Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1993 Sep 1;122(5):985–992. doi: 10.1083/jcb.122.5.985

Reversible effects of nuclear membrane permeabilization on DNA replication: evidence for a positive licensing factor

PMCID: PMC2119632  PMID: 8354698

Abstract

We have investigated the mechanism which prevents reinitiation of DNA replication within a single cell cycle by exploiting the observation that intact G2 HeLa nuclei do not replicate in Xenopus egg extract, unless their nuclear membranes are first permeabilized (Leno et al., 1992). We have asked if nuclear membrane permeabilization allows escape of a negative inhibitor from the replicated nucleus or entry of a positive activator as proposed in the licensing factor hypothesis of Blow and Laskey (1988). We have distinguished these possibilities by repairing permeabilized nuclear membranes after allowing soluble factors to escape. Membrane repair of G2 nuclei reverses the effects of permeabilization arguing that escape of diffusible inhibitors is not sufficient to allow replication, but that entry of diffusible activators is required. Membrane repair has no significant effect on G1 nuclei. Pre-incubation of permeable G2 nuclei in the soluble fraction of egg extract before membrane repair allows semiconservative DNA replication of these nuclei when incubated in complete extract. Addition of the same fraction after membrane repair has no effect. Our results provide direct evidence for a positively acting "licensing" activity which is excluded form the interphase nucleus by the nuclear membrane. Nuclear membrane permeabilization and repair can be used as an assay for licensing activity which could lead to its purification and subsequent analysis of its action within the nucleus.

Full Text

The Full Text of this article is available as a PDF (1.4 MB).

Selected References

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

  1. Alouf J. E. Streptococcal toxins (streptolysin O, streptolysin S, erythrogenic toxin). Pharmacol Ther. 1980;11(3):661–717. doi: 10.1016/0163-7258(80)90045-5. [DOI] [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. Preventing re-replication of DNA in a single cell cycle: evidence for a replication licensing factor. J Cell Biol. 1993 Sep;122(5):993–1002. doi: 10.1083/jcb.122.5.993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Blumenthal A. B., Kriegstein H. J., Hogness D. S. The units of DNA replication in Drosophila melanogaster chromosomes. Cold Spring Harb Symp Quant Biol. 1974;38:205–223. doi: 10.1101/sqb.1974.038.01.024. [DOI] [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. 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]
  9. D'Urso G., Marraccino R. L., Marshak D. R., Roberts J. M. Cell cycle control of DNA replication by a homologue from human cells of the p34cdc2 protein kinase. Science. 1990 Nov 9;250(4982):786–791. doi: 10.1126/science.2173140. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Duncan J. L., Schlegel R. Effect of streptolysin O on erythrocyte membranes, liposomes, and lipid dispersions. A protein-cholesterol interaction. J Cell Biol. 1975 Oct;67(1):160–174. doi: 10.1083/jcb.67.1.160. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dunn J. J., Krippl B., Bernstein K. E., Westphal H., Studier F. W. Targeting bacteriophage T7 RNA polymerase to the mammalian cell nucleus. Gene. 1988 Sep 7;68(2):259–266. doi: 10.1016/0378-1119(88)90028-5. [DOI] [PubMed] [Google Scholar]
  13. Dutta A., Din S., Brill S. J., Stillman B. Phosphorylation of replication protein A: a role for cdc2 kinase in G1/S regulation. Cold Spring Harb Symp Quant Biol. 1991;56:315–324. doi: 10.1101/sqb.1991.056.01.038. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Fotedar R., Roberts J. M. Association of p34cdc2 with replicating DNA. Cold Spring Harb Symp Quant Biol. 1991;56:325–333. doi: 10.1101/sqb.1991.056.01.039. [DOI] [PubMed] [Google Scholar]
  16. Fotedar R., Roberts J. M. Cell cycle regulated phosphorylation of RPA-32 occurs within the replication initiation complex. EMBO J. 1992 Jun;11(6):2177–2187. doi: 10.1002/j.1460-2075.1992.tb05277.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gibson S. I., Surosky R. T., Tye B. K. The phenotype of the minichromosome maintenance mutant mcm3 is characteristic of mutants defective in DNA replication. Mol Cell Biol. 1990 Nov;10(11):5707–5720. doi: 10.1128/mcb.10.11.5707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gurdon J. B. Injected nuclei in frog oocytes: fate, enlargement, and chromatin dispersal. J Embryol Exp Morphol. 1976 Dec;36(3):523–540. [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  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. McVey D., Brizuela L., Mohr I., Marshak D. R., Gluzman Y., Beach D. Phosphorylation of large tumour antigen by cdc2 stimulates SV40 DNA replication. Nature. 1989 Oct 12;341(6242):503–507. doi: 10.1038/341503a0. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. Prigent D., Alouf J. E. Interaction of steptolysin O with sterols. Biochim Biophys Acta. 1976 Aug 16;443(2):288–300. doi: 10.1016/0005-2736(76)90511-3. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. 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]
  29. 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]
  30. 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]

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

RESOURCES