Mechanisms of chromatin remodeling and roadblock clearance by DNA motor proteins

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。同源重组是一种无错误的DNA修复途径，从细菌到人类都是保守的。在重组的第一步，Sgs 1和其他专门的DNA马达蛋白沿着断裂的DNA移动，处理受损的DNA链进行修复。人类Sgs 1的缺失会导致毁灭性的疾病，如Bloom，Werner和Rothmund-Thomson综合征。Sgs 1和相关的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然没有解决。我们的假设是，DNA马达通过顺序置换和驱逐障碍物，从而使核小体和其他路障不稳定，从而允许其他修复酶进入DNA。 我已经开始解决DNA马达如何通过直接可视化这些碰撞在单分子水平上的路障。我观察到RecBCD，一种原核DNA修复马达，当它沿着沿着DNA移动时，会取代多种类型的障碍物。在K99阶段，我将扩展我的单分子测定来研究Sgs 1的马达特性。在R 00阶段，我将阐明Top3/Rmi 1和DNA 2在促进Sgs 1依赖的真核DNA修复中的作用。我在R 00阶段的第二个目标是确定Sgs 1/Top3/Rmi 1复合物如何处理染色质。这些实验将依赖于格林实验室开发的一项新技术，该技术使我们能够在真实的时间内直接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水平研究同源重组。我的最终职业目标是在一家研究机构担任终身教授。我在K99奖学金阶段开发的技能将使我能够成功地成为一名独立调查员。 公共卫生相关性：DNA断裂经常由于外部破坏剂和细胞复制过程中的自然现象而出现。该项目旨在描述细胞用于维持基因组稳定性的关键DNA修复途径。了解DNA修复机制的细节将有助于了解各种易患癌症的人类疾病。

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.