Analysis of JET1 mutant, which does not require CDK activity in the initiation of DNA replication

真核生物DNA复制起始于多个复制起点，它们由两种蛋白激酶CDK和Cdc7-Dbf4激活，DNA复制起始于S期。CDK在这一过程中的底物一直不为人所知，S1d2在2002年被证明是CDK起始的一个重要靶点。我已经证明了Sld2磷酸化是必要的，但不是起始的必要条件。因此，为了识别未知的CDK靶点，我建立了一个基因筛选。在筛选中，构建了Sld2的拟磷突变体（S1d2-D）并分离出新的突变体JET1，该突变体即使在缺乏CDK活性的情况下也能与S1d2-D一起启动DNA复制。这种JET1突变发生在CDC45上，这是起始和延伸所必需的。我们发现Cdc45不是CDK的底物，相反，Cdc45的结合伙伴Sld3是CDK起始的基本靶点。我们还发现，Sld2和Sld3的磷酸化增强了Dpb11的相互作用。有人认为这个反应触发了DNA复制的开始。此外，通过结合绕过Sld2和S1d3磷酸化的突变，我们可以绕过CDK对起始的要求。这些数据强烈提示Sid2和Sld3是CDK起始的最低基本要求。这些发现对于理解真核生物DNA复制的起始反应至关重要。

Eukaryotic DNA replication initiates from multiple origins of replication They are activated by two protein kinases, CDK and Cdc7-Dbf4, and DNA replication initiates in S phase. Substrates of CDK in this process was unknown for long time, S1d2 was shown as an essential target of CDK in the initiation in 2002. I have shown that Sld2 phosphorylation is necessary but not essential for the initiation. Therefore, to identify unknown CDK target, I set up a genetic screening. In the screening, phosphomimetic mutant of Sld2 (S1d2-D) was constructed and isolated the novel mutant JET1, which initiated DNA replication in conjunction with S1d2-D even in the absence of CDK activity. This JET1 mutation was occurred in CDC45, which required for the initiation and elongation. We revealed that Cdc45 is not the substrate of CDK, instead Sld3, the binding partner of Cdc45, is the essential target of CDK in initiation. We also showed that phosphorylations of Sld2 and Sld3 enhance the interaction Dpb11. It was suggested that this reaction triggers the initiation of DNA replication. Moreover by combining the mutations that bypass the phosphorylation of Sld2 and S1d3, we could bypass CDK requirements for initiation. These data strongly suggest that Sid2 and Sld3 constitute minimal essential requirements of CDK in initiation. These findings are crucial to understand the initiation reaction of eukaryotic DNA replication.