Cell cycle regulation of DNA replication licensing factor Cdt1

基因组的完整性是通过确保DNA在细胞周期中只复制一次来维持的。为了阐明这种控制，我们一直在研究DNA复制许可因子Cdt1.1。Cdt1蛋白结构域分析：我们构建了一系列Cdt1的n端或c端缺失结构，并检测了它们与Geminin和Cyclin-CDKs的相互作用。我们发现(a) Geminin与。Cdt1中间，(b) Cdt1与CyclinA结合，但不与Cyclin b和e结合。(C)我们还发现n端具有NLS，核定位信号。Cdt1蛋白水解的控制和细胞周期中Cdt1高表达的影响：我们构建了表达Cdt1的n端（1-189）或c端（160-末端）（与NLS融合）的稳定HeLa细胞系，并检测了它们在细胞周期中的稳定性。我们发现Cdt1的n端参与了它在s期的降解。CyclinA与Cdtl的n端结合，并使其磷酸化，导致Skp2结合，随后对Geminin进行泛素化和降解SiRNA实验表明，在s期没有Geminin的情况下，Cdt1被降解。另一方面，Cdt1在293T细胞中稳定（160-end）（与NLS融合）的高表达导致DNA含量增加。我们得出结论，Cdt1可以通过双胞蛋白结合和蛋白水解来阻断再复制而独立失活。

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.