Single-Molecule Assembly of Protein-DNA Complexes

描述（由申请人提供）： 这项计划的长期目标是在单独的蛋白质-DNA复合物水平上了解蛋白质的行为。该提案利用了一种新的单分子方法，该方法可以可视化蛋白质与DNA的单个复合物的行为和动态特性。将检查几种不同的蛋白质-DNA复合物;每一种都是DNA重组过程的重要组成部分。将被检查的蛋白质包括DNA链交换蛋白，RecA和Rad 51; RecA/Rad 51功能的介质和调节剂;以及最后的核蛋白和染色质重塑易位酶，Rad 54蛋白。具体目标是：1.观察并测量RecA和Rad 51核蛋白丝的组装、拆卸和极性。这一目标解决了RecA/Rad 51核蛋白丝的核心行为-其动态行为和发现DNA同源性的能力。2.确定RecA/Rad 51核蛋白丝的动态行为如何被竞争对手和介质改变。这一目标解决了竞争对手，调解员和马达蛋白如何调节RecA和Rad 51丝的行为的问题。3.明确Rad 54蛋白在双链DNA沿着易位中的作用。这一目标解决了Rad 54易位能力的功能及其重塑蛋白质-DNA复合物的能力。 这些蛋白质在单分子水平上的实时可视化为了解这些蛋白质的行为和功能提供了一个全新的窗口。这些蛋白质中的每一种都参与通过重组修复DNA断裂，这一过程的机制尚未完全了解。最近，已经开发了可视化这些修复酶对DNA单分子作用的新方法。这些方法可以提供对这些复杂过程的前所未有的理解。这些单分子方法将被用来定义一些分子事件，包括越来越复杂的生化过程，支持重组DNA修复。 公共卫生相关性： 基因重组是维持染色体完整性所必需的基本生物学过程。这项研究具有重要意义，因为它提供了关于蛋白质-蛋白质和蛋白质-DNA相互作用的基本信息，这些相互作用对于通过重组进行正常的DNA断裂修复至关重要。重组DNA断裂修复的主要功能是防止基因组重排。这些蛋白质负责保持所有生物体的遗传完整性，当人类有缺陷时，会导致各种疾病。如果不修复，这些DNA断裂会导致基因组不稳定，从而可能导致肿瘤发生。这些蛋白质的人类对应物的突变不仅导致癌症的易感性，而且引起异常减数分裂和胚胎死亡。了解这一过程的分子机制，将有助于阐明导致染色体易位和基因组不稳定的异常生物学事件。因此，对重组DNA修复的详细分子理解对人类健康是重要的。

DESCRIPTION (provided by applicant): The long-term objective of this proposal is to understand the behavior of proteins at the level of solitary protein-DNA complexes. This proposal takes advantage of a novel single-molecule approach that can visualize the behavior and dynamic properties of individual complexes of proteins with DNA. Several different protein-DNA complexes will be examined; each is an essential component of the DNA recombination process. The proteins that will be examined include the DNA strand exchange proteins, RecA and Rad51; the mediators and modulators of RecA/Rad51 function; and the finally the nucleoprotein- and chromatin-remodeling translocase, Rad54 protein. The specific aims are to: 1. Visualize and measure the assembly, disassembly, and polarity of RecA and Rad51 nucleoprotein filaments. This aim addresses the core behavior of the RecA/Rad51 nucleoprotein filament - its dynamic behavior and its ability to find DNA homology. 2. Determine how the dynamic behavior of RecA/Rad51 nucleoprotein filaments is modified by competitors and mediators. This aim addresses the question of how competitor, mediator, and motor proteins modulate the behavior of RecA and Rad51 filaments. 3. Define the role of translocation along dsDNA by Rad54 protein. This aim addresses the functions of Rad54 translocation capacity and its ability to remodel protein-DNA complexes. The visualization of these proteins acting at the single-molecule level, in real-time, affords a completely new window into the behavior and function of these proteins. Each of these proteins is involved in the repair of DNA breaks by recombination, a process whose mechanism is not fully understood. Recently, new methods of visualizing the action of these repair enzymes on single-molecules of DNA have been developed. These methods can provide an unprecedented level of understanding of these intricate processes. These single-molecule methods will be used to define some of the molecular events comprising increasingly complicated biochemical processes that underpin recombinational DNA repair. PUBLIC HEALTH RELEVANCE: Project Narrative Genetic recombination is a fundamental biological process that is necessary for the maintenance of chromosomal integrity. The research in this proposal is significant because it provides basic information about the protein-protein and protein-DNA interactions that are essential for normal DNA-break repair by recombination. A major function of recombinational DNA-break repair is the prevention of genomic rearrangement. These proteins are responsible for preserving genetic integrity in all organisms and, when defective in humans, are responsible for a variety of diseases. Left unrepaired, these DNA breaks can result in genomic instabilities that that might permit tumorigenesis. Mutations in the human counterparts of these proteins result not only in predispositions to cancer, but give rise to aberrant meiosis and embryonic lethality. Understanding the molecular mechanism of this process should shed light abnormal biological events that lead to chromosomal translocations and genome instability. Consequently, a detailed molecular understanding of recombinational DNA repair is important to human health.