Structural mechanism of DNA replication

项目摘要 人们普遍认为DNA复制进化了两次，分别在细菌和 古生菌/真核生物，因为复制机制的主要组成部分(如 复制解旋酶和DNA聚合酶)在这两个分支中没有进化上的关系 生活的一部分。在哺乳动物中，染色体复制错误或复制错误的纠正不足， 是癌症的一个主要原因。DNA复制的启动发生在细胞周期的G1期， 当复制启动子CDC6结合并激活起源识别复合体(ORC)以 招募与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是 很好理解。在G1到S的转变过程中，McM2-7双六角体被转换为两个 活跃的复制型解旋酶，cdc45-mcm2-7-gins(CMG)复合体。为了合成DNA， 底物酶和聚合酶以及十几种额外的蛋白质因子聚集在 CMG解旋酶形成复制体进程复合体(RPC)。因为它的巨大规模和 动态的性质，对真核复制体的结构知之甚少。然而， 冷冻-EM方法学的最新进展以及最新和引人注目的成功 在起源激活、前导链和滞后链DNA的体外重组中 综合，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复制方面的专家合作，确定了几个 复制复合体，包括OCCM，它是ORC-Cdc6-Cdt1-Mcm2-7负载 DNA上的中间体；DNA上的McM2-7双六聚体；以及叉形上的CMG解旋酶 DNA我们已经证明，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，这两个聚合酶位于解旋酶的两边，导致了深刻的 不对称复制体结构。在这些成功的基础上，国际刑警组织建议继续 复制源激活和复制体的协同机制研究 建筑。这项拟议的研究意义重大，因为复制是细胞的核心 生长，因为复制的失调会导致不受控制的增殖和 肿瘤发生学。

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.