Molecular mechanisms for regulated assembly and action of the metazoan replicative helicase

 描述(申请人提供)：DNA复制中的一个主要活动是由解旋酶解开互补的DNA链，以产生聚合酶因子的模板。在真核生物中，微小染色体维持复合体(MCM2-7)构成了复制解旋酶的核心；然而，尽管MCM2-7具有重要的作用，但对MCM2-7如何发挥作用的基本方面尚不清楚。拟议的研究将确定后生动物复制解旋酶生命周期中两个关键事件的分子机制：募集到 解旋酶活性的启动和激活。这项提议的力量既来自所提出问题的根本性质，也来自将用于回答这些问题的跨学科方法。在目标1中，我将利用果蝇遗传学和生物化学来确定伴侣MCM2-7与必不可少的解旋酶负载因子CDT1形成稳定的复合体的机制。虽然MCM2-7·CDT1复合体是负载反应中的一个已知中间体，但由于未知的调节机制，该复合体尚未从后生动物中分离出来。我将定义这一机制，它将为未来旨在体外完全重建后生动物解旋酶负载的努力提供关键的见解。在MCM2-7募集和装载之后，解旋酶通过与另外两个蛋白因子CDC45和GINS(CMG复合体)稳定地结合而被激活。CDC45和GINS如何激活MCM2-7的潜在解旋酶活性尚不清楚。在目标2中，我将使用低温电子显微镜来确定在解开周期中停滞的CMG的亚纳米结构。这种结构将为CDC45和GINS如何重塑MCM2-7和底物DNA以促进解旋酶激活提供直接的物理解释。这项拟议的工作总体上将对DNA复制领域产生重大影响，并将有助于阐明这些因素在复制起始缺陷导致的癌症和侏儒症等疾病中所起的作用。

 DESCRIPTION (provided by applicant): A major activity in DNA replication is the unwinding of complementary DNA strands by a helicase to produce template for polymerase factors. In eukaryotes, the Minichromosome Maintenance complex (MCM2-7) forms the core of the replicative helicase; however, despite its essential role, fundamental aspects of how MCM2-7 functions are not understood. The proposed studies will determine the molecular mechanism for two critical events in the life cycle of the metazoan replicative helicase: recruitment to sites of initiation and activation of helicase activity. The strength of this proposal draws from both the fundamental nature of the questions being asked and the interdisciplinary approach that will be used to answer them. In Aim 1, I will employ Drosophila genetics and biochemistry to identify the mechanism that chaperones MCM2-7 into a stable complex with Cdt1, an essential helicase loading factor. Although the MCM2-7•Cdt1 complex is a known intermediate in the loading reaction, this complex has not been isolated from metazoans due to an unrecognized regulatory mechanism. I will define this mechanism, which will provide critical insights for future efforts aimed at fully reconstituting metazoan helicase loading in vitro. Following MCM2-7 recruitment and loading, the helicase is activated through stable association with two additional protein factors, Cdc45 and GINS (CMG complex). How Cdc45 and GINS activate the latent helicase activity of MCM2-7 is unknown. In Aim 2, I will use cryo-EM to determine the sub-nanometer structure of the CMG stalled in the midst of an unwinding cycle. This structure will provide a direct physical explanation for how Cdc45 and GINS remodel MCM2-7 and substrate DNA to facilitate helicase activation. The proposed work overall will have significant implications for th field of DNA replication, and will help elaborate the role these factors play in diseases such as cancer and dwarfism that result from replication initiation defects.