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. 1993 Sep 1;122(5):993–1002. doi: 10.1083/jcb.122.5.993

Preventing re-replication of DNA in a single cell cycle: evidence for a replication licensing factor

PMCID: PMC2119628  PMID: 8354699

Abstract

Xenopus egg extracts treated with the protein kinase inhibitor 6- dimethylaminopurine (6-DMAP) are unable to support the initiation of DNA replication. Nuclei assembled in 6-DMAP extracts behave as though they are in G2, and will not undergo another round of DNA replication until passage through mitosis. 6-DMAP extracts are functionally devoid of a replication factor that modifies chromatin in early G1 before nuclear envelope assembly, but which is itself incapable of crossing the nuclear envelope. This chromatin modification is capable of supporting only a single round of semiconservative replication. The behavior of this replication factor is sufficient to explain why eukaryotic DNA is replicated once and only once in each cell cycle, and conforms to the previous model of a Replication Licensing Factor. Cell cycle analysis shows that this putative Licensing Factor is inactive during metaphase, but becomes rapidly activated on exit from metaphase when it can modify chromatin before nuclear envelope assembly is complete.

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Selected References

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  1. Bakken A., Morgan G., Sollner-Webb B., Roan J., Busby S., Reeder R. H. Mapping of transcription initiation and termination signals on Xenopus laevis ribosomal DNA. Proc Natl Acad Sci U S A. 1982 Jan;79(1):56–60. doi: 10.1073/pnas.79.1.56. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. 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]
  4. 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]
  5. Blow J. J., Sleeman A. M. Replication of purified DNA in Xenopus egg extract is dependent on nuclear assembly. J Cell Sci. 1990 Mar;95(Pt 3):383–391. doi: 10.1242/jcs.95.3.383. [DOI] [PubMed] [Google Scholar]
  6. Blow J. J., Watson J. V. 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. doi: 10.1002/j.1460-2075.1987.tb02463.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Broek D., Bartlett R., Crawford K., Nurse P. Involvement of p34cdc2 in establishing the dependency of S phase on mitosis. Nature. 1991 Jan 31;349(6308):388–393. doi: 10.1038/349388a0. [DOI] [PubMed] [Google Scholar]
  8. Coverley D., Downes C. S., Romanowski P., Laskey R. A. Reversible effects of nuclear membrane permeabilization on DNA replication: evidence for a positive licensing factor. J Cell Biol. 1993 Sep;122(5):985–992. doi: 10.1083/jcb.122.5.985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Coxon A., Maundrell K., Kearsey S. E. Fission yeast cdc21+ belongs to a family of proteins involved in an early step of chromosome replication. Nucleic Acids Res. 1992 Nov 11;20(21):5571–5577. doi: 10.1093/nar/20.21.5571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Crevel G., Cotterill S. DNA replication in cell-free extracts from Drosophila melanogaster. EMBO J. 1991 Dec;10(13):4361–4369. doi: 10.1002/j.1460-2075.1991.tb05014.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. 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]
  15. Finlay D. R., Newmeyer D. D., Price T. M., Forbes D. J. Inhibition of in vitro nuclear transport by a lectin that binds to nuclear pores. J Cell Biol. 1987 Feb;104(2):189–200. doi: 10.1083/jcb.104.2.189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Handeli S., Weintraub H. The ts41 mutation in Chinese hamster cells leads to successive S phases in the absence of intervening G2, M, and G1. Cell. 1992 Nov 13;71(4):599–611. doi: 10.1016/0092-8674(92)90594-3. [DOI] [PubMed] [Google Scholar]
  17. Harland R. M., Laskey R. A. Regulated replication of DNA microinjected into eggs of Xenopus laevis. Cell. 1980 Oct;21(3):761–771. doi: 10.1016/0092-8674(80)90439-0. [DOI] [PubMed] [Google Scholar]
  18. Hennessy K. M., Clark C. D., Botstein D. Subcellular localization of yeast CDC46 varies with the cell cycle. Genes Dev. 1990 Dec;4(12B):2252–2263. doi: 10.1101/gad.4.12b.2252. [DOI] [PubMed] [Google Scholar]
  19. Hennessy K. M., Lee A., Chen E., Botstein D. A group of interacting yeast DNA replication genes. Genes Dev. 1991 Jun;5(6):958–969. doi: 10.1101/gad.5.6.958. [DOI] [PubMed] [Google Scholar]
  20. Hutchison C. J., Cox R., Ford C. C. The control of DNA replication in a cell-free extract that recapitulates a basic cell cycle in vitro. Development. 1988 Jul;103(3):553–566. doi: 10.1242/dev.103.3.553. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. Lohka M. J., Masui Y. Effects of Ca2+ ions on the formation of metaphase chromosomes and sperm pronuclei in cell-free preparations from unactivated Rana pipiens eggs. Dev Biol. 1984 Jun;103(2):434–442. doi: 10.1016/0012-1606(84)90331-2. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. Luca F. C., Ruderman J. V. Control of programmed cyclin destruction in a cell-free system. J Cell Biol. 1989 Nov;109(5):1895–1909. doi: 10.1083/jcb.109.5.1895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Meijer L., Pondaven P. Cyclic activation of histone H1 kinase during sea urchin egg mitotic divisions. Exp Cell Res. 1988 Jan;174(1):116–129. doi: 10.1016/0014-4827(88)90147-4. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Minshull J., Blow J. J., Hunt T. Translation of cyclin mRNA is necessary for extracts of activated xenopus eggs to enter mitosis. Cell. 1989 Mar 24;56(6):947–956. doi: 10.1016/0092-8674(89)90628-4. [DOI] [PubMed] [Google Scholar]
  30. Minshull J., Golsteyn R., Hill C. S., 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. doi: 10.1002/j.1460-2075.1990.tb07476.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Neant I., Guerrier P. 6-Dimethylaminopurine blocks starfish oocyte maturation by inhibiting a relevant protein kinase activity. Exp Cell Res. 1988 May;176(1):68–79. doi: 10.1016/0014-4827(88)90121-8. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. 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]
  35. Rao P. N., Johnson R. T. Mammalian cell fusion: studies on the regulation of DNA synthesis and mitosis. Nature. 1970 Jan 10;225(5228):159–164. doi: 10.1038/225159a0. [DOI] [PubMed] [Google Scholar]
  36. Roberts J. M., Weintraub H. Cis-acting negative control of DNA replication in eukaryotic cells. Cell. 1988 Feb 12;52(3):397–404. doi: 10.1016/s0092-8674(88)80032-1. [DOI] [PubMed] [Google Scholar]
  37. Roberts J. M., Weintraub H. Negative control of DNA replication in composite SV40-bovine papilloma virus plasmids. Cell. 1986 Aug 29;46(5):741–752. doi: 10.1016/0092-8674(86)90350-8. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Strausfeld U., Labbé J. C., Fesquet D., Cavadore J. C., Picard A., Sadhu K., Russell P., Dorée M. Dephosphorylation and activation of a p34cdc2/cyclin B complex in vitro by human CDC25 protein. Nature. 1991 May 16;351(6323):242–245. doi: 10.1038/351242a0. [DOI] [PubMed] [Google Scholar]
  40. Thömmes P., Fett R., Schray B., Burkhart R., Barnes M., Kennedy C., Brown N. C., Knippers R. Properties of the nuclear P1 protein, a mammalian homologue of the yeast Mcm3 replication protein. Nucleic Acids Res. 1992 Mar 11;20(5):1069–1074. doi: 10.1093/nar/20.5.1069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Usui T., Yoshida M., Abe K., Osada H., Isono K., Beppu T. Uncoupled cell cycle without mitosis induced by a protein kinase inhibitor, K-252a. J Cell Biol. 1991 Dec;115(5):1275–1282. doi: 10.1083/jcb.115.5.1275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Yan H., Gibson S., Tye B. K. Mcm2 and Mcm3, two proteins important for ARS activity, are related in structure and function. Genes Dev. 1991 Jun;5(6):944–957. doi: 10.1101/gad.5.6.944. [DOI] [PubMed] [Google Scholar]
  43. de Roeper A., Smith J. A., Watt R. A., Barry J. M. Chromatin dispersal and DNA synthesis in G1 and G2 HeLa cell nuclei injected into Xenopus eggs. Nature. 1977 Feb 3;265(5593):469–470. doi: 10.1038/265469a0. [DOI] [PubMed] [Google Scholar]

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