Single-Molecule Assembly of Protein-DNA Complexes

描述(由申请人提供)： 这项提议的长期目标是在孤立蛋白质-DNA复合体的水平上了解蛋白质的行为。这一提议利用了一种新的单分子方法，可以可视化蛋白质与DNA的单个复合体的行为和动态性质。我们将研究几种不同的蛋白质-DNA复合体，每个复合体都是DNA重组过程的重要组成部分。将要检测的蛋白质包括DNA链交换蛋白RecA和RAD51；RecA/RAD51功能的介体和调节物；以及最后的核蛋白和染色质重塑转位酶Rad54蛋白。具体目的是：1.可视化和测量RecA和RAD51核蛋白细丝的组装、拆解和极性。这一目标研究了RecA/RAD51核蛋白细丝的核心行为--它的动态行为和寻找DNA同源性的能力。2.确定竞争对手和介体如何改变RecA/RAD51核蛋白细丝的动态行为。这个目的是解决竞争蛋白、介体蛋白和马达蛋白如何调节RecA和RAD51细丝的行为的问题。3.明确Rad54蛋白沿dsDNA易位的作用。本研究的目的是探讨RAD54转位能力的功能及其重塑蛋白质-DNA复合体的能力。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个都参与通过重组修复DNA断裂，这一过程的机制尚不完全清楚。最近，可视化这些修复酶在DNA单分子上的作用的新方法已经开发出来。这些方法可以对这些复杂的过程提供前所未有的理解。这些单分子方法将被用来定义一些分子事件，这些事件包括支持重组DNA修复的日益复杂的生化过程。

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.