Mechanisms of chromatin remodeling and roadblock clearance by DNA motor proteins

项目概要/摘要 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， 处理受损的股线进行修复。Sgs 1在人类中的缺失导致毁灭性的疾病，如Bloom， Werner和Rothmund-Thomson综合征。Sgs 1和相关DNA马达导航的过程 对高度浓缩的染色质和处理其他核蛋白的碰撞问题仍然没有解决。我们 一种假说是DNA马达通过顺序地破坏核小体和其他路障， 取代和驱逐障碍物，从而允许其他修复酶进入DNA。 我已经开始通过直接可视化这些碰撞来解决DNA马达如何克服路障的问题 在单分子水平上。我观察到RecBCD，一种原核DNA修复马达， 当它沿着DNA移动时，在K99阶段，我将扩展我的单分子测定来研究 Sgs 1的运动特性。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。我在R 00阶段的第二个目标是确定 Sgs 1/Top3/Rmi 1复合体加工染色质。这些实验将依赖于一种新的技术开发 在格林实验室，这使我们能够直接可视化数百个单独的DNA马达蛋白在真实的 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， 前所未有的机械细节水平。我的最终职业目标是在一所大学担任教授 研究机构。我在K99奖学金阶段培养的技能将使我能够成功 作为一名独立调查员

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.