真核生物DNA复制解旋酶激活过程中DDK和Sld3机制的研究

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.

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。

为了保证基因组的稳定性，真核生物DNA复制包含有一个复杂的复制起始调控机制，以保证在正确的时间和位置上起始复制。首先复制起点识别复合物ORC识别并结合到DNA复制起点，并招募解旋酶MCM，组装形成预起始复合物（pre-RC）。随着细胞周期进入S期，在细胞周期素依赖的蛋白激酶DDK和CDK的调控和多个激活因子的参与下，解旋酶的活性激活，复制体组装完成，DNA复制进入延伸阶段。按照研究计划，本项目对解旋酶激活过程中激酶DDK介导的MCM激活以及相关因子的工作机制进行了结构研究。结合生物化学和冷冻电镜结构生物学方法，成功纯化出了DDK结合解旋酶MCM的大分子复合物，并解析出该复合物3.3 Å的高分辨率结构，为DDK对MCM磷酸化机制的阐释提供了重要的结构信息。此外，在本项目的支持下，我们还对ORC识别复制起点的机制进行了研究，利用冷冻电镜解析了酵母ORC与起点DNA结合的3.0 Å分辨率的结构，成功阐释了酵母ORC特异性识别起点DNA的分子基础（Nature, 2018）。