Biochemical Mechanism and Structure of the Eukaryotic Replication Fork

真核生物复制叉的生化机制和结构

• 批准号: 9906902
• 负责人: MICHAEL E O'DONNELL
• 金额: $ 33.9万
• 依托单位: ROCKEFELLER 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 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9906902
• 关键词: ATP phosphohydrolase; Address; Antineoplastic Agents; BRCA2 gene; Bacteria; Biochemical; Biology; Cell Cycle; Cells; Chromosome abnormality; Chromosomes; Collaborations; Complex; Cryoelectron Microscopy; DNA; DNA Repair; DNA biosynthesis; DNA replication fork; DNA-Binding Proteins; DNA-Directed DNA Polymerase; DNA-Directed RNA Polymerase; Data; Development; Disease; Enzymatic Biochemistry; Enzymes; Eukaryota; Eye; Foundations; Future; Genes; Genome; Genome Stability; Health; Homo sapiens; Homologous Gene; Human; Institutes; Institution; Intelligence; Knowledge; Lead; Lesion; Life; MCM10 gene; Malignant Neoplasms; Mechanics; Modeling; Modification; Molecular Conformation; Mutagenesis; Mutation; Nucleosomes; Organism; Pharmaceutical Preparations; Polymerase; Prevention; Process; Proteins; Quality Control; Rad51 recombinase; Recombinants; Resolution; SMARCA3 gene; Saccharomyces cerevisiae; Saccharomycetales; Speed; Structure; System; Time; Titan; Tumor Suppressor Proteins; Universities; Work; Yeasts; anticancer treatment; cancer therapy; cancer type; cohesion; condensin; data sharing; design; detector; ds-DNA; experience; genome integrity; helicase; human disease; insight; operation; polypeptide; preservation; prevent; reconstitution; repaired; response; restoration; structural biology; symposium; translocase

PROJECT SUMMARY Duplication of the genome is essential to all cells. Yet very few labs have succeeded in reconstituting the enzymology of the eukaryotic replication fork from pure proteins for mechanistic studies. This is in part due to the numerous proteins required to drive replication and the difficulty in obtaining these many factors. Unlike bacteria, eukaryotes use different DNA polymerases to duplicate the leading and lagging strands, the eukaryotic helicase contains 11 distinct proteins, and there exist numerous eukaryotic replication proteins that have no homologue in bacteria. We have made several breakthrough studies using pure proteins to reconstitute the eukaryotic budding yeast (Saccharomyces cerevisiae) replisome (>30 different subunits). We have determined the organization of replisome proteins by EM, the mechanisms by which DNA polymerase (Pol) ε is directed to the leading strand and Pol δ is directed to the lagging strand, along with quality control mechanisms that prevent these Pols from working on the “wrong” strands. We also solved the orientation of the CMG helicase while translocating on DNA by cryoEM and the consequent profound implications for origin initiation. Questions to be addressed in this proposal include development of the Homo sapiens (H.s.) replisome machinery to address how metazoan replisomes function to perform fork regression, a genome stability process in higher eukaryotes. Thus far we have purified the difficult multisubunit recombinant H.s. factors, including 11-subunit H.s. CMG helicase and have reconstituted the H.s. leading strand replisome. We will characterize how the H.s. replisome functions, compare it to budding yeast and then examine metazoan specific ATPase fork remodelers that reverse stalled forks for genomic integrity. Fork remodelers have not been studied with replisome proteins, and we will address their action in the presence of H.s. replisome proteins and also the fork protection factors, H.s. RAD51 recombinase and the BRCA2 tumor suppressor. We will also examine how reversed forks with bound BRCA2- RAD51, are restored to enable replication restart. We also propose to continue our understanding of replisome structure through cryoEM of various replisome subcomplexes in both the human and budding yeast systems. In overview, the studies proposed here will provide a deep understanding of the workings of the eukaryotic DNA replication machinery, and its intimate involvement in DNA repair, mutagenesis, and human disease. Replication is also crucial for a positive response to many anticancer drugs that are currently in use. Hence detailed knowledge of this central and vital process to cellular life will provide important information useful to prevention and cure of human disease.

项目摘要 基因组的重复对所有细胞都是必不可少的。然而，很少有实验室成功地重组 来自纯蛋白质的真核复制叉的酶学用于机械研究。这部分是由于 驱动复制所需的众多蛋白质以及获得这些许多因素的困难。与众不同 细菌，真核生物使用不同的DNA聚合酶来复制领先和滞后链， 真核解旋酶包含11种不同的蛋白质，并且存在许多真核复制蛋白，这些蛋白质是 在细菌中没有同源物。 我们已经使用纯蛋白质进行了几项突破性研究，以重建真核生物萌芽的酵母 （酿酒酵母）复制体（> 30个不同的亚基）。我们已经确定了 EM的复制体蛋白质，DNA聚合酶（POL）ε的机制针对领先链 Polδ针对滞后链，以及质量控制机制，以防止这些pol 处理“错误”链。我们还解决了CMG解旋酶的方向 Cryoem的DNA以及随之而来的对起源启动的深刻含义。要解决的问题 该建议包括开发Homo Sapiens（H.S.）副本机械，以解决Metazoan的方式 复制体功能可以执行叉回归，这是高等真核生物中的基因组稳定过程。那我们很远 已经纯化了困难的多生产重组H.S.因素，包括11份H.S. CMG解旋酶和 重构了H.S.领先的链复制体。我们将表征H.S.复制函数， 将其与萌芽的酵母菌进行比较，然后检查反向停滞的后唑特异性ATPase叉远方 基因组完整性的叉子。叉子重塑剂尚未使用复制体蛋白研究，我们将 在H.S.在场的情况下解决他们的行动复制体蛋白质以及前叉保护因素H.S. RAD51 重组酶和BRCA2肿瘤抑制剂。我们还将研究如何具有绑定BRCA2-的逆转叉 RAD51恢复以启用复制重新启动。我们还建议继续我们对复制体的理解 人类和发芽酵母系统中各种复制体子复合物的结构。 在概述中，这里提出的研究将对真核的工作有深入的了解 DNA复制机制及其在DNA修复，诱变和人类疾病中的密切参与。 复制对于对当前正在使用的许多抗癌药物的积极反应也至关重要。因此 对蜂窝生活的这一中心和至关重要的过程的详细了解将为重要信息提供有用的重要信息 预防和治愈人类疾病。