Single-Molecule Assembly of Protein-DNA Complexes

描述(由申请人提供)：这项资助提案的主要长期目标是在单个蛋白质-DNA复合体的水平上了解参与重组DNA修复的蛋白质的行为。这一提议利用了一种新颖的单分子方法，可以从字面上可视化蛋白质和DNA单个复合体的动态和功能。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。将被检测的一类蛋白质是DNA链交换蛋白RecA和RAD51；第二类是改变RecA/RAD51核蛋白细丝动力学的蛋白质；第三类是核蛋白和染色质重塑易位酶Rad54蛋白。其具体目的是：1)可视化和测量RecA和RAD51核蛋白微丝的组装、拆解和极性；2)观察SSB/RPA、RecFOR、Rad52、RAD51 Paralog和BRCA2等中介蛋白在RecA/RAD51微丝组装中的作用；3)确定Rad54蛋白沿dsDNA移位的功能和后果。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。如果不加以修复，DNA断裂会导致基因组不稳定，从而导致癌症。这些蛋白质的人类对应物的突变导致癌症易感性、异常减数分裂和胚胎死亡。因此，对重组DNA的详细分子理解对于理解涉及的许多蛋白质的功能是必要的。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。这些方法可以提供对这些复杂过程的实时行为的前所未有的理解。这些单分子方法将被用来定义一些分子事件，这些分子事件包括通过重组修复DNA所涉及的日益复杂的生化过程。

DESCRIPTION (provided by applicant): The major long-term objective of this grant proposal is to understand the behavior of proteins involved in recombinational DNA repair at the level single protein-DNA complexes. This proposal takes advantage of a novel single-molecule approach that can literally visualize the dynamics and function of individual complexes of proteins and DNA. Several different protein-DNA complexes will be examined; each is an essential component of the DNA recombination process. One class of proteins that will be examined is the DNA strand exchange proteins, RecA and Rad51; and the second is the class of proteins that modify RecA/Rad51 nucleoprotein filament dynamics; and the third is 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 filament formation; 2) Observe the role of mediator proteins such as SSB/RPA, RecFOR, Rad52, Rad51 paralogs, and BRCA2 on RecA/Rad51 filament assembly; and 3) Define the function and consequences of Rad54 protein translocation along dsDNA. Each of these proteins is involved in the repair of DNA breaks by recombination, a process whose mechanism is not fully understood. Left unrepaired, DNA breaks result in genomic instabilities that give rise to cancers. Mutations in the human counterparts of these proteins result in predispositions to cancer, aberrant meiosis, and embryonic lethality. Consequently, a detailed molecular understanding of recombinational DNA is necessary to understand the functions of the many proteins involved. 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 the real-time behavior of these intricate processes. These single-molecule methods will be used to define some of the molecular events comprising increasingly complicated biochemical processes involved in the repair of DNA by recombination.