Structural mechanism of DNA replication

DNA复制的结构机制

• 批准号: 10786193
• 负责人: Huilin Li
• 金额: $ 22.1万
• 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/10786193
• 关键词: Archaea; Architecture; Bacteria; Binding; Biology; Cancer Etiology; Cell Cycle; Collaborations; Complex; Cryoelectron Microscopy; DNA; DNA Primase; DNA biosynthesis; DNA-Directed DNA Polymerase; Eukaryotic Cell; G1 Phase; In Vitro; Life; Malignant Neoplasms; Mammals; Methodology; Modeling; Molecular; Molecular Machines; Motor; Nature; Polymerase; Process; Proliferating; 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 通常认为DNA复制在细菌和中独立于 古细菌/eukarya，因为复制机械的主要组成部分（例如 复制性解旋酶和DNA聚合酶）在两个分支中与进化无关 生活。在哺乳动物中，染色体复制误差或对复制误差的校正不足， 是癌症的主要原因。 DNA复制的启动发生在细胞周期的G1阶段， 当复制引发剂CDC6结合并激活原点识别复合物（ORC）至 招募CDT1结合MCM2-7 Hexamer，从而组装了不活动的MCM2-7双己隔室 在双链DNA上。这个多步启动过程的分子机制不是 理解。在G1到S的过渡期间，MCM2-7双六聚体被转换为两个 活性复制解旋酶，cdc45-MCM2-7-吉尼木（CMG）配合物。为了合成DNA， 原始酶和聚合酶以及超过十几个其他蛋白质因子在周围组装 CMG解旋酶形成重新组合进程复合物（RPC）。因为它的尺寸很大， 动态的性质，对真核的重生体系结构知之甚少。然而， 冷冻EM方法的最新进展，以及最新和壮观的成功 在原点激活的体外重构中，前导链和滞后链DNA 合成使应对这些挑战的可行。在过去的十年中，我们有 与真核DNA复制专家合作，以确定几种原子模型 复制络合物，包括OCCM，这是ORC-CDC6-CDT1-MCM2-7加载 在DNA上中间； DNA上的MCM2-7双己甲；和叉子上的CMG解旋酶 脱氧核糖核酸。我们已经表明，铅链聚合酶epsilon与C层电动环结合， 而polα-蛋白酶由CTF4募集到CMG解旋酶的N层环侧。 因此，两种聚合酶骑在解旋酶的相对侧，导致了深刻的 不对称的重取体架构。在这些成功的基础上，PI建议继续 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，因为复制对于细胞是核心 增长和由于复制失调可能导致不受控制的增殖和 肿瘤发生。

项目成果

The Saccharomyces cerevisiae Yta7 ATPase hexamer contains a unique bromodomain tier that functions in nucleosome disassembly.

• DOI: 10.1016/j.jbc.2022.102852
• 发表时间: 2023-03
• 期刊: JOURNAL OF BIOLOGICAL CHEMISTRY
• 影响因子: 4.8
• 作者: Wang, Feng;Feng, Xiang;He, Qing;Li, Hua;Li, Huilin
• 通讯作者: Li, Huilin

Molecular mechanisms of eukaryotic origin initiation, replication fork progression, and chromatin maintenance.

• DOI: 10.1042/bcj20200065
• 发表时间: 2020-09-30
• 期刊: The Biochemical journal
• 作者: Yuan Z;Li H
• 通讯作者: Li H

Cdc6-induced conformational changes in ORC bound to origin DNA revealed by cryo-electron microscopy.

• DOI: 10.1016/j.str.2012.01.011
• 发表时间: 2012-03-07
• 期刊: STRUCTURE
• 影响因子: 5.7
• 作者: Sun, Jingchuan;Kawakami, Hironori;Zech, Juergen;Speck, Christian;Stillman, Bruce;Li, Huilin
• 通讯作者: Li, Huilin

Structure of the human UBR5 E3 ubiquitin ligase.

• DOI: 10.1016/j.str.2023.03.010
• 发表时间: 2023-05-04
• 期刊: STRUCTURE
• 影响因子: 5.7
• 作者: Wang, Feng;He, Qing;Zhan, Wenhu;Yu, Ziqi;Finkin-Groner, Efrat;Ma, Xiaojing;Lin, Gang;Li, Huilin
• 通讯作者: Li, Huilin

Water skating: How polymerase sliding clamps move on DNA.

• DOI: 10.1111/febs.15740
• 发表时间: 2021-12
• 期刊: The FEBS journal
• 作者: Li H;Zheng F;O'Donnell M
• 通讯作者: O'Donnell M