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.

项目摘要/摘要 染色体由一种名为复制体的复杂全酶复制。障碍是例行公事地谈判 通过具有确保基因组完整性的辅助机制的复制体，其异常可导致 染色体不稳定和包括癌症在内的一系列疾病。候选人的长期目标是 了解遗传和表观遗传保真度的分子基础，并有可能改进治疗 和/或预防疾病。在这项提案中，候选人将使用重建的全功能复制体 研究复制体如何绕过基因组中经常出现的障碍 同时在世代之间加强遗传和表观遗传的完整性。在第一个具体目标中，候选人的 目前关于复制体绕过病变的分子机制的工作将被详细阐述，重点是 检验点激酶Mec1和Rad53以及MRc1/Tof1/Csm3(MTC)复合体如何改变MEC1和RAD53的活性 调节病变旁路时的复制体。几个关键的复制体组件之间的相互作用 MTC络合物将通过微尺度热电泳法(MST)和交联质谱(XL-MS)进行探测。 Ms)。除了生化实验外，还将使用单分子方法来探索其机理。 MTC复合体对复制体的调节，在活动期间用荧光解析复制体成分 DNA上的复制。在独立阶段将使用单分子FRET实验来探索如何 MTC影响复制体的结构动力学。在第二个目标中，候选人将调查 复制体绕过核小体，关注复制后组蛋白在指导过程中的命运 相位。利用富含荧光的组蛋白，复制偶联的组蛋白沉积将通过 首次尝试通过组合光学组件在空间上分辨领先与落后的链产品 捕获、荧光和流动。与基于珠子的生化实验一起，结果将会有所帮助 区分表观遗传模式。在独立阶段， 复制体和组蛋白伴侣蛋白FACT将用MST和XL-MS确定。分子 不同分子伴侣的机制将通过单分子FRET实验监测 染色质重塑的实时空间动力学，也决定了组蛋白修饰在 改建。该项目的指导阶段将在Michael O‘Donnell博士的实验室进行 (导师)和刘士新博士(共同导师)在洛克菲勒大学，世界级的研究环境。这个 候选人提议的研究的成功关键取决于使用先进的集成单分子 技术以及XL-MS。因此，应聘者寻求高强度的操作和检测训练 合作导师最先进的设备中的单个分子，此外还有Brian Chait博士和 易师(合作者)。候选人还计划了一些活动，以改善指导、实验室管理、 科学沟通和专业技能，使成功过渡到独立的职业生涯。