Structural mechanism of DNA replication

项目摘要 人们普遍认为DNA复制进化了两次，分别是在细菌和古生代/真核生物中， 因为复制机制的主要组成部分(如复制解旋酶和DNA 聚合酶)在生命的两个分支中没有进化上的联系。在哺乳动物中，染色体复制 错误，或对复制错误的不充分纠正，是癌症的主要原因。启动DNA复制 发生在细胞周期的G1期，当复制启动子CDC6结合并激活起始点时 识别复合体(ORC)招募CDT1结合的Mcm2-7六聚体，从而组装失活的Mcm2-7 双链DNA上的双六聚体。这种多步骤引发过程的分子机制并不是 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跃的复制体 解旋酶，cdc45-mcm2-7-gins(CMG)复合体。合成DNA、底物酶和聚合酶 十多个额外的蛋白质因子聚集在CMG解旋酶周围形成复制体 进展复合体(RPC)。由于它的巨大规模和动态性质，人们对它的了解很少 真核复制体结构。然而，冷冻-EM方法学的最新进展，以及大多数 最近在起源激活、先导链和滞后链的体外重组方面取得了惊人的成功 链DNA合成使应对这些挑战成为可能。在过去的十年里，我们已经 与真核DNA复制专家合作，确定了几种复制的原子模型 络合物，包括OCCM，它是一种ORC-Cdc6-Cdt1-Mcm2-7负载DNA的中间体； DNA上的MCM2-7双六聚体和分叉DNA上的CMG解旋酶。我们已经表明，领先的 链聚合酶epsilon与C-层马达环结合，而Polα-Primase被Ctf4招募到 CMG解旋酶的N层环侧。因此，这两种聚合酶分别位于 解旋酶，导致深度不对称的复制体结构。在这些成功的基础上，PI 建议继续开展复制起点激活和复制体的协作和机制研究 建筑。这项拟议的研究意义重大，因为复制是细胞生长的核心，而且因为 复制的失调可能会导致不受控制的增殖和肿瘤形成。

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.