Biochemical Mechanism and Structure of the Eukaryotic Replication Fork

项目总结 基因组的复制对所有细胞来说都是必不可少的。然而，很少有实验室成功地重建了 用于机制研究的纯蛋白质真核复制叉酶。这部分归因于 驱动复制所需的大量蛋白质以及获得这些众多因素的难度。不像 细菌，真核生物使用不同的DNA聚合酶来复制领先链和滞后链，即 真核解旋酶包含11种不同的蛋白质，并且存在大量的真核复制蛋白 在细菌中没有同源物。 我们利用纯蛋白重组真核萌芽酵母已经取得了几项突破性的研究。 (酿酒酵母)复制体(&gt；30个不同亚基)。我们已经确定了 复制体蛋白的EM分析--DNA聚合酶(POL)ε被导向前导链的机制 而POLδ针对的是滞后的链，以及防止这些POL 在“错误的”线索上工作。解决了CMG解旋酶在转位过程中的定位问题。 冷冻电子显微镜检测DNA及其对起源起源的深远影响。要在中解决的问题 这项建议包括发展智人(H.S.)复制机制以解决后生动物如何 复制体的功能是执行分叉回归，这是高等真核生物中的一个基因组稳定过程。到目前为止我们 纯化了难度较大的多亚基重组H.S.因子，包括11个亚基H.S.CMG解旋酶和 已经重组了H.S.前导链复制体。我们将描述H.S.是如何。复制体功能， 将其与发芽酵母进行比较，然后检查后生动物特有的ATPase分叉改构器逆转停滞的情况 用于基因组完整性的叉子。叉子重建器还没有用复制体蛋白进行研究，我们将 在H.S.面前说明他们的行动。复制体蛋白和叉状保护因子，H.S.RAD51 重组酶和BRCA2肿瘤抑制因子。我们还将研究具有绑定的BRCA2的反向分叉- 恢复RAD51，以实现复制重启。我们还建议继续我们对复制体的理解 人类和萌芽酵母系统中各种复制体亚复合体的冷冻EM结构。 综上所述，这里提出的研究将提供对真核生物工作机制的深刻理解。 DNA复制机制及其与DNA修复、突变和人类疾病的密切关系。 复制对于对目前使用的许多抗癌药物的积极反应也是至关重要的。因此 详细了解这一对细胞生命至关重要的核心过程将提供有用的重要信息 防治人类疾病。

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.