Single-Molecule Assembly of Protein-DNA Complexes

描述（由申请人提供）：本资助提案的主要长期目标是了解在单个蛋白质-DNA复合物水平上参与重组DNA修复的蛋白质的行为。该提案利用了一种新的单分子方法，可以从字面上可视化蛋白质和DNA的单个复合物的动力学和功能。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。将被检查的一类蛋白质是DNA链交换蛋白，RecA和Rad 51;和第二类的蛋白质，修改RecA/Rad 51核蛋白丝动力学;和第三个是核蛋白和染色质重塑易位酶，Rad 54蛋白。具体目标是：1）观察和测量RecA和Rad 51核蛋白丝形成的组装、拆卸和极性; 2）观察介体蛋白如SSB/RPA、RecFOR、Rad 52、Rad 51旁系同源物和BRCA 2对RecA/Rad 51丝组装的作用; 3）定义Rad 54蛋白沿着沿着dsDNA移位的功能和后果。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。如果不修复，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中的突变导致癌症、异常减数分裂和胚胎致死的倾向。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，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实时行为的前所未有的理解。这些单分子方法将用于定义一些分子事件，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.