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: 8251196
• 负责人: ILYA J FINKELSTEIN
• 金额: $ 2.45万
• 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 2012-07-08
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8251196
• 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; public health relevance; 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）：修复断裂的 DNA 对于预防可能导致癌症等疾病的突变至关重要。同源重组是一种从细菌到人类都保守的无错误 DNA 修复途径。在重组的第一步中，Sgs1 和其他专门的 DNA 马达蛋白沿着断裂的 DNA 移动，处理受损的链以进行修复。人类中 Sgs1 的缺失会导致毁灭性的疾病，例如 Bloom、Werner 和 Rothmund-Thomson 综合征。 Sgs1 和相关 DNA 马达在高度浓缩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 马达通过顺序地取代和驱逐障碍物来协作破坏核小体和其他障碍的稳定，从而允许其他修复酶接触到 DNA。 我已经开始通过在单分子水平上直接可视化这些碰撞来解决 DNA 马达如何克服障碍。我观察到 RecBCD（一种原核 DNA 修复马达）在沿着 DNA 移动时会取代多种类型的障碍。在 K99 阶段，我将扩展我的单分子实验来研究 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 复合物如何处理染色质。这些实验将依赖于 Greene 实验室开发的一项新技术，该技术使我们能够直接实时可视化数百个单独的 DNA 马达蛋白。通过快速收集统计相关的数据集，我们可以以前所未有的机制细节来研究同源重组。我的最终职业目标是成为研究机构的终身教授。 The skills that I develop during the K99 phase of the fellowship will enable me to succeed as an independent investigator. 公共健康相关性：DNA 断裂经常因外部损伤剂而发生，并且在细胞复制过程中自然发生。该项目旨在表征细胞用来维持基因组稳定性的关键 DNA 修复途径。了解 DNA 修复机制的细节将有助于了解各种易患癌症的人类疾病。