Single-Molecule Assembly of Protein-DNA Complexes

蛋白质-DNA 复合物的单分子组装

• 批准号: 8338808
• 负责人: Stephen Charles Kowalczykowski
• 金额: $ 31.57万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-15 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8338808
• 关键词: ATP Hydrolysis; Address; Behavior; Binding Proteins; Biochemical; Biochemical Process; Biological; Complex; DNA; DNA Repair; Disease; Dissociation; Eukaryota; Event; Filament; Fluorescence; Genetic; Genetic Recombination; Heterogeneity; Human; Image; Imagery; Individual; Kinetics; Laboratories; Measures; Mediator of activation protein; Methods; Microscopy; Molecular; Motor; Nucleoproteins; Organism; Process; Prokaryotic Cells; Property; Proteins; Rec A Recombinases; Role; Solutions; Surface; System; Techniques; Time; chromatin remodeling; innovation; instrument; macromolecular assembly; novel; optical traps; protein complex; protein function; protein phosphatase inhibitor-2; repair enzyme; single molecule; translocase

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.

描述（由申请人提供）： 该建议的长期目标是了解蛋白质在孤立蛋白-DNA复合物水平上的行为。该建议利用了一种新型的单分子方法，该方法可以可视化蛋白质与DNA的单个复合物的行为和动态特性。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。将要检查的蛋白质包括DNA链交换蛋白，RECA和RAD51； RECA/RAD51功能的介体和调节剂；最后是核蛋白和染色质型转运酶RAD54蛋白。具体目的是：1。可视化和测量RECA和RAD51核蛋白丝的组装，拆卸和极性。该目的解决了RECA/RAD51核蛋白丝的核心行为 - 其动态行为及其找到DNA同源性的能力。 2。确定RECA/RAD51核蛋白丝的动态行为如何由竞争者和介体修饰。这个目的解决了竞争对手，调解器和运动蛋白如何调节RECA和RAD51细丝的行为的问题。 3。定义rad54蛋白沿dsDNA的易位作用。该目的解决了RAD54易位能力的功能及其重塑蛋白-DNA复合物的能力。 这些蛋白质在单分子水平上的可视化实时为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断裂的修复，该过程尚未完全理解其机制。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的过程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，其中包括越来越复杂的生化过程，这些过程是基于重组DNA修复的基础。