| Budget Amount *help |
¥4,110,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥510,000)
Fiscal Year 2007: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
Fiscal Year 2006: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Research Abstract |
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.
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