Cryo-EM of the Eukaryotic Replisome

项目摘要 一般认为，DNA复制在细菌和真核生物中独立进化了两次， 复制机制的主要组成部分，如复制解旋酶和DNA聚合酶 在进化上并不相关。在哺乳动物中，染色体复制错误或不足 复制错误的纠正是癌症的主要原因。因此，了解分子 真核生物DNA复制机制不仅对基础生物学，而且对理解 以及开发人类疾病的治疗方法。复制体进展复合物（RPC）由以下组成： 复制解旋酶Cdc 45-Mcm 2 -7-GINS（CMG）复合物，前导链DNA聚合酶P450， 滞后链DNA聚合酶δ，聚合酶α-引物酶，和十几种额外的蛋白质 因素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 架构然而，冷冻EM方法学的最新进展，沿着最新的体外重建， DNA合成的主导链和滞后链，使应对这一挑战变得可行。中 初步研究，PI的实验室已经使用冷冻EM来确定11蛋白CMG解旋酶的原子模型， 并绘制了复制体的几个关键组成部分，包括Pol α，Ctf 4和Pol α。因为 Mcm 2 -7是一个C-末端为环状的双环结构， AAA+电机环堆叠在N层环的顶部。EM工作表明，前导链聚合酶 结合到C-层马达环，而Pol α-引发酶被Ctf 4募集到C-层马达环的N-层环侧。 CMG解旋酶。因此，这两种聚合酶位于解旋酶的相对侧，从而导致解旋酶的解旋。 极其不对称的复制体架构。在这一成功的基础上，PI建议继续冷冻- EM结构和生化研究，关于CMG如何与分叉DNA相互作用，以及CMG支架如何 复制体PI已经与一位专家复制生物化学家合作;他们一起做好了准备， 阐明真核复制体的结构和功能。这项研究意义重大，因为 复制体在细胞生长中起中心作用，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 Archaea/Eukarya, as the principle 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 the replication error is a major cause of cancers. Therefore, understanding the molecular mechanism of eukaryotic DNA replication is important not only for fundamental biology but also for understanding and developing treatment for human diseases. The replisome progression complex (RPC) is composed of replicative helicase the Cdc45-Mcm2-7-GINS (CMG) complex, the leading strand DNA polymerase epsilon, the lagging strand DNA polymerase delta, the polymerase alpha-primase, and over a dozen additional protein factors. Because of its sheer size and dynamic nature, very little is known about the eukaryotic replisome architecture. However, recent advance in cryo-EM methodology, along with the most recent in vitro reconstitution of the leading strand and the lagging strand DNA synthesis, has made it feasible to tackle this challenge. In a preliminary study, the PI's lab has used cryo-EM to determine an atomic model of the 11-protein CMG helicase, and mapped a few key components of the replisome, including Pol epsilon, Ctf4, and Pol alpha. Because the individual Mcm proteins are bi-lobed and elongated, the Mcm2-7 is a double ring-like structure with the C-terminal AAA+ motor ring stacked on top of the N-tier ring. EM work has 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 reside on the opposite side of the helicase, resulting to a profoundly asymmetric replisome architecture. Building on that success, the PI proposes to continue the cryo- EM structural and biochemical investigation, on how CMG interacts with the forked DNA, and how CMG scaffolds the replisome. The PI has teamed up with an expert replication biochemist; together they are well poised to elucidate the structure and function of the eukaryotic replisome. The proposed research is significant because the replisome plays a central role in cellular growth, and dysregulation of replication can lead to uncontrolled proliferation and tumorigenesis.