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. 1996 Sep;16(9):5081–5090. doi: 10.1128/mcb.16.9.5081

Physical interactions among Mcm proteins and effects of Mcm dosage on DNA replication in Saccharomyces cerevisiae.

M Lei 1, Y Kawasaki 1, B K Tye 1
PMCID: PMC231509  PMID: 8756666

Abstract

Mcm2, Mcm3, and Mcm5/Cdc46 are conserved proteins essential for the initiation of DNA synthesis at replication origins in Saccharomyces cerevisiae. The accumulation of these proteins in the nucleus before the onset of DNA synthesis suggests that they play a role in restricting DNA synthesis to once per cell cycle. In this work, we show that Mcm2, Mcm3, and Mcm5 self-interact and interact with one another to form complexes. Mcm2 and Mcm3 are abundant proteins, present in approximately 4 X 10(4) and 2 X 10(5) copies per cell, respectively. Reducing the dosage of Mcm2 by half results in diminished usage of specific replication origins. These results together suggest that a significant molar excess of Mcm proteins relative to replication origins is required for the proper initiation of all replication origins.

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Selected References

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  1. Alfano C., McMacken R. Ordered assembly of nucleoprotein structures at the bacteriophage lambda replication origin during the initiation of DNA replication. J Biol Chem. 1989 Jun 25;264(18):10699–10708. [PubMed] [Google Scholar]
  2. Bell S. P., Stillman B. ATP-dependent recognition of eukaryotic origins of DNA replication by a multiprotein complex. Nature. 1992 May 14;357(6374):128–134. doi: 10.1038/357128a0. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. Broach J. R., Li Y. Y., Feldman J., Jayaram M., Abraham J., Nasmyth K. A., Hicks J. B. Localization and sequence analysis of yeast origins of DNA replication. Cold Spring Harb Symp Quant Biol. 1983;47(Pt 2):1165–1173. doi: 10.1101/sqb.1983.047.01.132. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Chan C. S., Tye B. K. Autonomously replicating sequences in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6329–6333. doi: 10.1073/pnas.77.11.6329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chen Y., Hennessy K. M., Botstein D., Tye B. K. CDC46/MCM5, a yeast protein whose subcellular localization is cell cycle-regulated, is involved in DNA replication at autonomously replicating sequences. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10459–10463. doi: 10.1073/pnas.89.21.10459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Chong J. P., Thömmes P., Blow J. J. The role of MCM/P1 proteins in the licensing of DNA replication. Trends Biochem Sci. 1996 Mar;21(3):102–106. [PubMed] [Google Scholar]
  11. 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]
  12. Diffley J. F., Cocker J. H. Protein-DNA interactions at a yeast replication origin. Nature. 1992 May 14;357(6374):169–172. doi: 10.1038/357169a0. [DOI] [PubMed] [Google Scholar]
  13. Dubey D. D., Davis L. R., Greenfeder S. A., Ong L. Y., Zhu J. G., Broach J. R., Newlon C. S., Huberman J. A. Evidence suggesting that the ARS elements associated with silencers of the yeast mating-type locus HML do not function as chromosomal DNA replication origins. Mol Cell Biol. 1991 Oct;11(10):5346–5355. doi: 10.1128/mcb.11.10.5346. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Ferguson B. M., Brewer B. J., Reynolds A. E., Fangman W. L. A yeast origin of replication is activated late in S phase. Cell. 1991 May 3;65(3):507–515. doi: 10.1016/0092-8674(91)90468-e. [DOI] [PubMed] [Google Scholar]
  15. Fields S., Song O. A novel genetic system to detect protein-protein interactions. Nature. 1989 Jul 20;340(6230):245–246. doi: 10.1038/340245a0. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  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. Huberman J. A., Zhu J. G., Davis L. R., Newlon C. S. Close association of a DNA replication origin and an ARS element on chromosome III of the yeast, Saccharomyces cerevisiae. Nucleic Acids Res. 1988 Jul 25;16(14A):6373–6384. doi: 10.1093/nar/16.14.6373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jarvik J., Botstein D. Conditional-lethal mutations that suppress genetic defects in morphogenesis by altering structural proteins. Proc Natl Acad Sci U S A. 1975 Jul;72(7):2738–2742. doi: 10.1073/pnas.72.7.2738. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. 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]
  25. Kornberg R. D., Klug A. The nucleosome. Sci Am. 1981 Feb;244(2):52–64. doi: 10.1038/scientificamerican0281-52. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Li J. J., Herskowitz I. Isolation of ORC6, a component of the yeast origin recognition complex by a one-hybrid system. Science. 1993 Dec 17;262(5141):1870–1874. doi: 10.1126/science.8266075. [DOI] [PubMed] [Google Scholar]
  28. Loo S., Fox C. A., Rine J., Kobayashi R., Stillman B., Bell S. The origin recognition complex in silencing, cell cycle progression, and DNA replication. Mol Biol Cell. 1995 Jun;6(6):741–756. doi: 10.1091/mbc.6.6.741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. 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]
  31. 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]
  32. Petes T. D., Williamson D. H. Fiber autoradiography of replicating yeast DNA. Exp Cell Res. 1975 Oct 1;95(1):103–110. doi: 10.1016/0014-4827(75)90614-x. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Reynolds A. E., McCarroll R. M., Newlon C. S., Fangman W. L. Time of replication of ARS elements along yeast chromosome III. Mol Cell Biol. 1989 Oct;9(10):4488–4494. doi: 10.1128/mcb.9.10.4488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Rivin C. J., Fangman W. L. Replication fork rate and origin activation during the S phase of Saccharomyces cerevisiae. J Cell Biol. 1980 Apr;85(1):108–115. doi: 10.1083/jcb.85.1.108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Starborg M., Hög C. The murine replication protein P1 is differentially expressed during spermatogenesis. Eur J Cell Biol. 1995 Oct;68(2):206–210. [PubMed] [Google Scholar]
  38. TAYLOR J. H. Asynchronous duplication of chromosomes in cultured cells of Chinese hamster. J Biophys Biochem Cytol. 1960 Jun;7:455–464. doi: 10.1083/jcb.7.3.455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. Treisman J. E., Follette P. J., O'Farrell P. H., Rubin G. M. Cell proliferation and DNA replication defects in a Drosophila MCM2 mutant. Genes Dev. 1995 Jul 15;9(14):1709–1715. doi: 10.1101/gad.9.14.1709. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. 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]
  43. 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]
  44. 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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