Mechanistic studies of stalled DNA replication fork rescue

母公司Grant GM10056摘要 在理解如何拯救停滞不前的DNA复制叉方面存在着根本性的差距。的继续存在 这一空白代表着一个重要的问题，因为在它被填补之前，对 将不会重新激活停滞的叉子。这一理解是至关重要的，因为这些修复机制在高等 生物体会导致突变的积累，从而导致癌症，因此拟议的研究是直接相关的 人类疾病。因此，我们的长期目标是了解DNA复制叉子停滞的机制 重新激活。这项提议的主要目的是了解单链DNA结合之间的相互作用 类核模板上的蛋白质(SSB)和关键分叉抢救酶以及导致 修复叉形结构。为了实现这一目标，本提案分为三个具体目标：1)确定 分叉回归的机理(S)；2)确定分叉障碍如何影响分叉回归；3)确定影响 在叉子抢救酶上的类核相关蛋白。在第一个目标下，磁镊子和原子力显微镜 (空中和缓冲区中的高速)将用于确定单边带加载和RecG的回归如何受以下因素影响 PriA，并确定RecA和RuvAB是否能够催化有效的单向叉形回归反应。 当目标1的拟议研究完成后，一个新生的、停滞不前的复制叉子上的事件的清晰图景将是 如果是这样的话。在第二个目标下，将使用相同的两个单DNA分子方法来提供对 复制体障碍对失速叉子救援的影响，具有高空间和时间分辨率。在结束时， 目标2的拟议研究将阐明DNA损伤和蛋白质-DNA复合体对叉子救援的影响，并 预计将获得取代叉子附近停滞的核糖核酸聚合酶的机制(S)。在……下面 最终的目标是使用磁性镊子来操纵DNA的单分子，以确定类核的影响。 叉子救援上的相关蛋白(NAP)。待目标3的拟议研究完成后，便可确定是否 NAP本身会催化退化，如果它们帮助或抑制叉状救援酶的话。建议的研究具有创新性。 因为采取了组合策略。它也是创新的，因为它令人兴奋和新颖的单分子 使用的方法，对类核模板的关注，以及对初级蛋白质屏障如何获得理解(S) 导致复制体停滞的情况被移除。最后，这项工作也是创新的，因为在阐明如何 重组解旋酶在SSB存在的情况下发挥作用。拟议的研究具有重要意义，因为它将允许 第一次，发展了发生在嵌在类核物质中的失速叉子上的机械事件的清晰模型 模板，它将提供对重新激活停滞的叉子所发生的事件范围的首次实时洞察 活着。

ABSTRACT OF THE PARENT GRANT GM10056 There is a fundamental gap in understanding how stalled DNA replication forks are rescued. The 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.