Biochemistry of Eukaryotic Replication Fork and DNA Repair

真核复制叉的生物化学和 DNA 修复

• 批准号: 10550045
• 负责人: MICHAEL E O'DONNELL
• 金额: $ 42.38万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2028-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10550045
• 关键词: ATR gene; Binding; Biochemical; Biochemistry; Cells; Closure by clamp; Collaborations; Complex; DNA; DNA Primase; DNA Repair; DNA biosynthesis; DNA damage checkpoint; DNA replication fork; DNA-Directed DNA Polymerase; Disease; Disseminated Malignant Neoplasm; Enzymes; Epigenetic Process; Genetic Materials; Histones; Human; Hybrids; Investigation; Left; Malignant Neoplasms; Methods; Mismatch Repair; Nuclear; Nucleosomes; Pathway interactions; Polymerase; Process; Proteins; RNA; Reaction; Saccharomyces cerevisiae; Signal Transduction; Site; Slide; Structure; System; Twin Multiple Birth; Universities; Visualization; Yeasts; dimer; helicase; human disease; insight; mutant; prevent; protein function; protein purification; reconstitution; scaffold; single molecule; superresolution microscopy; time use

Project Summary DNA replication is performed by numerous proteins that act as a dynamic machine, termed a replisome. The core components of the eukaryotic replisome consist of 1) an 11 subunit “CMG” helicase that separates the parental DNA strands, 2) The leading and lagging strand DNA polymerases (Pol), Pol d and Pol e, respectively,3) The PCNA sliding clamp that encircles DNA and tethers both Pols to DNA for high processivity, 4) the RFC clamp loader pentamer that loads PCNA onto DNA and 5) Pol a-primase that makes a hybrid RNA- DNA primed site for the Pols to initiate DNA synthesis. In addition to these “core” components, there are several ancillary proteins including RPA, Tof1, Mec1, Csm3, FACT, Mcm10, Ctf4, Ctf18-RFC. There are a host of proteins that assemble two CMGs around duplex DNA at origins. The CMG dimer unwinds the closed duplex in an unknown reaction and scaffolds enzymes to assemble replisomes. We have purified these proteins in the yeast (Saccharomyces cerevisiae; S.c.) system. In this proposal, we will extend our studies on the structure/function of the eukaryotic replisome. We will use biochemical and single-molecule methods to determine if PCNA accumulates on the lagging strand as expected, and whether PCNA may periodically be left on the leading strand for mismatch repair and assembly of naïve nucleosomes. We have solved numerous structures with our collaborator, Huilin Li (VanAndel Institute, MI), and have many more structures in progress and planned. We have purified the several factors of the ATR DNA damage checkpoint signaling system of which many replisome proteins are targets of this pathway. We plan biochemical studies that will clarify targets and their effect on replisomes. We will determine the mechanism of nucleosome inheritance during replication. In metazoans, epigenetic inheritance of nucleosomes, gone awry, can lead to cancer and other diseases. In yeast, cell studies have shown that a Mcm2 histone binding mutant prevents epigenetic transfer to lagging strands, and Pol e lacking the Dpb3/4 subunits does not transfer epigenetic marks to the leading strand. We plan to visualize nucleosome transfer during replication in real time using single-molecule studies with our newly acquired Q trap) in collaboration with Dr. Shixin Liu (Rockefeller University)). We have various nucleosome mobility factors and yeast nucleosomes having different fluorescently tagged histones for these studies. Replication occurs in nuclear foci, having “replication factories” with many DNA replication forks. Our recent biochemical and structural studies have defined the composition and atomic structure of the most basic unit of a replication factory, a dimeric replisome. We will employ super resolution microscopy to validate if our reconstituted factory is the same as that inside cells. We have insight into how duplex DNA at origins is opened into single strands from our recent finding that the twin CMG helicases encircling duplex DNA at an origin are directed inward, opposite the “outward” direction thought for a decade. We find that two inward directed CMG can shear DNA apart. We have plans to further this line of investigation.

项目摘要 DNA复制是由许多蛋白质，作为一个动态的机器，称为复制体。的 真核复制体的核心成分包括：1）一个11亚基的“CMG”解旋酶，用于分离 亲本DNA链，2）前导和滞后链DNA聚合酶（Pol），Pol d和Pol e， 3）围绕DNA并将两个Pol系于DNA以获得高持续合成能力的PCNA滑动夹， 4)将PCNA装载到DNA上RFC钳位装载剂五聚体和5）产生杂合RNA的Pol α-引发酶， Pol启动DNA合成的DNA引发位点。除了这些“核心”组件， 几种辅助蛋白包括RPA、Tof 1、Mec 1、Csm 3、FACT、Mcm 10、Ctf 4、Ctf 18-RFC。还有许多 在双链DNA周围组装两个CMG的蛋白质。CMG二聚体展开封闭的 双链体在未知的反应和支架酶组装复制体。我们净化了这些 酵母（Saccharomyces cerevisiae; S.c.）系统在这项建议中，我们会把研究扩展至 真核复制体的结构/功能。我们将使用生物化学和单分子方法， 确定PCNA是否如预期的那样在滞后链上积累，以及PCNA是否可以周期性地留在 用于错配修复和幼稚核小体的组装。我们解决了许多 我与我们的合作者Huilin Li（VanAndel Institute，MI）一起构建了一个新的结构，并且还有更多的结构正在进行中 计划好了我们纯化了ATR-DNA损伤检查点信号系统的几个因子， 许多复制体蛋白是这一途径的靶点。我们计划进行生化研究， 以及它们对复制体的影响。我们将确定复制过程中核小体遗传的机制。 在后生动物中，核小体的表观遗传发生错误，可能导致癌症和其他疾病。在 酵母，细胞研究表明，Mcm 2组蛋白结合突变体阻止表观遗传转移到落后的， 链，并且缺少Dpb 3/4亚基的Pole不将表观遗传标记转移到前导链。我们 计划使用我们的单分子研究，在真实的时间内可视化复制过程中的核小体转移。 新收购的Q trap）与刘世新博士（洛克菲勒大学）合作）。我们有不同的 核小体迁移因子和具有不同荧光标记的组蛋白的酵母核小体 问题研究复制发生在核灶中，具有带有许多DNA复制叉的“复制工厂”。我们 最近的生物化学和结构研究已经确定了最基本的组成和原子结构 复制工厂的一个单位，二聚体复制体。我们将采用超分辨率显微镜来验证我们的 重组后的工厂与细胞内的工厂相同。我们已经深入了解了双链DNA在起源是如何打开的， 我们最近发现，在起点环绕双链DNA的双CMG解旋酶是 向内，相反的“向外”方向认为了十年。我们发现，两个内向CMG 可以剪切DNA我们计划进一步调查。