| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2006: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2005: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
Loading of the origin recognition complex (ORC) onto the chromatin is essential for the assembly of the pre-replicative complex. To examine physiological functions of the mammalian ORC, we cloned cDNAs of Orc subunits from mouse NIH3T3 cells and found novel variant forms of Orc1, Orc2, and Orc3, each generated by alternative RNA splicing. Characterization of Orc1A and Orc1B allowed us to identify the domain of Orc1, which is essential for nuclear translocation, phosphorylation, and degradation. To further clarify the role of Orc1A phosphorylation, we generated an antibody against phospho-peptides containing Ser^<276> and Orc1A protein is predominantly phosphorylated during G2/M phase. In addition, small amount of phosphorylated Orc1A gradually increases during S phase, while most of Orc1A remains unphosphorylated and bound to chromatin. All of the phosphorylated Orc1A is extracted in chromatin-unbound fraction containing 100 mM NaC1, Transiently expressed Orc1A is partially phosphoryla
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ted in the nucleus while nuclear translocation-deficient Orc1 mutant is extensively phosphorylated, We found that phosphorylation of Ser^<276> is dependent on conserved cyclin-docking (Cy) motif located just beside amino-terminal bromo adjacent homology (BAH) domain. Orc1 BAH domain has been shown to act as a binding site with Sir1 in S. cerevisiae or HP1 in Drosophila and Human. Taken together, we conclude that mouse Orc1 is regulated at least by phosphorylation of Ser^<276> residue throughout the cell cycle. At G1 phase, unphosphorylated Orc1A bound to chromatin and localized mainly on heterochromatin. During S phase, Orc1A is gradually released from chromatin, and released Orc1A is phosphorylated by CDK/cyclin A through Cy motif. Phosphorylated Orc1 is degraded during S phase. In contrast, at G2/M phase, phsophorylated Orc1 is not degraded but re-associates with heterochromatin after dephosphorylation. We are currently trying to understand whether Cy motif is masked by amino-terminal BAH domain on chromatin through interaction with HP1 or other chromatin factors. Less
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