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（通过应用程序提供）：DNA复制的主要活性是通过解旋酶放松互补的DNA链，以产生聚合酶因子的模板。在真核生物中，微型浓度体维持络合物（MCM2-7）构成了复制性解旋酶的核心。但是，它的基本作用是如何不理会MCM2-7功能的基本方面。拟议的研究将确定后生复制酶生命周期中两个关键事件的分子机制：募集到 愈合活性的启动和激活。该提议的优势借鉴了所提出的问题的基本性质和将用于回答问题的跨学科方法。在AIM 1中，我将采用果蝇遗传学和生物化学来识别链链MCM2-7与CDT1稳定的复合物的机理，CDT1是必需的解旋酶负载因子。尽管MCM2-7•CDT1复合物是负载反应中的已知中间体，但由于无法识别的调节机制，该复合物尚未从后生动物中分离出来。我将定义这种机制，该机制将为未来的努力提供旨在充分重建后生动物酶负载的未来努力的关键见解。在MCM2-7募集和加载之后，通过与另外两个蛋白质因子CDC45和杜松子（CMG复合物）稳定关联来激活解旋酶。 CDC45和杜松子如何激活MCM2-7的潜在解旋酶活性是未知的。在AIM 2中，我将使用Cryo-EM来确定在放松周期中停滞的CMG的子纳米结构。该结构将为CDC45和GINS重塑MCM2-7和底物DNA如何促进解旋酶激活提供直接的物理解释。拟议的工作整体将对DNA复制领域产生重大影响，并将有助于阐述这些因素在癌症和诸如癌症和矮人之类的疾病中所起的作用，这是复制计划缺陷所致。