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Cdc 7-Dbf 4激活，DNA复制起始于S期。CDK在此过程中的作用底物一直不清楚，2002年首次发现S1 d2是CDK的重要作用靶点。我已经表明，Sld 2磷酸化是必要的，但不是必不可少的启动。因此，为了确定未知的CDK靶点，我建立了一个基因筛选。在筛选中，构建了Sld 2的磷酸化模拟突变体（S1 d2-D），并分离了新的突变体JET 1，其即使在没有CDK活性的情况下也能与S1 d2-D一起启动DNA复制。JET 1突变发生在CDC 45中，这是启动和延伸所必需的。我们发现Cdc 45不是CDK的底物，而Cdc 45的结合伴侣Sld 3是CDK启动的重要靶点。我们还表明，磷酸化的Sld 2和Sld 3增强相互作用Dpb 11。有人认为，这种反应触发了DNA复制的启动。此外，通过组合绕过Sld 2和S1 d3磷酸化的突变，我们可以绕过CDK启动的要求。这些数据有力地表明，Sid 2和Sld 3构成CDK启动的最低必需品。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.