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. 1996 Sep 17;93(19):10189–10194. doi: 10.1073/pnas.93.19.10189

XMCM7, a novel member of the Xenopus MCM family, interacts with XMCM3 and colocalizes with it throughout replication.

P Romanowski 1, M A Madine 1, R A Laskey 1
PMCID: PMC38359  PMID: 8816774

Abstract

A minichromosome maintenance (MCM) protein complex has been implicated in restricting DNA replication to once per cell cycle in Xenopus egg extracts, based on the behavior of a single protein, XMCM3. Using a two-hybrid screen with XMCM3, we have identified a novel member of the MCM family in Xenopus that is essential for DNA replication. The protein shows strong homology to Saccharomyces cerevisiae MCM7 (CDC47) and has thus been named XMCM7. XMCM7 is present in a multiprotein complex with other MCM proteins. It binds to chromatin and is displaced from chromatin by the act of replication. XMCM7 does not preferentially colocalize with sites of DNA replication but colocalizes with XMCM3 throughout replication. Immunodepletion of the MCM complex from Xenopus egg extract by anti-XMCM7 antibodies inhibits DNA replication of sperm and permeable HeLa G2 nuclei but not permeable HeLa G1 nuclei. Replication capacity of the Xenopus egg extract immunodepleted of the MCM complex by anti-XMCM7 antibody can be rescued by MCM proteins eluted from anti-XMCM3 antibody. We conclude that both proteins are present in the same complex in Xenopus egg extract throughout the cell cycle, that they remain together after binding to chromatin and during DNA replication, and that they perform similar functions.

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Selected References

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

  1. 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]
  2. Breeden L., Nasmyth K. Regulation of the yeast HO gene. Cold Spring Harb Symp Quant Biol. 1985;50:643–650. doi: 10.1101/sqb.1985.050.01.078. [DOI] [PubMed] [Google Scholar]
  3. Burkhart R., Schulte D., Hu D., Musahl C., Göhring F., Knippers R. Interactions of human nuclear proteins P1Mcm3 and P1Cdc46. Eur J Biochem. 1995 Mar 1;228(2):431–438. [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. Dalton S., Whitbread L. Cell cycle-regulated nuclear import and export of Cdc47, a protein essential for initiation of DNA replication in budding yeast. Proc Natl Acad Sci U S A. 1995 Mar 28;92(7):2514–2518. doi: 10.1073/pnas.92.7.2514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Harshman K. D., Moye-Rowley W. S., Parker C. S. Transcriptional activation by the SV40 AP-1 recognition element in yeast is mediated by a factor similar to AP-1 that is distinct from GCN4. Cell. 1988 Apr 22;53(2):321–330. doi: 10.1016/0092-8674(88)90393-5. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Hollenberg S. M., Sternglanz R., Cheng P. F., Weintraub H. Identification of a new family of tissue-specific basic helix-loop-helix proteins with a two-hybrid system. Mol Cell Biol. 1995 Jul;15(7):3813–3822. doi: 10.1128/mcb.15.7.3813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Kearsey S. E., Maiorano D., Holmes E. C., Todorov I. T. The role of MCM proteins in the cell cycle control of genome duplication. Bioessays. 1996 Mar;18(3):183–190. doi: 10.1002/bies.950180305. [DOI] [PubMed] [Google Scholar]
  12. Kimura H., Nozaki N., Sugimoto K. DNA polymerase alpha associated protein P1, a murine homolog of yeast MCM3, changes its intranuclear distribution during the DNA synthetic period. EMBO J. 1994 Sep 15;13(18):4311–4320. doi: 10.1002/j.1460-2075.1994.tb06751.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Koonin E. V. A common set of conserved motifs in a vast variety of putative nucleic acid-dependent ATPases including MCM proteins involved in the initiation of eukaryotic DNA replication. Nucleic Acids Res. 1993 Jun 11;21(11):2541–2547. doi: 10.1093/nar/21.11.2541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kozak M. An analysis of vertebrate mRNA sequences: intimations of translational control. J Cell Biol. 1991 Nov;115(4):887–903. doi: 10.1083/jcb.115.4.887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Krude T., Musahl C., Laskey R. A., Knippers R. Human replication proteins hCdc21, hCdc46 and P1Mcm3 bind chromatin uniformly before S-phase and are displaced locally during DNA replication. J Cell Sci. 1996 Feb;109(Pt 2):309–318. doi: 10.1242/jcs.109.2.309. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. 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]
  19. 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]
  20. Maine G. T., Sinha P., Tye B. K. Mutants of S. cerevisiae defective in the maintenance of minichromosomes. Genetics. 1984 Mar;106(3):365–385. doi: 10.1093/genetics/106.3.365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Maiorano D., Van Assendelft G. B., Kearsey S. E. Fission yeast cdc21, a member of the MCM protein family, is required for onset of S phase and is located in the nucleus throughout the cell cycle. EMBO J. 1996 Feb 15;15(4):861–872. [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Musahl C., Schulte D., Burkhart R., Knippers R. A human homologue of the yeast replication protein Cdc21. Interactions with other Mcm proteins. Eur J Biochem. 1995 Jun 15;230(3):1096–1101. doi: 10.1111/j.1432-1033.1995.tb20660.x. [DOI] [PubMed] [Google Scholar]
  24. Nakatsuru S., Sudo K., Nakamura Y. Isolation and mapping of a human gene (MCM2) encoding a product homologous to yeast proteins involved in DNA replication. Cytogenet Cell Genet. 1995;68(3-4):226–230. doi: 10.1159/000133918. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Ray A., Roy N., Maitra M., Sinha P. A 61-kb ring chromosome shows an ARS-dependent increase in its mitotic stability in the mcm2 mutant of yeast. Curr Genet. 1994 Nov-Dec;26(5-6):403–409. doi: 10.1007/BF00309926. [DOI] [PubMed] [Google Scholar]
  27. Robbins J., Dilworth S. M., Laskey R. A., Dingwall C. Two interdependent basic domains in nucleoplasmin nuclear targeting sequence: identification of a class of bipartite nuclear targeting sequence. Cell. 1991 Feb 8;64(3):615–623. doi: 10.1016/0092-8674(91)90245-t. [DOI] [PubMed] [Google Scholar]
  28. Romanowski P., Madine M. A. Mechanisms restricting DNA replication to once per cell cycle: MCMS, pre-replicative complexes and kinases. Trends Cell Biol. 1996 May;6(5):184–188. doi: 10.1016/0962-8924(96)10015-5. [DOI] [PubMed] [Google Scholar]
  29. Schiestl R. H., Gietz R. D. High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr Genet. 1989 Dec;16(5-6):339–346. doi: 10.1007/BF00340712. [DOI] [PubMed] [Google Scholar]
  30. Schulte D., Burkhart R., Musahl C., Hu B., Schlatterer C., Hameister H., Knippers R. Expression, phosphorylation and nuclear localization of the human P1 protein, a homologue of the yeast Mcm 3 replication protein. J Cell Sci. 1995 Apr;108(Pt 4):1381–1389. doi: 10.1242/jcs.108.4.1381. [DOI] [PubMed] [Google Scholar]
  31. Springer P. S., McCombie W. R., Sundaresan V., Martienssen R. A. Gene trap tagging of PROLIFERA, an essential MCM2-3-5-like gene in Arabidopsis. Science. 1995 May 12;268(5212):877–880. doi: 10.1126/science.7754372. [DOI] [PubMed] [Google Scholar]
  32. Takahashi K., Yamada H., Yanagida M. Fission yeast minichromosome loss mutants mis cause lethal aneuploidy and replication abnormality. Mol Biol Cell. 1994 Oct;5(10):1145–1158. doi: 10.1091/mbc.5.10.1145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Todorov I. T., Attaran A., Kearsey S. E. BM28, a human member of the MCM2-3-5 family, is displaced from chromatin during DNA replication. J Cell Biol. 1995 Jun;129(6):1433–1445. doi: 10.1083/jcb.129.6.1433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Yan H., Merchant A. M., Tye B. K. Cell cycle-regulated nuclear localization of MCM2 and MCM3, which are required for the initiation of DNA synthesis at chromosomal replication origins in yeast. Genes Dev. 1993 Nov;7(11):2149–2160. doi: 10.1101/gad.7.11.2149. [DOI] [PubMed] [Google Scholar]

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