Single-Molecule Assembly of Protein-DNA Complexes

蛋白质-DNA 复合物的单分子组装

基本信息

批准号：
7937183
负责人：
Stephen Charles Kowalczykowski
金额：
$ 25.89万
依托单位：
UNIVERSITY OF CALIFORNIA AT DAVIS
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-30 至 2011-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7937183
关键词：
Address Applications Grants BRCA2 gene Behavior Binding Proteins Biochemical Process Biological Complex DNA DNA Repair Detection Disease Dissociation Embryo Eukaryota Event Filament Fluorescence Genetic Genetic Recombination Genomic Instability Human Image Imagery Individual Kinetics Laboratories Left Malignant Neoplasms Measures Mediator of activation protein Meiosis Methods Molecular Multienzyme Complexes Mutation Nucleoproteins Organism Phase Predisposition Process Prokaryotic Cells Protein translocation Proteins Rec A Recombinases Role Time chromatin remodeling innovation instrument macromolecular assembly novel paralogous gene protein complex protein function repair enzyme repaired single molecule translocase

Address Applications Grants BRCA2 gene Behavior Binding Proteins Biochemical Process Biological Complex DNA DNA Repair Detection Disease Dissociation Embryo Eukaryota Event Filament Fluorescence Genetic Genetic Recombination Genomic Instability Human Image Imagery Individual Kinetics Laboratories Left Malignant Neoplasms Measures Mediator of activation protein Meiosis Methods Molecular Multienzyme Complexes Mutation Nucleoproteins Organism Phase Predisposition Process Prokaryotic Cells Protein translocation Proteins Rec A Recombinases Role Time chromatin remodeling innovation instrument macromolecular assembly novel paralogous gene protein complex protein function repair enzyme repaired single molecule translocase

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项目摘要

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.

描述（由申请人提供）：该赠款建议的主要长期目标是了解在水平的单蛋白DNA复合物下参与重组DNA修复的蛋白质的行为。该建议利用了一种新型的单分子方法，该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。每个都是DNA重组过程的重要组成部分。一类将要检查的蛋白质是DNA链交换蛋白，RECA和RAD51。第二个是修改RECA/RAD51核蛋白丝动力学的蛋白质类别。第三个是核蛋白和染色质 - 复发易生酶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）观察介质蛋白（例如SSB/RPA，RECFOR，RAD52，RAD51旁系同源物和BRCA2）在RECA/RAD51细丝组件上的作用； 3）定义沿dsDNA的Rad54蛋白易位的功能和后果。这些蛋白质中的每一个都参与通过重组修复DNA断裂的修复，该过程尚未完全理解其机制。剩下的未修复的DNA断裂导致基因组不稳定性引起癌症。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性。因此，必须对重组DNA进行详细的分子理解，以了解所涉及的许多蛋白质的功能。最近，已经开发了这些修复酶对DNA单分子的作用的新方法。这些方法可以为这些复杂过程的实时行为提供前所未有的理解水平。这些单分子方法将用于定义一些分子事件，其中包含越来越复杂的生化过程，涉及通过重组修复DNA。