Mechanistic studies of stalled DNA replication fork rescue

摘要 在理解停滞的DNA复制叉如何被拯救方面存在根本性的差距。继续存在 这一差距是一个重要的问题，因为，直到它被填补，一个完整和清晰的理解， 将缺乏失速叉重新激活的机制。这种理解是至关重要的，因为这些修复中的缺陷 在高等生物中的机制导致突变的积累，导致癌症，并提出了 因此，研究与人类疾病直接相关。因此，长期目标是了解 停滞的DNA复制叉再激活的机制。本提案的主要目的是了解 类核上单链DNA结合蛋白与关键叉拯救酶的相互作用 模板和随后的处理事件，导致分叉结构的恢复。实现这一 目标，本建议分为三个具体目标：1），确定分叉回归的机制; 2）确定分叉障碍物如何影响分叉消退; 3）确定类核相关的影响。 叉救援酶上的蛋白质。在第一个目标下，磁镊和原子力显微镜（两者都在 空气和高速缓冲）将用于确定SSB加载和RecG回归如何受到影响 并确定RecA和RuvAB是否能够催化一个有效的和单向的分叉 回归反应当目标1的拟议研究完成后， 将提供初期的、停止的复制分叉。在第二个目标下，相同的两个单个DNA分子 方法将用于提供深入了解复制体障碍对停滞的叉拯救的影响， 高空间和时间分辨率。在目标2的拟议研究结束时， 损伤和蛋白质-DNA复合物对叉拯救的作用将变得清晰，预计其机制 用于置换叉附近停滞的RNA聚合酶。在最后的目标下，磁性 操纵DNA单分子的镊子将被用来确定与类核相关的效应。 蛋白质（NAP）在叉救援。当目标3的拟议研究完成后，将确定是否 NAP本身催化退化，如果它们帮助或抑制叉拯救酶。拟议研究 是创新的，因为采取了组合策略。它的创新之处还在于它的刺激性和新颖性 使用的单分子方法，重点是类核模板和理解如何获得 除去引起复制体停滞的一级蛋白质屏障。最后，这项工作也是创新的，因为 在阐明重组解旋酶在SSB存在下如何起作用时所采取的谨慎态度。拟议 研究是重要的，因为它将允许，第一次，明确的模型的发展机制， 事件发生在一个停滞的叉嵌入类核模板，它将提供第一个实时洞察 在体内重新激活停滞的分叉的一系列事件。

Abstract There is a fundamental gap in understanding how stalled DNA replication forks are rescued. Continued existence of this gap represents an important problem because, until it is filled, a complete and clear understanding of the mechanism of stalled fork reactivation will be lacking. This understanding is crucial as defects in these repair mechanisms in higher organisms lead to the accumulation of mutations leading to cancer, and the proposed studies are therefore directly relevant to human disease. Consequently, the long term goal is to understand the mechanism of stalled DNA replication fork reactivation. The main objective of this proposal is to understand the interplay between the single stranded DNA binding protein (SSB) and key fork rescue enzymes on nucleoid templates and of the subsequent processing events leading to restoration of a fork structure. To achieve this objective, this proposal is divided into three specific aims: 1), Determine the mechanism(s) of fork regression; 2,) To determine how fork impediments affect fork regression; and 3), Ascertain the effects of nucleoid associated proteins on fork rescue enzymes. Under the first aim, magnetic tweezers and atomic force microscopy (both in air and high-speed in buffer) will be used to determine how SSB loading and regression by RecG are affected by PriA and to ascertain whether RecA and RuvAB are able to catalyze an efficient and unidirectional fork regression reaction. When the proposed studies for Aim 1 are complete, a clear picture of the events at a nascent, stalled replication fork will be provided. Under the second aim, the same two single DNA molecule approaches will be used to provide insight into the effects of replisome impediments on stalled fork rescue, with high spatial and temporal resolution. At the conclusion of the proposed studies for Aim 2, the effects of DNA lesions and protein-DNA complexes on fork rescue will be made clear and it is anticipated that the mechanism(s) for displacing stalled RNA polymerase in the vicinity of forks will be obtained. Under the final aim, magnetic tweezers to manipulate single molecules of DNA will be used to ascertain the effects of nucleoid associated proteins (NAPs) on fork rescue. When the proposed studies for Aim 3 are complete, it will be ascertained whether NAPs catalyze regression on their own and if they assist or inhibit fork rescue enzymes. The proposed research is innovative because of the combinatorial strategy taken. It is also innovative because of the exciting and novel single molecule approaches used, the focus on nucleoid templates and an understanding to be gained of how the primary protein barrier(s) causing replisome stalling are removed. Finally, the work is also innovative because of the care taken in elucidating how recombination helicases function in the presence of SSB. The proposed research is significant because it will allow, for the first time, the development of clear models of the mechanistic events occurring at a stalled fork embedded within nucleoid templates and, it will provide the first real-time insight into the range of events that transpire to reactivate a stalled fork in vivo.