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: 8090740
• 负责人: ILYA J FINKELSTEIN
• 金额: $ 9万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 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 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8090740
• 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; 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

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Breaks in DNA arise frequently as a result of external damaging agents and naturally during cell replication. This project aims to characterize a crucial DNA repair pathway used by cells to maintain genome stability. Understanding details of DNA repair mechanisms will shed light on various cancer-prone human diseases.

描述（由申请人提供）：修复破裂的DNA对于预防可能导致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。在重组的第一步中，SGS1和其他专门的DNA运动蛋白沿破裂的DNA移动以处理受损的链以进行修复。人类中SGS1的丧失导致毁灭性疾病，例如Bloom，Werner和Rothmund-Thomson综合征。 SGS1和相关的DNA电动机在高度冷凝的染色质上导航并处理其他核蛋白碰撞的过程仍未解决。我们的假设是，DNA电动机通过顺序置换和驱逐障碍物来协作，以破坏核小体和其他障碍，从而允许其他修复酶进入DNA。 我已经开始通过直接在单个分子水平上直接可视化这些碰撞来解决DNA电动机如何谈判障碍。我观察到，核核DNA修复电动机RecBCD在沿DNA移动时取代了多种类型的障碍物。在K99阶段，我将扩展单分子测定法以研究SGS1的运动特性。在R00阶段，我将阐明TOP3/RMI1和DNA2在促进SGS1依赖性真核DNA修复中的作用。我在R00阶段的第二个目标是确定SGS1/TOP3/RMI1复合过程如何染色质。这些实验将依靠Greene实验室中开发的新技术，这使我们能够实时直接可视化数百个单独的DNA运动蛋白。通过快速收集统计相关的数据集，我们可以使用前所未有的机械细节来研究同源重组。我的最终职业目标是在研究机构担任教授的任期。我在奖学金的K99阶段发展的技能将使我能够成功成为独立的研究者。 公共卫生相关性：由于外部破坏药物和细胞复制过程中自然而自然出现DNA中断。该项目旨在表征细胞用于维持基因组稳定性的关键DNA修复途径。了解DNA修复机制的细节将阐明各种容易发生癌症的人类疾病。