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

 描述（由申请人提供）：DNA复制的主要活动是通过解旋酶解旋互补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物（MCM 2 -7）形成了复制解旋酶的核心;然而，尽管它的重要作用，MCM 2 -7功能的基本方面还不清楚。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： 解旋酶活性的起始和活化。这一建议的力量来自所提出问题的基本性质和将用于回答这些问题的跨学科方法。目的一：利用果蝇遗传学和生物化学的方法研究分子伴侣MCM 2 -7与解旋酶负载因子Cdt 1形成稳定复合物的机制。虽然MCM 2 -7·Cdt 1复合物是负载反应中的已知中间体，但由于未识别的调节机制，该复合物尚未从后生动物中分离出来。我将定义这种机制，这将提供关键的见解，为今后的努力，旨在完全重建后生动物解旋酶加载体外。在MCM 2 -7募集和加载后，解旋酶通过与两种额外的蛋白因子Cdc 45和GINS（CMG复合物）稳定结合而被激活。Cdc 45和GINS如何激活MCM 2 -7的潜在解旋酶活性尚不清楚。在目标2中，我将使用cryo-EM来确定CMG的亚纳米结构，该CMG在解旋周期中停滞。这种结构将为Cdc 45和GINS如何重塑MCM 2 -7和底物DNA以促进解旋酶激活提供直接的物理解释。这项工作将对DNA复制领域产生重大影响，并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.