Mechanisms of chromatin remodeling and roadblock clearance by DNA motor proteins

项目摘要/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。 同源重组是一种从细菌到人类保守的无错误的DNA修复途径。在……里面 重组的第一步，SGS1和其他特殊的DNA马达蛋白沿着断裂的DNA移动到 处理损坏的线束以进行修复。人类中SGS1的缺失会导致像布卢姆这样的毁灭性疾病， Werner和Rothmund-Thomson综合征。SGS1和相关DNA马达导航的过程 关于高度浓缩的染色质和处理其他核蛋白碰撞的问题仍未解决。我们的 假说是DNA马达协同作用，通过下列方式破坏核小体和其他障碍的稳定性 取代和驱逐障碍，从而允许其他修复酶获得DNA。 我已经开始讨论DNA发动机是如何通过直接可视化这些碰撞来穿越路障的 在单分子水平上。我观察到，原核DNA修复马达RecBCD可以取代多种类型 当它沿着DNA移动时会有很多障碍。在K99阶段，我将扩展我的单分子分析来研究 SGS1的电机特性。在R00阶段，我将阐明top3/Rmi1和DNA2在促进 依赖SGS1的真核DNA修复。我在R00阶段的第二个目标是确定 SGS1/top3/Rmi1复合体处理染色质。这些实验将依赖于开发的一项新技术 在格林实验室，这使我们能够直接可视化真实的数百个单独的DNA马达蛋白质 时间到了。通过快速收集统计上相关的数据集，我们可以用一个 史无前例的机械化细节。我的最终职业目标是在一所大学获得终身教职 研究机构。我在K99奖学金阶段所培养的技能将使我能够取得成功 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.