Mechanistic studies of stalled DNA replication fork rescue

挽救停滞DNA复制叉的机制研究

基本信息

批准号：
10387612
负责人：
Piero R Bianco
金额：
$ 6.02万
依托单位：
UNIVERSITY OF NEBRASKA MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-06-07 至 2022-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10387612
关键词：
Affect Air Atomic Force Microscopy Automobile Driving Binding Proteins Biological Models Buffers Caring Complex Cruciform DNA DNA DNA Damage DNA Structure DNA biosynthesis DNA lesion DNA replication fork DNA-Directed RNA Polymerase Data Defect Development Discrimination Enzymes Escherichia coli Eukaryota Event Failure Fostering Genetic Recombination Genetic Transcription Genome Genome Stability Genomic Instability Goals Health Lead Lesion Magnetism Malignant Neoplasms Mission Modeling Mutation Organism Outcome Pathway interactions Play Process Prokaryotic Cells Proteins Public Health Publishing Reaction Research Role SS DNA BP Speed Structure Testing Time Translations United States National Institutes of Health Work combinatorial genome integrity helicase homologous recombination human disease improved in vivo innovation insight novel parent grant public health relevance repaired restoration single molecule temporal measurement

项目摘要

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.

父母GM10056的摘要了解如何挽救停滞的DNA复制叉存在根本的差距。持续存在该差距代表了一个重要的问题，因为直到填补它，对将缺乏停滞的叉子重新激活。这种理解至关重要，因为这些修复机制中的缺陷生物体导致突变的积累导致癌症，因此提出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。该提案的主要目的是了解单链DNA结合之间的相互作用蛋白质（SSB）和关键叉子营救酶在核苷模板上以及随后的加工事件导致修复叉结构。为了实现这一目标，该建议分为三个具体目标：1）确定叉回归的机理； 2，）确定叉子障碍如何影响叉子回归；和3），确定效果叉子拯救酶上与核苷相关的蛋白。在第一个目标下，磁性镊子和原子力显微镜（在空气和高速缓冲区中）都将用于确定RECG的SSB加载和回归如何受到RECG的影响 PRIA并确定Reca和Ruvab是否能够催化有效且单向的叉回归反应。当提议的目标1完成拟议的研究时，清楚地了解了新生，停滞的复制叉的事件假如。在第二个目标下，将使用相同的两种单个DNA分子方法来洞悉重置障碍对停滞的叉子救援的影响，并具有高空间和时间分辨率。在结束时拟议的AIM 2研究，将明确并明确DNA病变和蛋白-DNA复合物对分叉救援的影响可以预期，将获得将失速的RNA聚合酶置换在叉子附近的机制。在下面最终目的是操纵DNA的单分子的磁镊子将用于确定核苷 - 叉子救援上的相关蛋白质（午睡）。当提出的目标3的研究完成时，将确定是否确定是否存在小睡会自行催化回归，如果它们协助或抑制叉子救援酶。拟议的研究是创新的由于采取了组合策略。这也是创新的，因为令人兴奋和新颖的单分子使用的方法，关注核苷模板，以及对主要蛋白质屏障如何获得的理解导致重新插座失速被去除。最后，这项工作也具有创新性，因为阐明了如何阐明重组解旋酶在SSB存在下起作用。拟议的研究很重要，因为它将允许第一次，发生在嵌入核苷内的失速叉处的机械事件的清晰模型的发展模板以及它将提供第一个实时洞察力，以了解蒸蒸日上叉子的一系列事件范围体内。