2003 Fiscal Year Final Research Report Summary
Cell cycle regulation of DNA replication licensing factor Cdt1
| Project/Area Number |
14580683
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Molecular biology
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| Research Institution | Kyushu University |
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Principal Investigator |
NISHITANI Hideo Kyushu University, Grad.Schl.Medical Science, Research Assistant, 大学院・医学研究院, 助手 (40253455)
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| Project Period (FY) |
2002 – 2003
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| Keywords | Replication / Cell cycle / Cdt1 / Geminin / Proteolysis / CDK / Skp2 |
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| 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.
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Research Products
(10 results)
