| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2005: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 2004: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Research Abstract |
Eukaryotic DNA replication initiates from multiple origins. In G1 phase, protein-DNA complex called prereplicative complex (pre-RC) is formed at every origin and this reaction known as ‘licensing' give cells competency for the DNA replication. In S phase, two protein kinases, CDK (cyclin-dependent kinase) and Cdc7-Dbf4 activate pre-RC and replication forks are established. During the activation of pre-RC, although target(s) of CDK was unclear for long time, Sld2 phosphorylation by CDK is reported as an essential step in this process recently.
In this research project, I have first asked whether the phosphorylation of Sld2 by CDK is sufficient for the initiation or not. Using site-directed mutagenesis technique, I have constructed a phosphomimetic mutant of Sld2, Sld2-11D. Although this mutant was likely mimicked the phosphorylated active from of Sld2, it could not bypass CDK requirement in initiation. This indicate that Sld2 phosphorylation by CDK is essential but not sufficient for initiation and suggested that the existence of target(s) other than Sld2. Therefore, I started genetic screening to identify unknown targets. First, I screened mutants which is synthetically lethal with sld2-11D and obtained several mutants. Although these mutants were occurred in genes whose function is required for DNA replication, all of them showed defect in initiation and were not a desired ones. Then I performed another genetic screening with different rationale, finally I obtained one interest candidate. Increase of DNA contents was occurred in the mutant under the condition in which usually DNA replication does not occur, such as, G1 arrest, inhibition of S phase CDK or inhibition of whole CDK. Moreover, CsCl_2 density gradient experiment and two-dimentional agarose gel electrophoresis have shown that actual DNA replication occurred in the mutant. The mutant I have isolated will be serve as a very powerful tool to elucidate the mechanism of initiation of DNA replication.
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