BYPASS MECHANISMS IN EUKARYOTIC REPLICATION

真核复制中的旁路机制

• 批准号: 10017302
• 负责人: Grant Schauer
• 金额: $ 24.9万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10017302
• 关键词: ATR gene; Address; Affect; Biochemical; Bypass; Cell Death; Cell physiology; Cells; Chromatin; Chromosomal Duplication; Chromosomal Instability; Chromosomes; Communication; Communities; Complex; Coupled; DNA; DNA Damage; DNA biosynthesis; DNA damage checkpoint; DNA lesion; Daughter; Deposition; Detection; Disease; Ensure; Environment; Epigenetic Process; Escherichia coli; Failure; Fluorescence; Generations; Genetic; Genetic Code; Genome; Genomics; Goals; Hand; Histone H3; Histones; Holoenzymes; Human Pathology; Individual; Kinetics; Laboratories; Lead; Lesion; Light; Malignant Neoplasms; Mass Spectrum Analysis; Mediation; Mediator of activation protein; Medical Research; Mentors; Microfluidics; Modeling; Molecular; Molecular Chaperones; Molecular Machines; Monitor; Nucleosomes; Parents; Pathway interactions; Pattern; Phase; Phosphorylation; Phosphotransferases; Play; Polymerase; Positioning Attribute; Prevention; Process; Proteins; Regulation; Reporting; Research; Role; S Phase; Series; Signal Transduction; Speed; Structure; Techniques; Time; Training; Universities; Work; base; career; chromatin remodeling; chromosome replication; crosslink; disorder prevention; experimental study; genome integrity; helicase; histone modification; improved; insight; optical traps; polypeptide; preservation; pressure; prevent; reconstitution; single molecule; single-molecule FRET; skills; success

PROJECT SUMMARY/ABSTRACT Chromosomes are copied by a complex holoenzyme called the replisome. Obstacles are routinely negotiated by the replisome with auxiliary mechanisms that ensure genomic integrity, aberrance of which can lead to chromosome instability and a broad range of diseases including cancer. The candidate’s long term goal is to understand the molecular basis for genetic and epigenetic fidelity, with the potential to improve the treatment and/or prevention of disease. In this proposal, the candidate will use a fully functional replisome reconstituted from over 30 pure polypeptides to study how replisomes bypass obstacles that regularly occur in the genome while enforcing genetic and epigenetic integrity across generations. In the first specific aim the candidate’s current work on the molecular mechanisms of lesion bypass by the replisome will be elaborated, with a focus on how checkpoint kinases Mec1 and Rad53 and the Mrc1/Tof1/Csm3 (MTC) complex modify the activity of the replisome while regulating lesion bypass. Interactions between several key replisome components and the MTC complex will be probed by microscale thermophoresis (MST) and cross-linking mass spectrometry (XL- MS). In addition to biochemical experiments, single-molecule approaches will be used to probe the mechanism of replisome regulation by the MTC complex, resolving replisome components with fluorescence during active replication on DNA. Single-molecule FRET experiments will be used in the independent phase to probe how MTC affects the structural dynamics of the replisome. In the second aim, the candidate will investigate nucleosome bypass by the replisome, focusing on the post-replication fate of histones during the mentored phase. Using histones enriched for fluorescence, replication-coupled histone deposition will be tracked by a first-of-its-kind attempt at spatially resolving leading vs. lagging strand products with a combination of optical trapping, fluorescence, and flow. Along with bead-based biochemical experiments, the results will help differentiate between models of epigenetic inheritance. In the independent phase, interactions between the replisome and FACT, a histone chaperone, will be determined with MST and XL-MS. The molecular mechanisms of various chaperones will be probed using single-molecule FRET experiments monitoring the spatiokinetics of chromatin remodeling in real time, also determining the role of histone modifications in remodeling. The mentored phase of the project will be conducted in the laboratories of Dr. Michael O’Donnell (mentor) and Dr. Shixin Liu (co-mentor) at Rockefeller University, a world-class research environment. The success of the candidate’s proposed research depends critically on using advanced integrative single-molecule techniques, as well as XL-MS. Thus, the candidate seeks intensive training with manipulation and detection of individual molecules in the co-mentor’s state-of-the-art facilities, in addition to XL-MS with Drs. Brian Chait and Yi Shi (collaborators). The candidate also has also planned activities to improve mentoring, lab management, scientific communication, and professional skills, enabling a successful transition to an independent career.

项目概要/摘要 染色体由一种称为复制体的复杂全酶复制，通常会克服障碍。 通过具有辅助机制的复制体确保基因组完整性，其异常可能导致 染色体不稳定和包括癌症在内的多种疾病。 了解遗传和表观遗传保真度的分子基础，有可能改善治疗 和/或预防疾病 在本提案中，候选人将使用功能齐全的重组复制体。 从 30 多种纯多肽中研究复制体如何绕过基因组中经常出现的障碍 同时在第一个具体目标中加强候选人的遗传和表观遗传完整性。 将详细阐述目前关于复制体绕过病变的分子机制的工作，重点是 关于检查点激酶 Mec1 和 Rad53 以及 Mrc1/Tof1/Csm3 (MTC) 复合物如何改变 复制体同时调节病变旁路几个关键复制体成分和复制体之间的相互作用。 MTC 复合物将通过微尺度热泳 (MST) 和交联质谱 (XL- 除了生化实验外，还将使用单分子方法来探索其机制。 MTC 复合物对复制体的调节，在活性期间用荧光解析复制体成分 DNA 上的复制将在独立阶段使用单分子 FRET 实验来探究如何复制。 MTC 影响复制体的结构动力学 在第二个目标中，考生将进行研究。 复制体绕过核小体，重点关注指导期间组蛋白的复制后命运 使用荧光富集的组蛋白，复制偶联的组蛋白沉积将被跟踪。 首次尝试结合光学技术在空间上解析领先与落后的链产品 与基于微珠的生化实验一起，结果将有所帮助。 区分表观遗传模型在独立阶段，表观遗传之间的相互作用。 复制体和组蛋白伴侣 FACT 将通过 MST 和 XL-MS 进行分子测定。 将使用单分子 FRET 实验来探讨各种分子伴侣的机制，以监测 实时染色质重塑的空间动力学，还确定组蛋白修饰在 该项目的指导阶段将在迈克尔·奥唐纳博士的实验室进行。 洛克菲勒大学（导师）和刘世新博士（共同导师）拥有世界一流的研究环境。 候选人提出的研究的成功关键取决于使用先进的集成单分子 因此，候选人寻求对操作和检测进行强化培训。 除了与 Brian Chait 博士一起进行的 XL-MS 之外，在共同导师最先进的设施中研究单个分子。 石一（合作者）还计划了改善指导、实验室管理、 科学沟通和专业技能，使您能够成功过渡到独立职业。