Structural mechanism of DNA replication

项目摘要 一般认为DNA复制进化了两次，分别在细菌和真核生物中， 因为复制机制的主要成分（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， 错误或复制错误的纠正不足是癌症的主要原因。DNA复制起始 发生在细胞周期的G1期，此时复制起始物Cdc 6结合并激活起始点 识别复合物（ORC）以募集Cdt 1结合的Mcm 2 -7六聚体，从而组装无活性的Mcm 2 -7 双链DNA上的双六聚体。这种多步引发过程的分子机制不是 很好理解。在G1到S的转变过程中，Mcm 2 -7双六聚体被转化为两个活跃的复制型多聚体。 解旋酶，Cdc 45-Mcm 2 -7-GINS（CMG）复合物。为了合成DNA引物酶和聚合酶 超过12个额外的蛋白质因子围绕CMG解旋酶组装形成复制体 进行复合体（RPC）。由于其庞大的规模和动态的性质，很少有人知道 真核复制体结构。然而，冷冻EM方法学的最新进展，沿着大多数 最近在体外重组起始激活、前导链和滞后链方面取得了惊人的成功， 链DNA合成，使得解决这些挑战变得可行。在过去的十年里，我们 与真核DNA复制专家合作，确定了几种复制的原子模型， 复合物，包括OCCM，它是DNA上的ORC-Cdc 6-Cdt 1-Mcm 2 -7装载中间体; DNA上的Mcm 2 -7双六聚体;和分叉DNA上的CMG解旋酶。我们已经证明， 链聚合酶Ctf 4与C-层马达环结合，而Pol α-引发酶由Ctf 4募集， CMG解旋酶的N层环侧。因此，两种聚合酶骑在聚合物的相对侧上。 解旋酶，导致一个深刻的不对称复制体架构。在这些成功的基础上， 建议继续进行复制起点激活和复制体的合作和机制研究 架构这项研究之所以重要，是因为复制对细胞生长至关重要， 复制的失调可导致不受控制的增殖和肿瘤发生。

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.