Abstract
In eukaryotes, tight regulatory mechanisms ensure the ordered progression through the cell cycle phases. The mechanisms that prevent chromosomal DNA replication from taking place more than once each cell cycle are thought to involve the function of proteins of the minichromosome maintenance (MCM) family. Here, we demonstrate that Xenopus MCM4, a member of the MCM protein family related to Spcdc21/ ScCDC54, is part of a large protein complex comprising several other MCM proteins. MCM4 undergoes cell cycle-dependent phosphorylation both in cleaving embryos and in cell-free extracts. MCM4 phosphorylation starts concomitantly with the clearing of the MCM complex from the chromatin during S phase. Phosphorylation is carried out by cdc2/cyclinB protein kinase, which phosphorylates MCM4 in vitro at identical sites as the ones phosphorylated in vivo. Phosphorylation is specific for cdc2 protein kinase since MCM4 is not a substrate for other members of the cdk family. Furthermore, phosphorylation of MCM4 dramatically reduces its affinity for the chromatin. We propose that the cell cycle-dependent phosphorylation of MCM4 is a mechanism which inactivates the MCM complex from late S phase through mitosis, thus preventing illegitimate DNA replication during that period of the cell cycle.
Full text
PDF





Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Adachi Y., Laemmli U. K. 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. doi: 10.1002/j.1460-2075.1994.tb06733.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- 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]
- Chong J. P., Mahbubani H. M., Khoo C. Y., Blow J. J. Purification of an MCM-containing complex as a component of the DNA replication licensing system. Nature. 1995 Jun 1;375(6530):418–421. doi: 10.1038/375418a0. [DOI] [PubMed] [Google Scholar]
- Coué M., Kearsey S. E., Méchali M. Chromotin binding, nuclear localization and phosphorylation of Xenopus cdc21 are cell-cycle dependent and associated with the control of initiation of DNA replication. EMBO J. 1996 Mar 1;15(5):1085–1097. [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- Gautier J., Maller J. L. Cyclin B in Xenopus oocytes: implications for the mechanism of pre-MPF activation. EMBO J. 1991 Jan;10(1):177–182. doi: 10.1002/j.1460-2075.1991.tb07934.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gautier J., Matsukawa T., Nurse P., Maller J. Dephosphorylation and activation of Xenopus p34cdc2 protein kinase during the cell cycle. Nature. 1989 Jun 22;339(6226):626–629. doi: 10.1038/339626a0. [DOI] [PubMed] [Google Scholar]
- Harper J. W., Elledge S. J., Keyomarsi K., Dynlacht B., Tsai L. H., Zhang P., Dobrowolski S., Bai C., Connell-Crowley L., Swindell E. Inhibition of cyclin-dependent kinases by p21. Mol Biol Cell. 1995 Apr;6(4):387–400. doi: 10.1091/mbc.6.4.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hayles J., Fisher D., Woollard A., Nurse P. Temporal order of S phase and mitosis in fission yeast is determined by the state of the p34cdc2-mitotic B cyclin complex. Cell. 1994 Sep 9;78(5):813–822. doi: 10.1016/s0092-8674(94)90542-8. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- Hu B., Burkhart R., Schulte D., Musahl C., Knippers R. The P1 family: a new class of nuclear mammalian proteins related to the yeast Mcm replication proteins. Nucleic Acids Res. 1993 Nov 25;21(23):5289–5293. doi: 10.1093/nar/21.23.5289-a. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kubota Y., Mimura S., Nishimoto S., Takisawa H., Nojima H. Identification of the yeast MCM3-related protein as a component of Xenopus DNA replication licensing factor. Cell. 1995 May 19;81(4):601–609. doi: 10.1016/0092-8674(95)90081-0. [DOI] [PubMed] [Google Scholar]
- Madine M. A., Khoo C. Y., Mills A. D., Laskey R. A. MCM3 complex required for cell cycle regulation of DNA replication in vertebrate cells. Nature. 1995 Jun 1;375(6530):421–424. doi: 10.1038/375421a0. [DOI] [PubMed] [Google Scholar]
- Madine M. A., Khoo C. Y., Mills A. D., Musahl C., Laskey R. A. The nuclear envelope prevents reinitiation of replication by regulating the binding of MCM3 to chromatin in Xenopus egg extracts. Curr Biol. 1995 Nov 1;5(11):1270–1279. doi: 10.1016/s0960-9822(95)00253-3. [DOI] [PubMed] [Google Scholar]
- Moir D., Stewart S. E., Osmond B. C., Botstein D. Cold-sensitive cell-division-cycle mutants of yeast: isolation, properties, and pseudoreversion studies. Genetics. 1982 Apr;100(4):547–563. doi: 10.1093/genetics/100.4.547. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Moreno S., Nurse P. Regulation of progression through the G1 phase of the cell cycle by the rum1+ gene. Nature. 1994 Jan 20;367(6460):236–242. doi: 10.1038/367236a0. [DOI] [PubMed] [Google Scholar]
- Su T. T., Feger G., O'Farrell P. H. Drosophila MCM protein complexes. Mol Biol Cell. 1996 Feb;7(2):319–329. doi: 10.1091/mbc.7.2.319. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tye B. K. The MCM2-3-5 proteins: are they replication licensing factors? Trends Cell Biol. 1994 May;4(5):160–166. doi: 10.1016/0962-8924(94)90200-3. [DOI] [PubMed] [Google Scholar]
- Whitebread L. A., Dalton S. Cdc54 belongs to the Cdc46/Mcm3 family of proteins which are essential for initiation of eukaryotic DNA replication. Gene. 1995 Mar 21;155(1):113–117. doi: 10.1016/0378-1119(94)00925-i. [DOI] [PubMed] [Google Scholar]
- 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]