|Budget Amount *help
¥15,200,000 (Direct Cost : ¥15,200,000)
Fiscal Year 2004 : ¥6,900,000 (Direct Cost : ¥6,900,000)
Fiscal Year 2003 : ¥8,300,000 (Direct Cost : ¥8,300,000)
The aim of this study is to clarify the molecular mechanism to couple DNA replication with the avoidance of DNA lesions by analyzing the processes in which RecQ family helicases and WRNIP1 (Werner helicase interacting protein 1) are involved and to elucidate the mechanism to induce cancer and aging due to the defect in the coupling.
The analyses using yeast indicated that Wrnip1 is functionally related to DNA polymerase δ (Polδ), PCNA, and RFC and interacts directly with Polδ. In addition, by using many mutants having mutations in the subunit of Polδ, pol31, which we isolated, it was revealed that Wrnip1 and Sgs1 (WRN homologue in yeast) play very important roles when Polδ has some defects. Biochemical analysis using purified human WRNIP1, Polδ, PCNA, and RFC indicated that WRNIP1 binds to Polδ and stimulates its activity. The mechanism of the stimulation was the enhancement of initiation frequency of DNA synthesis. To analyze the pathways in which WRN and WRNIP1 are involved, we constructed various gene knockout cells using chicken DT40 cells. The analyses using these cells suggested that WRN and WRNIP1 function in different repair pathways, and WRN functions in a recombination repair pathway in which Rad52 is involved but Xrcc3 is not. Furthermore, it was suggested that WRN somehow functionally interacts with Ku70, which is involved in nonhomologous end-joining.