Mechanistic studies of stalled DNA replication fork rescue

挽救停滞DNA复制叉的机制研究

• 批准号: 8812888
• 负责人: Piero R Bianco
• 金额: $ 44万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 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 2016-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8812888
• 关键词: Affinity; Animal Model; Atomic Force Microscopy; Binding; Biochemical; Biochemistry; Caring; Complex; DNA; DNA biosynthesis; DNA replication fork; DNA-Directed RNA Polymerase; Data; Defect; Development; Discrimination; Enzymes; Escherichia coli; Eukaryota; Event; Failure; Fostering; Genetic Recombination; Genome; Genome Stability; Genomic Instability; Goals; Health; Lead; Malignant Neoplasms; Mediating; Mission; Modeling; Molecular; Mutation; Organism; Outcome; Pathway interactions; Phase; Play; Process; Prokaryotic Cells; Proteins; Public Health; Publishing; Reaction; Research; Role; SS DNA BP; Staging; Structure; Testing; Time; Training; Visual; Work; base; combinatorial; enzyme mechanism; genome integrity; helicase; human disease; improved; in vivo; innovation; insight; movie; novel; nuclease; prevent; public health relevance; repaired; restoration; single molecule; temporal measurement

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.

描述（由申请人提供）：了解停滞的DNA复制叉如何回归并随后恢复存在基本差距。该差距的持续存在代表了一个重要的问题，因为在填补之前，将缺乏对停滞的叉子重新激活机制的完整而清晰的理解。这种理解至关重要，因为较高生物体中这些修复机制的缺陷导致突变的积累导致癌症，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复制叉重新激活的机制。该提案的目的是了解酶催化，分叉回归的机制以及随后的酶促加工事件，导致恢复叉子结构。为了实现这一目标，该提案分为两个具体的目的：1）确定通过关键重组解酶处理叉子底物的处理机制； 2）确定DNA单分子上叉重新激活的生化机制。在第一个目的下，将使用散装相生物化学和原子力显微镜来确定重组解旋酶如何与模拟模仿叉子的叉子和模仿叉子的底物结合和处理底物。当提出的针对AIM 1的研究完成时，将清楚地了解每种酶在分叉重新激活中的作用。在第二个目标下，将使用两种单个DNA分子方法来产生在具有高空间和时间分辨率的停滞DNA复制叉处发生的分子事件的实时电影。在拟议的AIM 2研究结束时，将提供对t驱散t在体内停滞叉子的事件范围的第一个视觉和实时洞察力。拟议的研究具有创新性，因为采用了组合策略，所使用的新型单分子方法以及在SSB存在下重新组合解旋酶如何发挥作用时所接受的护理。拟议的研究很重要，因为它将首次允许的开发，即清晰的模型o在停滞的叉子上发生的机械事件，它将提供第一个视觉和现实的洞察力，以洞悉跨越体内停滞的叉子的一系列事件范围。