Cryo-EM of the Eukaryotic Replisome

真核复制体的冷冻电镜

• 批准号: 9365915
• 负责人: Huilin Li
• 金额: $ 37.29万
• 依托单位: VAN ANDEL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9365915
• 关键词: Address; Archaea; Architecture; Bacteria; Binding; Biochemical; Biochemistry; Biology; C-terminal; Cancer Etiology; Cartoons; Cell physiology; Collaborations; Complex; DNA; DNA Polymerase II; DNA Polymerase III; DNA Primase; DNA Primers; DNA Structure; DNA biosynthesis; DNA-Directed DNA Polymerase; DNA-Directed RNA Polymerase; Development; Eukaryota; Eukaryotic Cell; Face; Foundations; Genomic Instability; Health; Holoenzymes; Homo; Human; In Vitro; Individual; Investigation; Label; Lead; Life; MCM Protein; Malignant Neoplasms; Mammals; Maps; Methodology; Methods; Modeling; Molecular; Molecular Conformation; Motor; Mutagenesis; Nature; Play; Polymerase; Process; Proteins; RNA; Recruitment Activity; Replication Error; Reporting; Research; Resolution; Ribosomal RNA; Role; Saccharomyces cerevisiae; Side; Single-Stranded DNA; Site; Streptavidin; Structure; System; Testing; Viral; Work; base; cancer therapy; cell growth; chromosome replication; crosslink; ds-DNA; fly; helicase; human disease; magnetic beads; novel; reconstitution; repaired; scaffold; success; tumorigenesis

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.

项目摘要 通常认为DNA复制在细菌和古细菌/Eukarya中独立演变为两次 复制机制的原理成分，例如复制性解旋酶和DNA聚合酶 在生活的两个分支中与进化无关。在哺乳动物中，染色体复制错误或不足 纠正复制误差是癌症的主要原因。因此，了解分子 真核DNA复制的机制不仅对基本生物学很重要，而且对理解也很重要 并开发人类疾病的治疗方法。重新组合进程复合物（RPC）由 复制解旋酶CDC45-MCM2-7-吉尼斯（CMG）复合物，铅链DNA聚合酶Epsilon， 滞后链DNA聚合酶三角洲，聚合酶α-蛋白酶和多种额外的蛋白质 因素。由于其巨大的大小和动态性，对真核生物的补充知之甚少 建筑学。但是，最近的Cryo-EM方法论以及最新的体外重构 在领先的链和滞后链DNA合成中，可以解决这一挑战。在 初步研究，PI的实验室已使用冷冻EM确定11-蛋白CMG解旋酶的原子模型，即 并绘制了重置体的一些关键组成部分，包括Pol Epsilon，CTF4和Pol Alpha。因为 单个MCM蛋白是双裂和拉长的，MCM2-7是一种双环状结构，具有C末端 AAA+电动环堆叠在N层环的顶部。 EM工作表明，领先的链聚合酶epsilon 与C层电动环结合，而CTF4将POL Alpha-primase募集到该环的N层环侧 CMG解旋酶。因此，两个聚合酶位于解旋酶的另一侧，导致 深刻的不对称重建体系结构。在成功的基础上，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支架的相互作用 重新质体。 PI与专家复制生物化学家合作；他们在一起很适合 阐明真核生物重现的结构和功能。拟议的研究很重要，因为 重壳体在细胞生长中起着核心作用，复制失调可能导致不受控制 增殖和肿瘤发生。