真核生物DNA复制解旋酶激活过程中DDK和Sld3机制的研究

真核生物DNA复制起始包括DNA复制解旋酶的加载和激活两个过程。解旋酶加载发生在细胞周期的G1期，起点识别复合物识别DNA复制起点，在Cdc6和Cdt1的协助下，招募解旋酶MCM2-7，形成解旋酶活性未激活的MCM2-7双六聚体复合物。解旋酶的激活发生在S期，受到DDK和CDK两种细胞周期依赖激酶的调控：DDK磷酸化解旋酶，Sld3识别这些磷酸化位点，并在Sld7的参与下招募Cdc45；接着CDK磷酸化Sld3，招募GINS，形成完整的具有解旋酶活性的CMG复合物。.作为激活过程的第一步，DDK介导的解旋酶激活是细胞周期调控的关键靶标。虽然已经有很多生化方面的分析，但DDK和Sld3的机制研究的并不明确。本项目计划利用冷冻电镜方法，解析DDK-MCM2-7和Sld3-Sld7-Cdc45-MCM2-7两种复合物的高分辨结构，对解旋酶激活过程中DDK和Sld3的机制进行探究。

Initiation of eukaryotic DNA replication could be divided into two steps: loading and activation of DNA replicative helicase. The loading of helicase occurs at the G1 phase of the cell cycle: the origin recognition complex (ORC) recognize the DNA replication origin; With the help of Cdc6 and Cdt1, the helicase MCM2-7 complex is recruited to form the inactive double-hexamer MCM2-7 complex. Activation of helicase occurs at S phase and is regulated by two cell cycle-dependent kinase, DDK and CDK: DDK phosphorylates the inactive helicase, Sld3 recognizes these phosphorylation sites and recruits Cdc45 with the participation of Sld7, followed by CDK-mediated phosphorylation of Sld3, the recruitment of GINS, and the formation of the complete CMG complex with helicase activity..As the first step in the activation process, DDK-mediated helicase activation is a key target in cell cycle regulation. Though with lots of study based on biochemical method published, the mechanism of DDK and Sld3 is still not very clear. In this project, we would apply cryo-EM technique to acquire the high resolution structure of DDK-MCM2-7 and Sld3-Sld7-Cdc45-MCM2-7 complexes, and explore the mechanism of DDK and Sld3 in the process of helicase activation based structural analysis and mechanism-verified biochemical experiments.