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复制是由多种充当动态机的蛋白质进行的，称为复制体。这 真核复制体的核心成分由1）11个亚基“ CMG”解旋酶分开 亲本DNA链，2）铅和滞后链DNA聚合酶（POL），pol d和pol E， 3）PCNA滑动夹环绕DNA和将两个POR的螺旋带到DNA，以进行高处理， 4）将PCNA加载到DNA和5）POL A-primase的RFC夹具装载机五聚体，该酶使杂种RNA- DNA启动位点用于启动DNA合成。除了这些“核心”组件，还有 几种辅助蛋白，包括RPA，TOF1，MEC1，CSM3，FACT，MCM10，CTF4，CTF18-RFC。有一个主机 在起源处聚集了两个CMG的蛋白质。 CMG二聚体闭合了 复式反应中的双链体和脚手架酶以组装复制品。我们已经净化了这些 酵母中的蛋白质（酿酒酵母； S.C。）系统。在此提案中，我们将扩展有关 真核复制体的结构/功能。我们将使用生化和单分子方法 确定PCNA是否如预期的 在幼稚核小体的不匹配修复和组装的领先链上。我们解决了许多 与我们的合作者Huilin Li（密歇根州Vanandel Institute）的结构，并进行了更多的结构 并计划。我们已经纯化了ATR DNA损伤检查点信号系统的几个因素 许多复制组蛋白是该途径的靶标。我们计划生化研究，以阐明目标 以及它们对复制品的影响。我们将确定复制过程中核遗传的机制。 在后生动物中，核小体的表观遗传遗传遗传性，出了问题，会导致癌症和其他疾病。 酵母，细胞研究表明，MCM2 Hisstone结合突变体可防止表观遗传转移到滞后 缺乏DPB3/4亚基的链和pol E不会将表观遗传标记传递到领先的链。我们 计划使用我们的单分子研究在实时复制过程中可视化核小体传递 与Shixin Liu博士（Rockefeller University）合作，新收购了Q Trap）。我们有很多 核小体迁移率因子和酵母核小体具有不同的荧光标记组蛋白 研究。复制发生在核灶中，具有许多DNA复制叉具有“复制工厂”。我们的 最近的生化和结构研究定义了最基本的组成和原子结构 复制工厂的单位，一个二聚体复制体。我们将采用超级分辨率显微镜来验证我们的 重构的工厂与内部单元格相同。我们对如何打开双链DNA有洞察力 从我们最近发现的单一链中，即包围双链DNA的双CMG解旋酶是起源的 向内定向，在“向外”方向的对面十年。我们发现两个向内指向CMG 可以剪切DNA。我们计划进一步调查。