Mechanistic studies of stalled DNA replication fork rescue

专利授权摘要GM 10056 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。的继续存在 这一差距是一个重要的问题，因为在填补这一差距之前， 将缺乏停滞的分叉重新激活。这种理解是至关重要的，因为这些修复机制中的缺陷在更高的 生物体导致突变的积累，导致癌症，因此，拟议的研究直接相关 人类疾病。因此，长期目标是了解停滞的DNA复制叉的机制 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， 蛋白质（SSB）和关键叉拯救酶对类核模板和随后的加工事件，导致 恢复一个叉结构。为实现这一目标，本建议分为三个具体目标：1）确定 分叉回归的机制; 2）确定分叉障碍如何影响分叉回归; 3）确定影响 类核蛋白对叉拯救酶的影响。在第一个目标下，磁镊和原子力显微镜 (both在空气中和高速缓冲）将用于确定SSB加载和RecG回归如何受到以下因素的影响： PriA和确定RecA和RuvAB是否能够催化有效和单向的叉回归反应。 当目标1的拟议研究完成时，将清楚地了解新生的、停滞的复制叉的事件。 提供了在第二个目标下，将使用相同的两个单DNA分子方法来提供对 复制体障碍对停滞叉拯救的影响，具有高空间和时间分辨率。结束时 目标2的拟议研究中，DNA损伤和蛋白质-DNA复合物对叉拯救的影响将得到明确， 预期将获得置换叉附近停滞的RNA聚合酶的机制。下 最终的目标，操纵DNA单分子的磁镊子将被用来确定类核的作用， 相关蛋白（NAP）在叉救援中的作用。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。该研究具有创新性 因为采取了组合策略。它也是创新的，因为令人兴奋的和新颖的单分子 所使用的方法，对类核模板的关注，以及对初级蛋白质屏障如何被破坏的理解。 导致复制体停滞的基因被去除。最后，这项工作也是创新的，因为在阐明如何照顾 重组解旋酶在SSB存在下起作用。拟议的研究是重要的，因为它将允许， 第一次，发展明确的模型的机械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的事件范围，发生重新激活一个停滞的分叉， vivo.

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.