of Xenopus in vitro systems. Exp Cell Res. 1993 Mar;205(1):1–15. doi: 10.1006/excr.1993.1051. [DOI] [PubMed] [Google Scholar]
  5. Blow J. J., Laskey R. A. A role for the nuclear envelope in controlling DNA replication within the cell cycle. Nature. 1988 Apr 7;332(6164):546–548. doi: 10.1038/332546a0. [DOI] [PubMed] [Google Scholar]
  6. Blow J. J., Laskey R. A. 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. doi: 10.1016/0092-8674(86)90622-7. [DOI] [PubMed] [Google Scholar]
  7. Blow J. J., 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. doi: 10.1016/0092-8674(90)90261-c. [DOI] [PubMed] [Google Scholar]
  8. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  9. Bramhill D., Kornberg A. Duplex opening by dnaA protein at novel sequences in initiation of replication at the origin of the E. coli chromosome. Cell. 1988 Mar 11;52(5):743–755. doi: 10.1016/0092-8674(88)90412-6. [DOI] [PubMed] [Google Scholar]
  10. Brill S. J., Stillman B. Replication factor-A from Saccharomyces cerevisiae is encoded by three essential genes coordinately expressed at S phase. Genes Dev. 1991 Sep;5(9):1589–1600. doi: 10.1101/gad.5.9.1589. [DOI] [PubMed] [Google Scholar]
  11. Brill S. J., Stillman B. Yeast replication factor-A functions in the unwinding of the SV40 origin of DNA replication. Nature. 1989 Nov 2;342(6245):92–95. doi: 10.1038/342092a0. [DOI] [PubMed] [Google Scholar]
  12. Callan H. G. Replication of DNA in the chromosomes of eukaryotes. Proc R Soc Lond B Biol Sci. 1972 Apr 18;181(1062):19–41. doi: 10.1098/rspb.1972.0039. [DOI] [PubMed] [Google Scholar]
  13. Cardoso M. C., 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. doi: 10.1016/0092-8674(93)90721-2. [DOI] [PubMed] [Google Scholar]
  14. Carle G. F., Frank M., Olson M. V. Electrophoretic separations of large DNA molecules by periodic inversion of the electric field. Science. 1986 Apr 4;232(4746):65–68. doi: 10.1126/science.3952500. [DOI] [PubMed] [Google Scholar]
  15. Challberg M. D., Kelly T. J. Animal virus DNA replication. Annu Rev Biochem. 1989;58:671–717. doi: 10.1146/annurev.bi.58.070189.003323. [DOI] [PubMed] [Google Scholar]
  16. Coverley D., Kenny M. K., Munn M., Rupp W. D., Lane D. P., Wood R. D. Requirement for the replication protein SSB in human DNA excision repair. Nature. 1991 Feb 7;349(6309):538–541. doi: 10.1038/349538a0. [DOI] [PubMed] [Google Scholar]
  17. Cox L. S. 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. doi: 10.1242/jcs.101.1.43. [DOI] [PubMed] [Google Scholar]
  18. Desai D., Gu Y., Morgan D. O. Activation of human cyclin-dependent kinases in vitro. Mol Biol Cell. 1992 May;3(5):571–582. doi: 10.1091/mbc.3.5.571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Din S., Brill S. J., Fairman M. P., Stillman B. Cell-cycle-regulated phosphorylation of DNA replication factor A from human and yeast cells. Genes Dev. 1990 Jun;4(6):968–977. doi: 10.1101/gad.4.6.968. [DOI] [PubMed] [Google Scholar]
  20. Dornreiter I., Erdile L. F., Gilbert I. U., von Winkler D., Kelly T. J., Fanning E. Interaction of DNA polymerase alpha-primase with cellular replication protein A and SV40 T antigen. EMBO J. 1992 Feb;11(2):769–776. doi: 10.1002/j.1460-2075.1992.tb05110.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Dunphy W. G., Brizuela L., Beach D., Newport J. The Xenopus cdc2 protein is a component of MPF, a cytoplasmic regulator of mitosis. Cell. 1988 Jul 29;54(3):423–431. doi: 10.1016/0092-8674(88)90205-x. [DOI] [PubMed] [Google Scholar]
  22. Dutta A., Ruppert J. M., Aster J. C., Winchester E. Inhibition of DNA replication factor RPA by p53. Nature. 1993 Sep 2;365(6441):79–82. doi: 10.1038/365079a0. [DOI] [PubMed] [Google Scholar]
  23. Dutta A., Stillman B. cdc2 family kinases phosphorylate a human cell DNA replication factor, RPA, and activate DNA replication. EMBO J. 1992 Jun;11(6):2189–2199. doi: 10.1002/j.1460-2075.1992.tb05278.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Fairman M. P., Stillman B. Cellular factors required for multiple stages of SV40 DNA replication in vitro. EMBO J. 1988 Apr;7(4):1211–1218. doi: 10.1002/j.1460-2075.1988.tb02933.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Fang F., Newport J. W. Distinct roles of cdk2 and cdc2 in RP-A phosphorylation during the cell cycle. J Cell Sci. 1993 Nov;106(Pt 3):983–994. doi: 10.1242/jcs.106.3.983. [DOI] [PubMed] [Google Scholar]
  26. Fang F., Newport J. W. 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. doi: 10.1016/0092-8674(91)90117-h. [DOI] [PubMed] [Google Scholar]
  27. Felix M. A., Pines J., Hunt T., Karsenti E. A post-ribosomal supernatant from activated Xenopus eggs that displays post-translationally regulated oscillation of its cdc2+ mitotic kinase activity. EMBO J. 1989 Oct;8(10):3059–3069. doi: 10.1002/j.1460-2075.1989.tb08457.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Finlay D. R., Forbes D. J. Reconstitution of biochemically altered nuclear pores: transport can be eliminated and restored. Cell. 1990 Jan 12;60(1):17–29. doi: 10.1016/0092-8674(90)90712-n. [DOI] [PubMed] [Google Scholar]
  29. Fox M. H., Arndt-Jovin D. J., Jovin T. M., Baumann P. H., Robert-Nicoud M. Spatial and temporal distribution of DNA replication sites localized by immunofluorescence and confocal microscopy in mouse fibroblasts. J Cell Sci. 1991 Jun;99(Pt 2):247–253. doi: 10.1242/jcs.99.2.247. [DOI] [PubMed] [Google Scholar]
  30. Grieco F., Hay J. M., Hull R. An improved procedure for the purification of protein fused with glutathione S-transferase. Biotechniques. 1992 Dec;13(6):856–858. [PubMed] [Google Scholar]
  31. Gu Y., Turck C. W., Morgan D. O. Inhibition of CDK2 activity in vivo by an associated 20K regulatory subunit. Nature. 1993 Dec 16;366(6456):707–710. doi: 10.1038/366707a0. [DOI] [PubMed] [Google Scholar]
  32. Harper J. W., Adami G. R., Wei N., Keyomarsi K., Elledge S. J. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell. 1993 Nov 19;75(4):805–816. doi: 10.1016/0092-8674(93)90499-g. [DOI] [PubMed] [Google Scholar]
  33. He Z., Brinton B. T., Greenblatt J., Hassell J. A., Ingles C. J. The transactivator proteins VP16 and GAL4 bind replication factor A. Cell. 1993 Jun 18;73(6):1223–1232. doi: 10.1016/0092-8674(93)90650-f. [DOI] [PubMed] [Google Scholar]
  34. Heyer W. D., Rao M. R., Erdile L. F., Kelly T. J., Kolodner R. D. An essential Saccharomyces cerevisiae single-stranded DNA binding protein is homologous to the large subunit of human RP-A. EMBO J. 1990 Jul;9(7):2321–2329. doi: 10.1002/j.1460-2075.1990.tb07404.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Hovanessian A. G., Galabru J., Rivière Y., Montagnier L. Efficiency of poly(A).poly(U) as an adjuvant. Immunol Today. 1988 Jun;9(6):161–162. doi: 10.1016/0167-5699(88)91288-1. [DOI] [PubMed] [Google Scholar]
  36. Hunter T. Braking the cycle. Cell. 1993 Dec 3;75(5):839–841. doi: 10.1016/0092-8674(93)90528-x. [DOI] [PubMed] [Google Scholar]
  37. Hutchison C. J., Cox R., Drepaul R. S., Gomperts M., Ford C. C. Periodic DNA synthesis in cell-free extracts of Xenopus eggs. EMBO J. 1987 Jul;6(7):2003–2010. doi: 10.1002/j.1460-2075.1987.tb02464.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Hyrien O., Méchali M. Chromosomal replication initiates and terminates at random sequences but at regular intervals in the ribosomal DNA of Xenopus early embryos. EMBO J. 1993 Dec;12(12):4511–4520. doi: 10.1002/j.1460-2075.1993.tb06140.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Jenkins H., Hölman T., Lyon C., Lane B., Stick R., Hutchison C. Nuclei that lack a lamina accumulate karyophilic proteins and assemble a nuclear matrix. J Cell Sci. 1993 Sep;106(Pt 1):275–285. doi: 10.1242/jcs.106.1.275. [DOI] [PubMed] [Google Scholar]
  40. Kenny M. K., Lee S. H., Hurwitz J. Multiple functions of human single-stranded-DNA binding protein in simian virus 40 DNA replication: single-strand stabilization and stimulation of DNA polymerases alpha and delta. Proc Natl Acad Sci U S A. 1989 Dec;86(24):9757–9761. doi: 10.1073/pnas.86.24.9757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Kenny M. K., Schlegel U., Furneaux H., Hurwitz J. The role of human single-stranded DNA binding protein and its individual subunits in simian virus 40 DNA replication. J Biol Chem. 1990 May 5;265(13):7693–7700. [PubMed] [Google Scholar]
  42. Kim C., Snyder R. O., Wold M. S. Binding properties of replication protein A from human and yeast cells. Mol Cell Biol. 1992 Jul;12(7):3050–3059. doi: 10.1128/mcb.12.7.3050. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Käs E., Laemmli U. K. In vivo topoisomerase II cleavage of the Drosophila histone and satellite III repeats: DNA sequence and structural characteristics. EMBO J. 1992 Feb;11(2):705–716. doi: 10.1002/j.1460-2075.1992.tb05103.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Labbé J. C., Cavadore J. C., Dorée M. M phase-specific cdc2 kinase: preparation from starfish oocytes and properties. Methods Enzymol. 1991;200:291–301. doi: 10.1016/0076-6879(91)00147-o. [DOI] [PubMed] [Google Scholar]
  45. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  46. Leno G. H., Downes C. S., Laskey R. A. 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. doi: 10.1016/0092-8674(92)90126-w. [DOI] [PubMed] [Google Scholar]
  47. Leno G. H., Laskey R. A. The nuclear membrane determines the timing of DNA replication in Xenopus egg extracts. J Cell Biol. 1991 Feb;112(4):557–566. doi: 10.1083/jcb.112.4.557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Li R., Botchan M. R. The acidic transcriptional activation domains of VP16 and p53 bind the cellular replication protein A and stimulate in vitro BPV-1 DNA replication. Cell. 1993 Jun 18;73(6):1207–1221. doi: 10.1016/0092-8674(93)90649-b. [DOI] [PubMed] [Google Scholar]
  49. Lohka M. J., Maller J. L. Induction of nuclear envelope breakdown, chromosome condensation, and spindle formation in cell-free extracts. J Cell Biol. 1985 Aug;101(2):518–523. doi: 10.1083/jcb.101.2.518. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Lohka M. J., Masui Y. Formation in vitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic components. Science. 1983 May 13;220(4598):719–721. doi: 10.1126/science.6601299. [DOI] [PubMed] [Google Scholar]
  51. Lohka M. J., 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. doi: 10.1083/jcb.98.4.1222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Mahbubani H. M., Paull T., Elder J. K., Blow J. J. DNA replication initiates at multiple sites on plasmid DNA in Xenopus egg extracts. Nucleic Acids Res. 1992 Apr 11;20(7):1457–1462. doi: 10.1093/nar/20.7.1457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Meier J., Campbell K. H., Ford C. C., Stick R., Hutchison C. J. The role of lamin LIII in nuclear assembly and DNA replication, in cell-free extracts of Xenopus eggs. J Cell Sci. 1991 Mar;98(Pt 3):271–279. doi: 10.1242/jcs.98.3.271. [DOI] [PubMed] [Google Scholar]
  54. Miake-Lye R., Newport J., Kirschner M. Maturation-promoting factor induces nuclear envelope breakdown in cycloheximide-arrested embryos of Xenopus laevis. J Cell Biol. 1983 Jul;97(1):81–91. doi: 10.1083/jcb.97.1.81. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Mills A. D., Blow J. J., White J. G., Amos W. B., Wilcock D., Laskey R. A. Replication occurs at discrete foci spaced throughout nuclei replicating in vitro. J Cell Sci. 1989 Nov;94(Pt 3):471–477. doi: 10.1242/jcs.94.3.471. [DOI] [PubMed] [Google Scholar]
  56. Murray A. W. Cell cycle extracts. Methods Cell Biol. 1991;36:581–605. [PubMed] [Google Scholar]
  57. Méchali M., Harland R. M. DNA synthesis in a cell-free system from Xenopus eggs: priming and elongation on single-stranded DNA in vitro. Cell. 1982 Aug;30(1):93–101. doi: 10.1016/0092-8674(82)90015-0. [DOI] [PubMed] [Google Scholar]
  58. Nakamura H., Morita T., Sato C. Structural organizations of replicon domains during DNA synthetic phase in the mammalian nucleus. Exp Cell Res. 1986 Aug;165(2):291–297. doi: 10.1016/0014-4827(86)90583-5. [DOI] [PubMed] [Google Scholar]
  59. Nakayasu H., Berezney R. Mapping replicational sites in the eucaryotic cell nucleus. J Cell Biol. 1989 Jan;108(1):1–11. doi: 10.1083/jcb.108.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Newmeyer D. D., Finlay D. R., Forbes D. J. In vitro transport of a fluorescent nuclear protein and exclusion of non-nuclear proteins. J Cell Biol. 1986 Dec;103(6 Pt 1):2091–2102. doi: 10.1083/jcb.103.6.2091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Newport J. W., Wilson K. L., Dunphy W. G. A lamin-independent pathway for nuclear envelope assembly. J Cell Biol. 1990 Dec;111(6 Pt 1):2247–2259. doi: 10.1083/jcb.111.6.2247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Newport J. Nuclear reconstitution in vitro: stages of assembly around protein-free DNA. Cell. 1987 Jan 30;48(2):205–217. doi: 10.1016/0092-8674(87)90424-7. [DOI] [PubMed] [Google Scholar]
  63. 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. doi: 10.1016/0092-8674(87)90425-9. [DOI] [PubMed] [Google Scholar]
  64. O'Keefe R. T., Henderson S. C., Spector D. L. Dynamic organization of DNA replication in mammalian cell nuclei: spatially and temporally defined replication of chromosome-specific alpha-satellite DNA sequences. J Cell Biol. 1992 Mar;116(5):1095–1110. doi: 10.1083/jcb.116.5.1095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Schnos M., Zahn K., Inman R. B., Blattner F. R. Initiation protein induced helix destabilization at the lambda origin: a prepriming step in DNA replication. Cell. 1988 Feb 12;52(3):385–395. doi: 10.1016/s0092-8674(88)80031-x. [DOI] [PubMed] [Google Scholar]
  66. Sheehan M. A., Mills A. D., Sleeman A. M., Laskey R. A., Blow J. J. 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. doi: 10.1083/jcb.106.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Stillman B. Initiation of eukaryotic DNA replication in vitro. Annu Rev Cell Biol. 1989;5:197–245. doi: 10.1146/annurev.cb.05.110189.001213. [DOI] [PubMed] [Google Scholar]
  68. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Wilcock D., Lane D. P. Localization of p53, retinoblastoma and host replication proteins at sites of viral replication in herpes-infected cells. Nature. 1991 Jan 31;349(6308):429–431. doi: 10.1038/349429a0. [DOI] [PubMed] [Google Scholar]
  70. Wold M. S., Kelly T. Purification and characterization of replication protein A, a cellular protein required for in vitro replication of simian virus 40 DNA. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2523–2527. doi: 10.1073/pnas.85.8.2523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  71. Wold M. S., Weinberg D. H., Virshup D. M., Li J. J., Kelly T. J. Identification of cellular proteins required for simian virus 40 DNA replication. J Biol Chem. 1989 Feb 15;264(5):2801–2809. [PubMed] [Google Scholar]
  72. Xiong Y., Hannon G. J., Zhang H., Casso D., Kobayashi R., Beach D. p21 is a universal inhibitor of cyclin kinases. Nature. 1993 Dec 16;366(6456):701–704. doi: 10.1038/366701a0. [DOI] [PubMed] [Google Scholar]

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