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

真核生物的DNA复制起始于多个复制起点，它们被CDK和CDC7-Dbf4两种蛋白激酶激活，DNA复制起始于S期。CDK在这一过程中的底物长期未知，S1d2在2002年的启动中被认为是CDK的一个重要靶点。我已经证明，SLD2的磷酸化是必要的，但不是启动所必需的。因此，为了识别未知的CDK靶点，我建立了基因筛查。在筛选过程中，构建了S1d2的拟磷突变体(S1d2-D)，并分离到了新的突变体JET1，该突变体即使在没有CDK活性的情况下也能与S1d2-D一起启动DNA复制。该JET1突变发生在CDC45中，这是启动和延伸所必需的。我们揭示了CDC45不是CDK的底物，而是CDC45的结合伙伴SLD3是CDK启动的主要靶点。我们还表明，SLD2和SLD3的磷酸化增强了Dpb11的相互作用。推测这一反应触发了DNA复制的启动。此外，通过结合绕过sld2和s1d3磷酸化的突变，我们可以绕过启动CDK的要求。这些数据有力地表明，SID2和SLD3在启动过程中构成了CDK的最低基本要求。这些发现对于理解真核细胞DNA复制的启动反应是至关重要的。

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.