Mechanistic studies of stalled DNA replication fork rescue

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。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 停滞的叉子重新激活机制将会缺失。这一理解是至关重要的，因为这些修复中的缺陷 高等生物体中导致突变积累导致癌症的机制，以及建议的 因此，研究与人类疾病直接相关。因此，长期目标是理解 停滞的DNA复制叉重新激活的机制。这项建议的主要目标是了解 单链DNA结合蛋白(SSB)与类核上的关键分叉抢救酶的相互作用 模板以及导致叉形结构恢复的后续处理事件。要做到这一点 目的，本建议分为三个具体目的：1)确定分叉回归的机制(S)； 2)确定分叉障碍如何影响分叉回归；以及3)确定与核相关的影响 叉子抢救酶上的蛋白质。在第一个目标下，磁镊子和原子力显微镜(都在 空气和缓冲区中的高速)将用于确定单边带加载和RecG的回归如何受到影响 以确定RecA和RuvAB是否能够催化高效的单向分叉 回归反应。当目标1的拟议研究完成后，将清楚地了解 将提供新生的、停滞不前的复制叉子。在第二个目标下，相同的两个单一DNA分子 方法将用于深入了解复制体障碍对停滞不前的叉子救援的影响， 高空间和时间分辨率。在目标2的拟议研究结束时，DNA的影响 抢救叉子上的损伤和蛋白质-DNA复合体将被阐明，并预计其机制(S) 为了取代叉子附近停滞的RNA聚合酶，将获得。在最终的目标下，磁性 操纵DNA单分子的镊子将被用来确定与类核相关的影响 蛋白质(NAP)在叉子救援上的作用。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。拟议的研究 是创新的，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 单分子方法的使用，对类核模板的关注，以及对如何获得的理解 导致复制体停滞的主要蛋白质屏障(S)被移除。最后，这项工作也是创新的，因为 在阐明重组解旋酶如何在SSB存在的情况下发挥作用时所采取的谨慎态度。建议数 这项研究意义重大，因为它将第一次允许发展清楚的机械模型 事件发生在嵌入类核模板中的停滞分叉上，它将提供第一个实时洞察 进入体内重新激活停滞的叉子的事件范围。

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.