Cell cycle regulation of DNA replication licensing factor Cdt1

DNA复制许可因子Cdt1的细胞周期调控

• 批准号: 14580683
• 负责人: NISHITANI Hideo
• 金额: $ 2.62万
• 依托单位: Kyushu University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14580683/
• 关键词: Replication; Cell cycle; Cdt1; Geminin; Proteolysis; CDK; Skp2; ライセンス化; 染色体

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.

通过在细胞周期中仅确保仅一次DNA复制来维持基因组完整性。为了阐明这种对照，我们一直在研究DNA复制许可因子CDT1.1.CDT1蛋白的域分析：我们制作了一系列的N末端或C-terminus或C-terminus删除CDT1的构建体，并检查了它们与Geminin和Cycinin and Cycinin和Cycinin和cyclin的相互作用-cdks。我们表明（a）双子素与。 CDT1，（b）CDT1的中间与细胞周期蛋白A相关，而与细胞周期蛋白B和E. Cyclina则与CDT1的N末端结合。 2. CDT1蛋白水解的控制和CDT1在细胞周期中的高表达的影响：我们构建了表达N末端（1-189）或C-termin US（160-End）（与NLS）（与NLS）（融合NLS）（融合）的稳定的HELA细胞系（与CDT1的NLS），并检查了它们在细胞箱中的稳定性。我们表明，CDT1的N末端参与其在S期中的降解。 Cyclina与cdtl和磷酸化的N端相关，从而导致SKP2结合，并在泛素化和降解的siRNA实验后，表明CDT1在S-CHASE中没有Geminin的情况下降解了CDT1。另一方面，293T细胞中CDT1的稳定（160端）（与NLS融合）的高表达导致DNA含量增加。我们得出的结论是，CDT1通过双子素结合和蛋白水解独立地灭绝以阻止重新复制。

项目成果

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Nishijima H. 等人：“咖啡因模拟腺嘌呤和 2-脱氧腺苷，两者都会抑制 RCC1 的鸟嘌呤核苷酸交换活性和 ATR 的激酶活性。”Genes Cells.. 8. 423-435 (2003)

Nishitani H.et al.: "Control of DNA replication licensing in a cell cycle"Genes Cells. 7. 523-534 (2002)

Nishitani H.等人：“细胞周期中 DNA 复制许可的控制”Genes Cells。

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)

Takahashi T.et al.：“裂殖酵母中有效的 MCM 加载和起始激发需要多个 ORC 结合位点”EMBO J.. 22. 964-974 (2003)

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)

Nishitani H.、Lygerou Z.、Nishimoto T.：“S 期 DNA 复制许可因子 Cdt1 的蛋白水解是通过其 N4errninal 区域独立于 Geminin 进行的”J.Biol.Chem..（出版中）。

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)

Seino H. 等人：“两种泛素结合酶，UbcP1/Ubc4 和 UbcP4/Ubc11，对有丝分裂周期蛋白泛素化具有不同的功能”Mol Cell Biol。