酿酒酵母染色质组装复合体CAF-1激活DNA复制检验点的分子机制

真核生物DNA的精准复制受到DNA复制检验点(S期检验点)的调控，其通过对DNA复制叉行进过程的实时监控，确保基因组的稳定、细胞周期正常和防止细胞癌变等。近年来，研究S期检验点关键因子的分子机制极其受到重视，因其对阐明DNA复制调控机制具有重要科学意义。基于前期工作，本项目拟用酿酒酵母为研究材料，对初步筛选到的激活S期检验点的新关键因子染色质组装复合体CAF-1的调节作用分子机制进行研究。拟结合高通量遗传互作图谱方法(E-MAP)、分子生物学、生化、生物信息学分析技术等多种手段，研究CAF-1在激活S期检验点的过程中对核心激酶Mec1的招募和激活，CAF-1新功能的关键功能域及位点，CAF-1的磷酸化修饰是否对Mec1的活性存在反馈调节等，以揭示CAF-1参与S期检验点的调节机制及其与染色质组装的偶联机制，为分析DNA复制应对损伤机制、损伤信号通路和诊断遗传疾病提供科学依据和应用技术基础。

In eukaryotes, accurate DNA replication is regulated by DNA replication checkpoint (the S-phase checkpoint), which ensures the stability of the genome, normal cell cycle and prevents cell carcinogenesis by real-time monitoring of the process of DNA replication fork. In recent years, the study of the molecular mechanism of the key factors that activate DNA replication checkpoint has attracted great attention, because it is of great scientific significance to elucidate the mechanism of DNA replication regulation of eukaryotes. On the basis of the previous work, the applicant proposed to use Saccharomyces cerevisiae as the research material to study the molecular regulatory mechanism of the new key factor of the S-phase checkpoint, CAF-1(chromatin assembly factor), which was hit by previous screen. By means of high-throughput genetic interaction mapping (E-MAP), molecular biological, biochemical and bioinformatics analysis, to investigate the recruitment and activation of core kinase Mec1 by CAF-1 and the key functional domains and important sites corresponding to the new function and whether phosphorylation modification has feedback regulation on Mec1 activity. It is expected to reveal the regulatory mechanism of CAF-1 in the S-phase checkpoint and the coupling mechanism of chromatin assembly, and to provide scientific basis and applied technology for the analysis of DNA replication coping mechanism in the presence of injury, damage response signaling pathway and diagnosis of genetic diseases.