Mechanisms of chromatin remodeling and roadblock clearance by DNA motor proteins

描述(申请人提供)：修复断裂的DNA对于防止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保守的无错误的DNA修复途径。在重组的第一步，SGS1和其他专门的DNA马达蛋白沿着断裂的DNA移动，处理受损的链进行修复。人类中SGS1的缺失会导致布鲁姆、沃纳和罗斯蒙德-汤姆森综合征等破坏性疾病。SGS1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未解决。我们的假设是，DNA发动机通过顺序移动和驱逐障碍来协作破坏核小体和其他路障的稳定，从而允许其他修复酶获得DNA。我已经开始讨论DNA发动机是如何通过在单分子水平上直接可视化这些碰撞来克服障碍的。我观察到，原核生物DNA修复马达RecBCD在沿着DNA移动时会移动多种类型的障碍。在K99阶段，我将扩展我的单分子分析来研究SGS1的运动特性。在R00阶段，我将阐明top3/Rmi1和DNA2在促进依赖SGS1的真核DNA修复中的作用。我在R00阶段的第二个目标是确定SGS1/top3/Rmi1复合体如何处理染色质。这些实验将依赖格林实验室开发的一项新技术，该技术允许我们实时直接可视化数百个单独的DNA马达蛋白。通过快速收集统计上相关的数据集，我们可以以前所未有的机械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获得教授终身教职。我在K99奖学金阶段培养的技能将使我成为一名成功的独立调查员。 与公共卫生相关：DNA断裂经常由于外部破坏性因素和细胞复制过程中的自然原因而出现。该项目旨在描述细胞用来维持基因组稳定的关键DNA修复途径。了解DNA修复机制的细节将有助于揭示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.