Structural mechanism of DNA replication

DNA复制的结构机制

• 批准号: 10414082
• 负责人: Huilin Li
• 金额: $ 71.25万
• 依托单位: VAN ANDEL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-07 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10414082
• 关键词: Archaea; Architecture; Bacteria; Binding; Biology; Cancer Etiology; Cell Cycle; Complex; Cryoelectron Microscopy; DNA; DNA Primase; DNA biosynthesis; DNA-Directed DNA Polymerase; Eukaryotic Cell; G1 Phase; In Vitro; Lead; Life; Malignant Neoplasms; Mammals; Methodology; Modeling; Molecular; Molecular Machines; Motor; Nature; Polymerase; Process; Proteins; Replication Error; Replication Initiation; Replication Origin; Research; Side; Structure; cancer therapy; cell growth; chromosome replication; ds-DNA; helicase; human disease; origin recognition complex; reconstitution; recruit; repaired; success; tumorigenesis

Project Summary It is generally thought that DNA replication evolved twice, independently in Bacteria and in Archaea/Eukarya, because the principal components of the replication machinery (such as the replicative helicase and the DNA polymerases) are not evolutionarily related in the two branches of life. In mammals, chromosome replication error, or insufficient correction of a replication error, is a major cause of cancers. Initiation of DNA replication occurs in G1 phase of the cell cycle, when the replication initiator Cdc6 binds and activates the origin recognition complex (ORC) to recruit Cdt1-bound Mcm2-7 hexamer, thereby assembling an inactive Mcm2-7 double hexamer on double-stranded DNA. The molecular mechanism of this multistep initiation process is not well understood. During G1-to-S transition, the Mcm2-7 double hexamer is converted to two active replicative helicases, the Cdc45-Mcm2-7-GINS (CMG) complexes. To synthesize DNA, the primases and polymerases and over a dozen additional protein factors assemble around the CMG helicase to form the replisome progression complex (RPC). Because of its sheer size and dynamic nature, very little is known about the eukaryotic replisome architecture. However, recent advances in cryo-EM methodology, along with the most recent and spectacular success in in vitro reconstitutions of origin activation, the leading strand and the lagging strand DNA synthesis, have made it feasible to tackle these challenges. Over the past decade, we have collaborated with experts in eukaryotic DNA replication to determine atomic models of several replication complexes, including the OCCM, which is an ORC-Cdc6-Cdt1-Mcm2-7 loading intermediate on DNA; the Mcm2-7 double-hexamer on DNA; and the CMG helicase on a forked DNA. We have shown that the leading strand polymerase epsilon binds to the C-tier motor ring, whereas the Pol alpha-primase is recruited by Ctf4 to the N-tier ring side of the CMG helicase. Therefore, the two polymerases ride on opposite sides of the helicase, resulting in a profoundly asymmetric replisome architecture. Building on these successes, the PI proposes to continue the collaborative and mechanistic study of replication origin activation and replisome architecture. The proposed research is significant because replication is central to cellular growth and because dysregulation of replication can lead to uncontrolled proliferation and tumorigenesis.

项目摘要 人们普遍认为，DNA复制在细菌和古细菌/Eukarya中独立发展了两次 因为复制机械的主要成分（例如复制旋转酶和DNA 聚合酶）在生命的两个分支中与进化无关。在哺乳动物中，染色体复制 错误或对复制错误的校正不足是癌症的主要原因。启动DNA复制 当复制引发剂CDC6结合并激活原点时，发生在细胞周期的G1阶段中 识别综合体（ORC）招募CDT1结合的MCM2-7六角形，从而组装了不活跃的MCM2-7 双链DNA上的双六聚体。这个多步启动过程的分子机制不是 理解。在G1到S过渡期间，MCM2-7双六聚体被转换为两个主动复制性 解旋酶，cdc45-MCM2-7-吉尼斯（CMG）配合物。为了合成DNA，原始酶和聚合酶 以及在CMG解旋酶周围组装的十几个其他蛋白质因子以形成重新构体 进展复合物（RPC）。由于其纯粹的大小和动态性，对 真核生物补充体系结构。但是，Cryo-Em方法论的最新进展以及最多 在原始激活，领先链和落后的体外重组中的最新和壮观 Strand DNA合成使得应对这些挑战是可行的。在过去的十年中，我们有 与真核DNA复制专家合作，以确定几种复制的原子模型 复合物，包括OCCM，它是DNA上的OCCM-CDC6-CDT1-MCM2-7加载中间体；这 DNA上的MCM2-7双杂种;和分叉DNA上的CMG解旋酶。我们已经证明了领导 链聚合酶Epsilon与C层电动环结合，而CTF4募集了pol alpha-primase to CMG解旋酶的N层环侧。因此，两个聚合酶在 解旋酶，导致深刻不对称的重新构体结构。在这些成功的基础上，PI 建议继续对复制起源激活和重新构体的协作和机械研究 建筑学。拟议的研究很重要，因为复制对于细胞生长和 复制失调可能导致不受控制的增殖和肿瘤发生。