Proteolysis of DNA replication licensing factor Cdt1 in S-phase is performed independently of Geminin through its N-terminal region
Proteolysis of DNA replication licensing factor Cdt1 in S-phase is performed independently of Geminin through its N-terminal region
复制标题
DOI：
10.1074/jbc.m312644200
复制
发表时间：
2004-07-16
影响因子：
4.8
通讯作者：
Nishimoto, T
中科院分区：
文献类型：
--
作者：
Nishitani, H;Lygerou, Z;Nishimoto, T
复制起点的许可在细胞周期中受到仔细调节，以维持基因组完整性。使用体内泛素化测定和温度敏感细胞系，我们证明哺乳动物细胞中的 Cdt1 通过 S 期泛素依赖性蛋白水解作用被降解。 Geminin 的 siRNA 实验表明，在没有 Geminin 的情况下，Cdt1 会被降解。 Cdt1 的 N 末端是其核定位所必需的，与细胞周期蛋白 A 相关，但对于 Cdt1 与细胞中 Geminin 的结合来说是可有可无的。该区域负责 S 期 Cdt1 的蛋白水解。另一方面，N 末端截短的 Cdt1 在 S 期稳定，并与许可抑制剂 Geminin 结合。这种形式的 Cdt1 的高水平表达使细胞具有更高的 DNA 含量。蛋白酶体抑制剂将 Cdt1 稳定在 S 期，并且在细胞核中检测到该蛋白质与 Geminin 形成复合物。当没有检测到 Geminin 时，这种形式的 Cdt1 与染色质的结合与 G1 细胞的结合紧密。我们的数据表明，在 S 期开始后，蛋白水解和 Geminin 结合独立地使 Cdt1 失活，以防止再复制。
Licensing of replication origins is carefully regulated in a cell cycle to maintain genome integrity. Using an in vivo ubiquitination assay and temperature-sensitive cell lines we demonstrate that Cdt1 in mammalian cells is degraded through ubiquitin-dependent proteolysis in S-phase. siRNA experiments for Geminin indicate that Cdt1 is degraded in the absence of Geminin. The N terminus of Cdt1 is required for its nuclear localization, associates with cyclin A, but is dispensable for the association of Cdt1 with Geminin in cells. This region is responsible for proteolysis of Cdt1 in S-phase. On the other hand, the N terminus-truncated Cdt1 is stable in S-phase, and associates with the licensing inhibitor, Geminin. High level expression of this form of Cdt1 brings about cells having higher DNA content. Proteasome inhibitors stabilize Cdt1 in S-phase, and the protein is detected in the nucleus in a complex with Geminin. This form of Cdt1 associates with chromatin as tightly as that of G1-cells, when no Geminin is detected. Our data show that proteolysis and Geminin binding independently inactivate Cdt1 after the onset of S-phase to prevent re-replication.