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

后生动物复制解旋酶调节组装和作用的分子机制

• 批准号: 9118764
• 负责人: Matthew W. Parker
• 金额: $ 5.61万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9118764
• 关键词: Address; Baculovirus Expression System; Binding; Biochemical; Biochemistry; Cell Cycle; Cell Fractionation; Cell division; Cells; Chromatin; Complementary DNA; Complex; DNA; DNA Replication Factor; DNA biosynthesis; DNA replication fork; Defect; Disease; Drosophila genus; Dwarfism; Embryo; Ensure; Eukaryota; Event; Failure; Future; Genetic; Genome; Genomic Instability; Health; Human; Image; In Vitro; Lead; Licensing; Life Cycle Stages; Literature; MCM2 gene; Maintenance; Malignant Neoplasms; Methods; Modification; Molecular; Molecular Chaperones; N-terminal; Nature; Negative Staining; Nucleotides; Organism; Pathway interactions; Peptide Initiation Factors; Play; Polymerase; Pre-Replication Complex; Process; Proteins; Reaction; Recruitment Activity; Replication Initiation; Replication Origin; Resolution; Role; S Phase; Saccharomycetales; Secure; Site; Staging; Structure; Work; Yeasts; base; carcinogenesis; cofactor; fly; genome integrity; helicase; in vivo; insight; interdisciplinary approach; nanometer; origin recognition complex; public health relevance; reconstitution; stem; structural biology; treatment strategy

 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.

 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，微型染色体维持复合物 (MCM2-7) 形成复制解旋酶的核心；然而，尽管 MCM2-7 发挥着重要作用，但其功能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：招募到 解旋酶活性的启动和激活。该提案的优势在于所提出问题的基本性质以及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分子伴侣 MCM2-7 与 Cdt1（一种重要的解旋酶负载因子）形成稳定复合物的机制。尽管MCM2-7•Cdt1复合物是加载反应中已知的中间体，但由于未知的调节机制，该复合物尚未从后生动物中分离出来。我将定义这种机制，这将为未来旨在完全重建体外后生动物解旋酶负载的努力提供重要的见解。 MCM2-7 募集和加载后，解旋酶通过与另外两种蛋白质因子 Cdc45 和 GINS（CMG 复合物）稳定结合而被激活。 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，我将使用冷冻电镜来确定在解卷周期中停滞的 CMG 的亚纳米结构。该结构将为 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。总体而言，拟议的工作将对DNA复制领域产生重大影响，并将有助于阐明这些因素在由复制起始缺陷引起的癌症和侏儒症等疾病中所发挥的作用。