Single-Molecule Assembly of Protein-DNA Complexes

蛋白质-DNA 复合物的单分子组装

• 批准号: 7467279
• 负责人: Stephen Charles Kowalczykowski
• 金额: $ 28.88万
• 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 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7467279
• 关键词: Address; Applications Grants; BRCA2 gene; Behavior; Binding Proteins; Biochemical Process; Biological; Class; Complex; DNA; DNA Repair; Detection; Disease; Dissociation; Embryo; Eukaryota; Eukaryotic Cell; 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 function; repair enzyme; repaired; single molecule; translocase

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）：本拨款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颖的单分子方法，可以真实地可视化蛋白质和 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；每一个都是 DNA 重组过程的重要组成部分。将要检查的一类蛋白质是 DNA 链交换蛋白、RecA 和 Rad51；第二类是改变 RecA/Rad51 核蛋白丝动力学的蛋白质；第三个是核蛋白和染色质重塑转位酶，Rad54 蛋白。具体目标是： 1) 可视化并测量 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 paralogs、BRCA2等介导蛋白对RecA/Rad51丝组装的作用； 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物的突变会导致癌症、减数分裂异常和胚胎死亡。因此，有必要对重组 DNA 进行详细的分子了解，以了解所涉及的许多蛋白质的功能。最近，开发了可视化这些修复酶对单分子 DNA 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，包括通过重组修复 DNA 所涉及的日益复杂的生化过程。