Mechanistic studies of stalled DNA replication fork rescue

挽救停滞DNA复制叉的机制研究

• 批准号: 10291961
• 负责人: Piero R Bianco
• 金额: $ 45.14万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-06-07 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10291961
• 关键词: Mechanistic; studies; stalled; DNA; replication

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.

抽象的 了解如何挽救停滞的DNA复制叉存在根本的差距。继续存在 这一差距代表了一个重要的问题，因为在填补之前，对 停滞的叉子重新激活的机制将缺乏。这种理解至关重要，因为这些修复中的缺陷 较高生物体的机制导致突变的积累导致癌症，并提出 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复制叉重新激活的机理。该提议的主要目的是了解 单链DNA结合蛋白（SSB）和核心上的关键叉子挽救酶之间的相互作用 模板和随后的处理事件，导致叉结构的恢复。实现这一目标 目的，该提议分为三个特定的目的：1），确定叉回归的机制； 2，）确定叉子障碍如何影响叉子回归；和3），确定核苷相关的影响 叉子营救酶上的蛋白质。在第一个目标下，磁性镊子和原子力显微镜（都在 空气和高速缓冲区）将用于确定RECG的SSB加载和回归如何受到影响 通过Pria并确定Reca和Ruvab是否能够催化有效且单向叉 回归反应。当提议的目标1完成拟议的研究时，清楚地了解了事件 将提供新生，停滞的复制叉。在第二个目标下，相同的两个单个DNA分子 方法将用于洞悉重壳障碍对停滞的叉子救援的影响，并与 高空间和时间分辨率。在拟议的AIM 2研究结束时，DNA的影响 叉子救援上的病变和蛋白质-DNA复合物将清楚，预计该机制是 为了将失速的RNA聚合酶移位到叉子附近。在最终目标下，磁性 镊子操纵DNA的单分子将用于确定核苷相关的影响 叉子救援上的蛋白质（午睡）。当提出的目标3的研究完成时，将确定是否确定是否存在 小睡会自行催化回归，如果它们协助或抑制叉子救援酶。拟议的研究 由于采取了组合策略，因此具有创新性。这也是创新的，因为令人兴奋和新颖 所使用的单分子方法，关注核苷模板的重点，并了解如何获得 导致重新降低的主要蛋白质屏障被去除。最后，这项工作也是创新的，因为 在阐明SSB存在下重组解旋酶如何发挥作用的护理。提议 研究很重要，因为它将首次允许开发机械的清晰模型 发生在嵌入核苷模板中的失速叉子上的事件，它将提供第一个实时洞察力 进入跨性体内停滞叉子的一系列事件。