Animal cells and systems

The Korean Society for Integrative Biology (한국통합생물학회)
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Biochemical Properties of the Minichromosomal Maintenance Complex after the Phosphorylation by Cdc7 Kinase

  • Lee, Joon-Kyu (Department of Biology Education, Seoul National University)
  • Published : 2006.03.31
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Abstract

Previous studies showed that Cdc7 kinase of Schizosaccharomyces pombe phosphorylated the minichromosome maintenance (Mcm) complex efficiently in the presence of spMcm10 protein. The biochemical properties of the phosphorylated Mcm complexes were examined to understand the activation mechanism of the Mcm complex by Cdc7 kinase. The phosphorylation of Mcm complex in the presence of spMcm10 by Cdc7 kinase did not affect the stability of the Mcm complex containing all six subunits, and the changes in the sedimentation properties were not observed after the phosphorylation. The reconstitution of the Mcm complex using the purified proteins showed that the phosphorylation of Mcm2 proteins did not affect the interactions between Mcm proteins. The phosphorylation of the Mcm2-7 complex at the same condition also did not activate the other biochemical activities such as DNA helicase and single stranded (ss) DNA binding activities. On the other hand, spMcm10 protein that was used for the stimulation of Mcm phosphorylation showed single stranded DNA binding activity, and inhibited the DNA helicase activity of the Mcm4/6/7 complex. These inhibitory effects were reduced by the addition of Cdc7 kinase, suggesting that the phosphorylation by Cdc7 kinase decreased the interactions between spMcm10 and the Mcm complex. Taken together, these results suggested that the phosphorylation by Cdc7 kinase alone is not sufficient for the remodeling and the activation of the Mcm complex, and the additional factors or the phosphorylations might be required for the activation of the Mcm complex.

Keywords

References

  1. Aparicio OM, Weinstein DM and Bell SP (1997) Components and dynamics of DNA replication complexes in S. cerevisiae: redistribution of MCM proteins and Cdc45p during S phase. Cell 91: 59-69 https://doi.org/10.1016/S0092-8674(01)80009-X
  2. Bell, SP and Dutta A (2002) DNA replication in eukaryotic cells. Annu Rev Biochem 71: 333-374 https://doi.org/10.1146/annurev.biochem.71.110601.135425
  3. Hardy CF, Dryga O, Seematter S, Pahl PM, and Sclafani RA (1997) mcm5/cdc46-bob1 bypasses the requirement for the S phase activator Cdc7p. Proc Natl Acad Sci USA 94: 3151-3155 https://doi.org/10.1073/pnas.94.7.3151
  4. Ishimi Y (1997) A DNA helicase activity is associated with an MCM4, -6, and -7 protein complex. J Biol Chem 272: 24508-24513 https://doi.org/10.1074/jbc.272.39.24508
  5. Kelly TJ and Brown GW (2000) Regulation of chromosome replication. Annu Rev Biochem 69: 829-880 https://doi.org/10.1146/annurev.biochem.69.1.829
  6. Kihara M, Nakai W, Asano S, Suzuki A, Kitada K, Kawasaki Y, Johnston LH, and Sugino A (2000) Characterization of the yeast Cdc7p/Dbf4p complex purified from insect cells. Its protein kinase activity is regulated by Rad53p. J Biol Chem 275:35051-35062 https://doi.org/10.1074/jbc.M003491200
  7. Labib K, Tercero JA, and Diffley JF (2000) Uninterrupted MCM2-7 function required for DNA replication fork progression. Science 288: 1643-1647 https://doi.org/10.1126/science.288.5471.1643
  8. Lee JK and Hurwitz J (2000) Isolation and characterization of various complexes of the minichromosome maintenance proteins of Schizosaccharomyces pombe. J Biol Chem 275: 18871-18878 https://doi.org/10.1074/jbc.M001118200
  9. Lee JK and Hurwitz J (2001) Processive DNA helicase activity of the minichromosome maintenance proteins 4, 6, and 7 complex requires forked DNA structures. Proc Natl Acad Sci USA 98: 54-59 https://doi.org/10.1073/pnas.98.1.54
  10. Lee JK, Seo YS, and Hurwitz J (2003) The Cdc23 (Mcm10) protein is required for the phosphorylation of minichromosome maintenance complex by the Dfp1-Hsk1 kinase. Proc Natl Acad Sci U S A 100: 2334-2339 https://doi.org/10.1073/pnas.0237384100
  11. Lei M, Kawasaki Y, Young MR, Kihara M, Sugino A, and Tye BK (1997) Mcm2 is a target of regulation by Cdc7-Dbf4 during the initiation of DNA synthesis. Genes Dev 11: 3365-3374 https://doi.org/10.1101/gad.11.24.3365
  12. Masai H and Arai K (2002) Cdc7 kinase complex: a key regulator in the initiation of DNA replication. J Cell Physiol 190: 287-296 https://doi.org/10.1002/jcp.10070
  13. Masai H, Matsui E, You Z, Ishimi Y, Tamai K, and Arai K (2000) Human Cdc7-related kinase complex. In vitro phosphorylation of MCM by concerted actions of Cdks and Cdc7 and that of a criticial threonine residue ofCdc7 by Cdks. J Biol Chem 275: 29042-29052 https://doi.org/10.1074/jbc.M002713200
  14. Thommes P, Kubota Y, Takisawa H, and Blow JJ (1997) The RLF-M component of the replication licensing system forms complexes containing all six MCM/P1 polypeptides. EMBO J 16: 3312-3319 https://doi.org/10.1093/emboj/16.11.3312
  15. Tye BK (1999) MCM proteins in DNA replication. Annu Rev Biochem 68: 649-686 https://doi.org/10.1146/annurev.biochem.68.1.649
  16. Weinreich M and Stillman B (1999) Cdc7p-Dbf4p kinase binds to chromatin during S phase and is regulated by both the APC and the RAD53 checkpoint pathway. EMBO J 18: 5334-5346 https://doi.org/10.1093/emboj/18.19.5334
  17. You Z, Ishimi Y, Mizuno T, Sugasawa K, Hanaoka F, and Masai H (2003) Thymine-rich single-stranded DNA activates Mcm4/6/7 helicase on Y-fork and bubble-like substrates. EMBO J 22: 6148-6160 https://doi.org/10.1093/emboj/cdg576