Biochemical Mechanism and Structure of the Eukaryotic Replication Fork

项目摘要 基因组的复制对所有细胞都是必不可少的。然而，很少有实验室成功地重建了 酶的真核生物复制叉从纯蛋白质的机制研究。这部分是由于 驱动复制所需的大量蛋白质以及获得这些因子的难度。不像 细菌，真核生物使用不同的DNA聚合酶来复制前导链和滞后链， 真核生物解旋酶包含11种不同的蛋白质，并且存在许多真核生物复制蛋白， 在细菌中没有同源物。 我们已经取得了几个突破性的研究，使用纯蛋白质，以重建真核芽殖酵母 （酿酒酵母）复制体（>30种不同亚基）。我们已经决定组织 复制体蛋白的EM，DNA聚合酶（Pol）ε被定向到前导链的机制 Pol δ被导向滞后链，沿着的质量控制机制防止这些Pol 在“错误”的链上工作。我们还解决了CMG解旋酶的方向，而易位上 DNA的cryoEM和随之而来的起源启动的深刻影响。拟解决的问题 这一提议包括开发智人（Homo sapiens，H.s.）复制体机制来解决后生动物 复制体的功能是进行分叉回归，这是高等真核生物中的基因组稳定性过程。到目前为止， 纯化了难分离的多亚基重组H.因子，包括11-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.复制体功能， 将其与芽殖酵母进行比较，然后检查后生动物特异性ATP酶叉重塑， forks for genomic基因组integrity完整性.叉重塑还没有研究与复制体蛋白，我们将 在H.S.在场的情况下解决他们的行动。复制体蛋白以及叉保护因子，H.s. RAD51 重组酶和BRCA 2肿瘤抑制因子。我们还将研究如何反向分叉结合BRCA 2- RAD 51，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 在人类和芽殖酵母系统中，通过冷冻电镜观察各种复制体亚复合物的结构。 总的来说，这里提出的研究将提供对真核生物的工作机制的深刻理解。 DNA复制机制及其与DNA修复、诱变和人类疾病的密切关系。 复制对于目前使用的许多抗癌药物的积极反应也至关重要。因此 对细胞生命的这一中心和重要过程的详细了解将提供重要的信息， 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.