Structural mechanism of DNA replication

DNA复制的结构机制

基本信息

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
项目类别：
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

项目摘要

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.

项目概要人们普遍认为 DNA 复制进化了两次，在细菌中和在古细菌/真核生物，因为复制机器的主要组成部分（例如复制解旋酶和 DNA 聚合酶）在这两个分支中没有进化相关性的生活。在哺乳动物中，染色体复制错误或复制错误纠正不充分，是癌症的一个主要原因。 DNA复制的起始发生在细胞周期的G1期，当复制起始子 Cdc6 结合并激活起源识别复合物 (ORC) 招募 Cdt1 结合的 Mcm2-7 六聚体，从而组装无活性的 Mcm2-7 双六聚体在双链DNA上。这种多步骤引发过程的分子机制并不很好理解。在 G1 到 S 的转变过程中，Mcm2-7 双六聚体转化为两个活性复制解旋酶，Cdc45-Mcm2-7-GINS (CMG) 复合物。为了合成DNA，引物酶和聚合酶以及十多种其他蛋白质因子聚集在 CMG 解旋酶形成复制体进展复合物 (RPC)。由于其庞大的规模和动态性质，人们对真核复制体结构知之甚少。然而，冷冻电镜方法的最新进展，以及最近取得的巨大成功体外重构起始激活、前导链和滞后链 DNA 综合，使应对这些挑战成为可能。过去十年，我们与真核 DNA 复制领域的专家合作，确定了多种基因的原子模型复制复合物，包括 OCCM，它是一个 ORC-Cdc6-Cdt1-Mcm2-7 负载 DNA 中间体； DNA 上的 Mcm2-7 双六聚体；和分叉上的 CMG 解旋酶脱氧核糖核酸。我们已经证明前导链聚合酶 epsilon 与 C 层电机环结合，而 Pol α-引物酶被 Ctf4 招募到 CMG 解旋酶的 N 层环侧。因此，两种聚合酶位于解旋酶的相反两侧，从而产生了深刻的影响。不对称复制体结构。在这些成功的基础上，PI 建议继续复制起点激活和复制体的协作和机制研究建筑学。拟议的研究意义重大，因为复制是细胞的核心生长，因为复制失调会导致不受控制的增殖和肿瘤发生。

项目成果

期刊论文数量（38）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

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
影响因子：
0
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.