Replication-dependent destruction of Cdt1 limits DNA replication to a single round per cell cycle in Xenopus egg extracts
Replication-dependent destruction of Cdt1 limits DNA replication to a single round per cell cycle in Xenopus egg extracts
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DOI：
10.1101/gad.1255805
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发表时间：
2005-01-01
影响因子：
10.5
通讯作者：
Walter, JC
中科院分区：
文献类型：
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作者：
Arias, EE;Walter, JC
在真核生物中，含有ORC、Cdc 6、Cdt 1和MCM 2 -7的复制前复合物（pre-RC）在G1期的染色质上组装。在S期，当DNA复制开始时，pre-RC被分解，并且新的pre-RC组装被限制，直到随后的G1期。因此，DNA复制被限制在每个细胞周期的一轮。在非洲爪蟾中发现了一种前RC组装抑制剂geminin，它在S期结合并使Cdt 1失活。然而，从爪蟾卵提取物中去除双生蛋白不足以引起再复制，这表明存在其他防止再复制的保障措施。在这里，我们表明，Cdt 1是完全降解的泛素介导的蛋白水解过程中的第一轮的DNA复制在非洲爪蟾卵提取物。降解依赖于Cdk 2/细胞周期蛋白E、Cdc 45、RPA和聚合酶a，表明需要复制起始。cdt 1在染色质上被泛素化，这一过程也需要复制起始。一旦复制开始，Cdk 2/细胞周期蛋白E被Cdt 1降解。当新鲜的Cdt 1提供后，第一轮的DNA复制，显着的再复制的结果，再复制增强的情况下，双生。我们的研究结果确定了一个复制依赖性的蛋白水解途径，目标Cdt 1和冗余的行为与双生蛋白，以阻止Cdt 1在S期。
In eukaryotes, prereplication complexes (pre-RCs) containing ORC, Cdc6, Cdt1, and MCM2-7 are assembled on chromatin in the G1 phase. In S phase, when DNA replication initiates, pre-RCs are disassembled, and new pre-RC assembly is restricted until the following G1 period. As a result, DNA replication is limited to a single round per cell cycle. One inhibitor of pre-RC assembly, geminin, was discovered in Xenopus, and it binds and inactivates Cdt1 in S phase. However, removal of geminin from Xenopus egg extracts is insufficient to cause rereplication, suggesting that other safeguards against rereplication exist. Here, we show that Cdt1 is completely degraded by ubiquitin-mediated proteolysis during the course of the first round of DNA replication in Xenopus egg extracts. Degradation depends on Cdk2/Cyclin E, Cdc45, RPA, and polymerase a, demonstrating a requirement for replication initiation. Cdt1 is ubiquitinated on chromatin, and this process also requires replication initiation. Once replication has initiated, Cdk2/Cyclin E is dispensable for Cdt1 degradation. When fresh Cdt1 is supplied after the first round of DNA replication, significant rereplication results, and rereplication is enhanced in the absence of geminin. Our results identify a replication-dependent proteolytic pathway that targets Cdt1 and that acts redundantly with geminin to inactivate Cdt1 in S phase.