Cell cycle regulation of DNA replication licensing factor Cdt1

• Project/Area Number: 14580683
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Molecular biology
• Research Institution: Kyushu University
• Principal Investigator: NISHITANI Hideo Kyushu University, Grad.Schl.Medical Science, Research Assistant, 大学院・医学研究院, 助手 (40253455)
• Project Period (FY): 2002 – 2003
• Project Status: Completed (Fiscal Year 2003)
• Budget Amount: ¥4,100,000 (Direct Cost: ¥4,100,000); Fiscal Year 2003: ¥2,000,000 (Direct Cost: ¥2,000,000); Fiscal Year 2002: ¥2,100,000 (Direct Cost: ¥2,100,000)
• Keywords: Replication / Cell cycle / Cdt1 / Geminin / Proteolysis / CDK / Skp2 / ライセンス化 / 染色体

Research Abstract

The genome integrity is maintained by ensuring a DNA replication only once in a cell cycle. To elucidate this control, we have been working on DNA replication licensing factor Cdt1.
1.Domain analysis of Cdt1 protein : We made a series of N-terminus or C-terminus -deleted constructs of Cdt1, and examined their interaction with Geminin and Cyclin-CDKs. We showed that (a) Geminin binds to. the middle of Cdt1, (b) Cdt1 associates with Cyclin A, but not with Cyclins B and E. CyclinA binds to the N-terminus of Cdt1.(C) We also found that N-terminus has an NLS, nuclear localization signal.
2.Control of Cdt1 proteolysis and effect of high expression of Cdt1 in cell cycle : We constructed stable HeLa cell lines expressing N-terminus(1-189) or C-termin us (160-end) (fused with NLS) of Cdt1, and examined their stability during a cell cyle. We showed that N-terminus of Cdt1 is involved in its degradation in S-phase. CyclinA associates with N-terminus of, Cdtl, and phosphorylates it, leading to Skp2 binding and following ubiquitination and degradation SiRNA experiments for Geminin shows that Cdt1 is degraded in the absence of Geminin in S-phase. On the other hand, high expression of stable (160-end) (fused with NLS) of Cdt1 in 293T cells resulted in increase in DNA content. We concluded that Cdt1 is inactiveated independently by Geminin binding and proteolysis to block re-replication.

Research Products

• [Publications] Nishitani H.et al.: "Proteolysis of DNA replication licensing factor Cdt1 in S-phase is performed independently of Geminin through its N-terminal region"J Biol, Chem.. (In press). (2004)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Nishitani H.et al.: "DNA Replication Licensing"Frontiers in Bioscience. (In press). (2004)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Sugimoto N.et al.: "Cdt1 phosphorylation by cyclin A-dependent kinases negatively regulates its function without affecting geminin binding"J Biol, Chem.. 279. 19691-19697 (2004)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Takahashi T.et al.: "Multiple ORC- binding sites are required for efficient MCM loading and origin firing in fission yeast"EMBO J.. 22. 964-974 (2003)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Nishitani H.et al.: "Control of DNA replication licensing in a cell cycle"Genes Cells. 7. 523-534 (2002)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Nishitani H., Lygerou Z., Nishimoto T.: "Proteolysis of DNA replication licensing factor Cdt1 in S-phase is performed independently of Geminin through its N4errninal region"J.Biol.Chem.. (in press). (2004)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Nishitani H., Lygerou Z.: "DNA replication licensing"Frontiers in Bioscience. (in press). (2004)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Sugimoto N, Tatsumi Y, Tsurumi T, Matsukage A, Kiyono T, Nishitani H, Fujita M.: "Cdt1 phosphorylation by cyclin A-dependent kinases negatively regulates its function without affecting geminin binding"J.Biol.Chem.. 279. 19691-19697 (2004)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Takahashi T, Ohara B, Nishitani H, Masukata H.: "Multiple ORC-binding sites are required for efficient MCM Loading and origin firing in fission yeast"EMBO J.. 22. 964-974 (2003)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Nishitani, H, Lygerou Z.: "Control of DNA replication licensing in a cell cycle"Genes Cells. 7. 523-534 (2002)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Nishitani H. et al.: "DNA Replication Licensing"Frontier Bioscience. (In press).
• [Publications] Seino H. et al.: "Two ubiquitin-conjugating enzymes, UbcP1/Ubc4 and UbcP4/Ubc11, have distinct functions for ubiquitination of mitotic cyclin"Mol Cell Biol. 23. 3497-3505 (2003)
• [Publications] Nishijima H. et al.: "Caffeine mimics adenine and 2'-deoxyadenosine, both of which inhibit the guanine-nucleotide exchange activity of RCC1 and the kinase activity of ATR."Genes Cells.. 8. 423-435 (2003)
• [Publications] Takahashi T. et al.: "ORC-binding sites are required for efficient MCM loading and origin firing in fission yeast"EMBO J.. 22. 964-974 (2003)
• [Publications] Umeda M. et al.: "A novel nuclear protein, Twa1, and Muskelin comprise a complex with RanBPM."Gene. 303. 47-54 (2003)
• [Publications] Nishitani H. et al.: "Control of DNA replication licensing in a cell cycle"Genes to Cells. 7. 523-534 (2002)
• [Publications] Takahashi T. et al.: "Multiple ORC-binding sites are required for efficient MCM loading and origin firing in fission yeast"EMBO J.. 22. 964-974 (2003)