Mechanisms of chromatin remodeling and roadblock clearance by DNA motor proteins

DNA 运动蛋白的染色质重塑和路障清除机制

• 批准号: 8616481
• 负责人: ILYA J FINKELSTEIN
• 金额: $ 24.9万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-05 至 2016-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8616481
• 关键词: Address; Bacteria; Binding; Biochemical; Biochemistry; Biological Assay; Biology; Cells; Chromatin; Complex; Crowding; DNA; DNA Repair; DNA Repair Pathway; DNA-Binding Proteins; Data Set; Deoxyribonucleases; Disease; Enzymes; Escherichia coli; Event; Excision; Fellowship; Genetic Recombination; Genome Stability; Genomics; Goals; Human; Individual; Institution; Laboratories; Light; Malignant Neoplasms; Mediation; Motor; Motor Activity; Mutation; Nucleoproteins; Nucleosomes; Obstruction; Outcome; Phase; Physics; Process; Property; Proteins; Reaction; Research; Research Personnel; Role; Rothmund-Thomson syndrome; S cerevisiae DNA2 protein; Staging; System; Technology; Testing; Time; Work; Yeasts; abstracting; career; chromatin remodeling; design; helicase; homologous recombination; human disease; in vivo; nanoscale; new technology; nuclease; prevent; professor; protein complex; repair enzyme; repaired; research study; single molecule; skills; tool; translocase

Project Summary/Abstract Repairing broken DNA is essential for preventing mutations that can cause diseases such as cancer. Homologous recombination is an error-free DNA repair pathway that is conserved from bacteria to human. In the first step of recombination, Sgs1 and other specialized DNA motor proteins move along the broken DNA to process damaged strands for repair. Loss of Sgs1 in humans leads to devastating diseases such as Bloom, Werner and Rothmund-Thomson syndromes. The process by which Sgs1 and related DNA motors navigate on highly condensed chromatin and deal with other nucleoprotein collisions remains unresolved. Our hypothesis is that DNA motors collaborate to destabilize nucleosomes and other roadblocks by sequentially displacing and evicting the obstacles, thereby allowing other repair enzymes to gain access to the DNA. I have begun to address how DNA motors negotiate roadblocks by directly visualizing these collisions at the single molecule level. I observed that RecBCD, a prokaryotic DNA repair motor, displaces multiple types of obstacles as it moves along DNA. In the K99 phase, I will extend my single molecule assay to study the motor properties of Sgs1. During the R00 phase, I will elucidate the role of Top3/Rmi1 and Dna2 in facilitating Sgs1-dependent eukaryotic DNA repair. My second aim in the R00 phase is to determine how the Sgs1/Top3/Rmi1 complex processes chromatin. These experiments will rely on a new technology developed in the Greene laboratory, which allows us to directly visualize hundreds of individual DNA motor proteins in real time. By rapidly gathering statistically relevant datasets, we can study homologous recombination with an unprecedented level of mechanistic detail. My ultimate career goal is to achieve tenure as a professor at a research institution. The skills that I develop during the K99 phase of the fellowship will enable me to succeed as an independent investigator.

项目摘要/摘要 修复破裂的DNA对于预防可能引起癌症等疾病的突变至关重要。 同源重组是一种无错误的DNA修复途径，从细菌到人都保守。在 重组的第一步，SGS1和其他专门的DNA运动蛋白沿破碎的DNA移动到 处理损坏的链以进行维修。人类中SGS1的损失导致毁灭性疾病，例如Bloom， Werner和Rothmund-Thomson综合征。 SGS1和相关DNA电动机导航的过程 在高度冷凝的染色质上，处理其他核蛋白碰撞仍未解决。我们的 假设是DNA电动机合作通过顺序破坏核小体和其他障碍 置换和驱逐障碍，从而使其他修复酶进入DNA。 我已经开始通过直接可视化这些碰撞来解决DNA电动机如何谈判障碍 在单分子水平。我观察到核核DNA修复电机RecBCD取代了多种类型 障碍物沿着DNA移动时的障碍物。在K99阶段，我将扩展我的单分子分析以研究 SGS1的电机特性。在R00阶段，我将阐明TOP3/RMI1和DNA2在促进中的作用 SGS1依赖性真核DNA修复。我在R00阶段的第二个目标是确定 SGS1/TOP3/RMI1复合过程染色质。这些实验将依靠开发的新技术 在Greene实验室中，这使我们能够直接可视化数百种单独的DNA运动蛋白 时间。通过快速收集统计相关的数据集，我们可以研究同源重组 机械细节的前所未有的水平。我的最终职业目标是在任职期间担任教授 研究机构。我在奖学金的K99阶段发展的技能将使我成功 作为独立研究员。