Molecular mechanism to couple DNA replication with avoidance of DNA lesions, the defect in which induces cancer and aging

本研究的目的是通过分析RecQ家族解旋酶和Werner解旋酶相互作用蛋白1 WRNIP1 （Werner helicase interacting protein 1）的参与过程，阐明DNA复制与DNA病变避免耦合的分子机制，并阐明这种耦合缺陷导致癌症和衰老的机制。酵母分析表明，Wrnip1在功能上与DNA聚合酶δ (Polδ)、PCNA和RFC相关，并与Polδ直接相互作用。此外，利用我们分离到的多个Polδ、pol31亚基突变体，发现当Polδ存在缺陷时，Wrnip1和Sgs1（酵母中WRN的同源物）发挥了重要的作用。利用纯化的人WRNIP1、Polδ、PCNA和RFC进行生化分析表明，WRNIP1与Polδ结合并刺激其活性。刺激的机制是提高DNA合成起始频率。为了分析WRN和WRNIP1参与的途径，我们利用鸡DT40细胞构建了各种基因敲除细胞。对这些细胞的分析表明，WRN和WRNIP1在不同的修复途径中起作用，WRN在Rad52参与而Xrcc3不参与的重组修复途径中起作用。此外，WRN与Ku70在一定程度上相互作用，参与了非同源末端连接。

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.