Mechanistic studies of stalled DNA replication fork rescue

描述（由申请人提供）：在理解停滞的DNA复制叉如何退化并随后恢复方面存在根本差距。这一差距的持续存在是一个重要的问题，因为在填补这一差距之前，将缺乏对停滞的分叉重新激活机制的完整和清晰的理解。这种理解是至关重要的，因为高等生物中这些修复机制的缺陷会导致导致癌症的突变积累，因此拟议的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。本提案的目的是了解酶催化的，叉回归和随后的酶处理事件导致恢复的叉结构的机制。为了实现这一目标，本提案分为两个具体目标：1）确定关键重组解旋酶处理叉底物的机制; 2）确定单分子DNA上叉再激活的生化机制。在第一个目标下，体相生物化学和原子力显微镜将用于确定重组解旋酶如何结合和处理模拟失速叉和模拟退化叉的底物。当目标1的拟议研究完成时，将提供每种酶在叉再激活中的作用的清晰图像。在第二个目标下，两个单DNA分子方法将用于以高空间和时间分辨率产生在停滞的DNA复制叉处发生的分子事件的实时电影。在目标2的拟议研究结束时，将提供对体内重新激活失速叉的事件范围的第一个视觉和实时见解。拟议的研究是创新的，因为所采取的组合策略，使用的新的单分子方法和在阐明重组解旋酶如何在SSB的存在下发挥作用的照顾。所提出的研究是重要的，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子，它将提供第一个视觉和真实的- tme洞察到的事件范围内，蒸发重新激活一个停滞的叉子在体内。

DESCRIPTION (provided by applicant): There is a fundamental gap in understanding how stalled DNA replication forks are regressed and subsequently restored. 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 objective of this proposal is to understand the mechanisms of enzyme-catalyzed, fork regression and of the subsequent enzymatic processing events leading to restoration of a fork structure. To achieve this objective, this proposal is divided into two specific aims: 1) Determine the mechanism of processing of fork substrates by key recombination helicases; and 2) Determine the biochemical mechanisms of fork reactivation on single molecules of DNA. Under the first aim, bulk-phase biochemistry and atomic force microscopy will be used to determine how recombination helicases bind to and process substrates that mimic stalled forks and substrates that mimic regressed forks. When the proposed studies for Aim 1 are complete, a clear picture of the role of each enzyme in fork reactivation will be provided. Under the second aim, two single DNA molecule approaches will be used to produce real-time movies of the molecular events occurring at stalled DNA replication forks with high spatial and temporal resolution. At the conclusion of the proposed studies for aim 2, the first visual and real-time insight into the range of events that transpire t reactivate a stalled fork in vivo will be provided. The proposed research is innovative because of the combinatorial strategy taken, the novel single molecule approaches used and 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 o the mechanistic events occurring at a stalled fork and it will provide the first visual and real- tme insight into the range of events that transpire to reactivate a stalled fork in vivo.