Identification of nuclear pre-replication centers poised for DNA synthesis in Xenopus egg extracts: immunolocalization study of replication protein A (original) (raw)

Abstract

We demonstrate by immunofluorescence that a 70-kD protein (P70) purified from Xenopus egg extracts is associated with subnuclear foci (about 200) which we propose to be an assembly of DNA pre-replication centers (preRCs). A cDNA encoding this protein reveals that P70 is the Xenopus homologue of replication protein A (RPA also called RF-A). RPA is know to be a cellular, three-subunit single-stranded DNA binding protein, which assists T-antigen in the assembly of the pre-priming complex in the SV40 replication system. The punctated preRCs exist transiently; they form post-mitotically during the period of nuclear membrane breakdown and disappear during ongoing DNA replication. P70 is homogeneously associated with chromatin at the later stages of the S- phase and is displaced from chromatin post replication, so that P70 cannot be detected on mitotic chromosomes. Double-immunofluorescence studies using biotin-dUTP demonstrate that initiation of DNA synthesis is confined to preRCs, resulting in the punctated replication pattern observed previously by others. PreRCs form efficiently on decondensed chromatin in membrane-free egg extracts if ATP and divalent cations are present. Our results suggest that preRCs are composed of an assembly of a large number of pre-initiation replication complexes poised for initiation at discreet subnuclear regions prior to nuclear reconstruction and initiation of DNA synthesis.

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Selected References

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  1. 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]
  2. Adachi Y., Yanagida M. Higher order chromosome structure is affected by cold-sensitive mutations in a Schizosaccharomyces pombe gene crm1+ which encodes a 115-kD protein preferentially localized in the nucleus and its periphery. J Cell Biol. 1989 Apr;108(4):1195–1207. doi: 10.1083/jcb.108.4.1195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Aebersold R. H., Leavitt J., Saavedra R. A., Hood L. E., Kent S. B. Internal amino acid sequence analysis of proteins separated by one- or two-dimensional gel electrophoresis after in situ protease digestion on nitrocellulose. Proc Natl Acad Sci U S A. 1987 Oct;84(20):6970–6974. doi: 10.1073/pnas.84.20.6970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Arndt-Jovin D. J., Jovin T. M., Bähr W., Frischauf A. M., Marquardt M. Covalent attachment of DNA to agarose. Improved synthesis and use in affinity chromatography. Eur J Biochem. 1975 Jun;54(2):411–418. doi: 10.1111/j.1432-1033.1975.tb04151.x. [DOI] [PubMed] [Google Scholar]
  6. Berezney R., Coffey D. S. Nuclear protein matrix: association with newly synthesized DNA. Science. 1975 Jul 25;189(4199):291–293. doi: 10.1126/science.1145202. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. 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]
  10. Bravo R., Macdonald-Bravo H. Existence of two populations of cyclin/proliferating cell nuclear antigen during the cell cycle: association with DNA replication sites. J Cell Biol. 1987 Oct;105(4):1549–1554. doi: 10.1083/jcb.105.4.1549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. 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]
  14. Cook P. R. The nucleoskeleton and the topology of replication. Cell. 1991 Aug 23;66(4):627–635. doi: 10.1016/0092-8674(91)90109-c. [DOI] [PubMed] [Google Scholar]
  15. Cox L. S., Laskey R. A. DNA replication occurs at discrete sites in pseudonuclei assembled from purified DNA in vitro. Cell. 1991 Jul 26;66(2):271–275. doi: 10.1016/0092-8674(91)90617-8. [DOI] [PubMed] [Google Scholar]
  16. Earnshaw W. C., Halligan B., Cooke C. A., Heck M. M., Liu L. F. Topoisomerase II is a structural component of mitotic chromosome scaffolds. J Cell Biol. 1985 May;100(5):1706–1715. doi: 10.1083/jcb.100.5.1706. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Erdile L. F., Heyer W. D., Kolodner R., Kelly T. J. 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. 1991 Jun 25;266(18):12090–12098. [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. 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]
  21. Gasser S. M., Laroche T., Falquet J., Boy de la Tour E., Laemmli U. K. Metaphase chromosome structure. Involvement of topoisomerase II. J Mol Biol. 1986 Apr 20;188(4):613–629. doi: 10.1016/s0022-2836(86)80010-9. [DOI] [PubMed] [Google Scholar]
  22. Gassmann M., Thömmes P., Weiser T., Hübscher U. Efficient production of chicken egg yolk antibodies against a conserved mammalian protein. FASEB J. 1990 May;4(8):2528–2532. doi: 10.1096/fasebj.4.8.1970792. [DOI] [PubMed] [Google Scholar]
  23. Hand R. Eucaryotic DNA: organization of the genome for replication. Cell. 1978 Oct;15(2):317–325. doi: 10.1016/0092-8674(78)90001-6. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. Hutchison C., Kill I. Changes in the nuclear distribution of DNA polymerase alpha and PCNA/cyclin during the progress of the cell cycle, in a cell-free extract of Xenopus eggs. J Cell Sci. 1989 Aug;93(Pt 4):605–613. doi: 10.1242/jcs.93.4.605. [DOI] [PubMed] [Google Scholar]
  27. Jackson D. A., Cook P. R. Replication occurs at a nucleoskeleton. EMBO J. 1986 Jun;5(6):1403–1410. doi: 10.1002/j.1460-2075.1986.tb04374.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Kill I. R., Bridger J. M., Campbell K. H., Maldonado-Codina G., Hutchison C. J. The timing of the formation and usage of replicase clusters in S-phase nuclei of human diploid fibroblasts. J Cell Sci. 1991 Dec;100(Pt 4):869–876. doi: 10.1242/jcs.100.4.869. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. 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]
  32. Li J. J., Kelly T. J. Simian virus 40 DNA replication in vitro. Proc Natl Acad Sci U S A. 1984 Nov;81(22):6973–6977. doi: 10.1073/pnas.81.22.6973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. 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]
  35. 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]
  36. 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]
  37. 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]
  38. Murray A. W. Cell cycle extracts. Methods Cell Biol. 1991;36:581–605. [PubMed] [Google Scholar]
  39. Murray A. W., Kirschner M. W. Cyclin synthesis drives the early embryonic cell cycle. Nature. 1989 May 25;339(6222):275–280. doi: 10.1038/339275a0. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. Nakagawa J., Kitten G. T., Nigg E. A. A somatic cell-derived system for studying both early and late mitotic events in vitro. J Cell Sci. 1989 Nov;94(Pt 3):449–462. doi: 10.1242/jcs.94.3.449. [DOI] [PubMed] [Google Scholar]
  42. 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]
  43. 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]
  44. Newmeyer D. D., Lucocq J. M., Bürglin T. R., De Robertis E. M. Assembly in vitro of nuclei active in nuclear protein transport: ATP is required for nucleoplasmin accumulation. EMBO J. 1986 Mar;5(3):501–510. doi: 10.1002/j.1460-2075.1986.tb04239.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. 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]
  46. 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]
  47. 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]
  48. Pfaller R., Smythe C., Newport J. W. Assembly/disassembly of the nuclear envelope membrane: cell cycle-dependent binding of nuclear membrane vesicles to chromatin in vitro. Cell. 1991 Apr 19;65(2):209–217. doi: 10.1016/0092-8674(91)90155-r. [DOI] [PubMed] [Google Scholar]
  49. Philpott A., Leno G. H., Laskey R. A. Sperm decondensation in Xenopus egg cytoplasm is mediated by nucleoplasmin. Cell. 1991 May 17;65(4):569–578. doi: 10.1016/0092-8674(91)90089-h. [DOI] [PubMed] [Google Scholar]
  50. Quinlan M. P., Chen L. B., Knipe D. M. The intranuclear location of a herpes simplex virus DNA-binding protein is determined by the status of viral DNA replication. Cell. 1984 Apr;36(4):857–868. doi: 10.1016/0092-8674(84)90035-7. [DOI] [PubMed] [Google Scholar]
  51. Rebagliati M. R., Weeks D. L., Harvey R. P., Melton D. A. Identification and cloning of localized maternal RNAs from Xenopus eggs. Cell. 1985 Oct;42(3):769–777. doi: 10.1016/0092-8674(85)90273-9. [DOI] [PubMed] [Google Scholar]
  52. Ruyechan W. T. The major herpes simplex virus DNA-binding protein holds single-stranded DNA in an extended configuration. J Virol. 1983 May;46(2):661–666. doi: 10.1128/jvi.46.2.661-666.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. 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]
  55. Smith D. E., Fisher P. A. Identification, developmental regulation, and response to heat shock of two antigenically related forms of a major nuclear envelope protein in Drosophila embryos: application of an improved method for affinity purification of antibodies using polypeptides immobilized on nitrocellulose blots. J Cell Biol. 1984 Jul;99(1 Pt 1):20–28. doi: 10.1083/jcb.99.1.20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Stillman B. W., Gluzman Y. Replication and supercoiling of simian virus 40 DNA in cell extracts from human cells. Mol Cell Biol. 1985 Aug;5(8):2051–2060. doi: 10.1128/mcb.5.8.2051. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. 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]
  58. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  59. 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]
  60. Tsurimoto T., Melendy T., Stillman B. Sequential initiation of lagging and leading strand synthesis by two different polymerase complexes at the SV40 DNA replication origin. Nature. 1990 Aug 9;346(6284):534–539. doi: 10.1038/346534a0. [DOI] [PubMed] [Google Scholar]
  61. Tsurimoto T., Stillman B. Multiple replication factors augment DNA synthesis by the two eukaryotic DNA polymerases, alpha and delta. EMBO J. 1989 Dec 1;8(12):3883–3889. doi: 10.1002/j.1460-2075.1989.tb08567.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Vaughn J. P., Dijkwel P. A., Mullenders L. H., Hamlin J. L. Replication forks are associated with the nuclear matrix. Nucleic Acids Res. 1990 Apr 25;18(8):1965–1969. doi: 10.1093/nar/18.8.1965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Wobbe C. R., Dean F., Weissbach L., Hurwitz J. In vitro replication of duplex circular DNA containing the simian virus 40 DNA origin site. Proc Natl Acad Sci U S A. 1985 Sep;82(17):5710–5714. doi: 10.1073/pnas.82.17.5710. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Wobbe C. R., Weissbach L., Borowiec J. A., Dean F. B., Murakami Y., Bullock P., Hurwitz J. Replication of simian virus 40 origin-containing DNA in vitro with purified proteins. Proc Natl Acad Sci U S A. 1987 Apr;84(7):1834–1838. doi: 10.1073/pnas.84.7.1834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. 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]
  66. Wold M. S., Li J. J., Kelly T. J. Initiation of simian virus 40 DNA replication in vitro: large-tumor-antigen- and origin-dependent unwinding of the template. Proc Natl Acad Sci U S A. 1987 Jun;84(11):3643–3647. doi: 10.1073/pnas.84.11.3643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. 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]
  68. Wu M., Gerhart J. C. Partial purification and characterization of the maturation-promoting factor from eggs of Xenopus laevis. Dev Biol. 1980 Oct;79(2):465–477. doi: 10.1016/0012-1606(80)90131-1. [DOI] [PubMed] [Google Scholar]
  69. de Bruyn Kops A., Knipe D. M. Formation of DNA replication structures in herpes virus-infected cells requires a viral DNA binding protein. Cell. 1988 Dec 2;55(5):857–868. doi: 10.1016/0092-8674(88)90141-9. [DOI] [PubMed] [Google Scholar]