Single-Molecule Assembly of Protein-DNA Complexes

描述（由申请人提供）：本拨款提案的主要长期目标是在单蛋白-DNA复合物水平上了解参与重组DNA修复的蛋白质的行为。这项提议利用了一种新颖的单分子方法，可以从字面上可视化单个蛋白质和DNA复合物的动态和功能。几种不同的蛋白质- dna复合物将被检查；每一个都是DNA重组过程的重要组成部分。我们要研究的一类蛋白质是DNA链交换蛋白，RecA和Rad51；二是修饰RecA/Rad51核蛋白丝动力学的一类蛋白；第三种是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核蛋白丝形成的组装、拆卸和极性；2)观察SSB/RPA、RecFOR、Rad52、Rad51同源物、BRCA2等中介蛋白在RecA/Rad51丝组装中的作用；3)定义Rad54蛋白沿dsDNA易位的功能和后果。这些蛋白质中的每一种都参与了DNA断裂的重组修复，这一过程的机制尚不完全清楚。如果不进行修复，DNA断裂会导致基因组不稳定，从而引发癌症。这些蛋白质的人类对应体的突变导致癌症、减数分裂异常和胚胎死亡的易感性。因此，对重组DNA的详细分子理解对于理解许多相关蛋白质的功能是必要的。最近，研究人员开发了可视化这些修复酶对DNA单分子作用的新方法。这些方法可以为了解这些复杂过程的实时行为提供前所未有的水平。这些单分子方法将用于定义一些分子事件，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.