Single-Molecule Assembly of Protein-DNA Complexes

蛋白质-DNA 复合物的单分子组装

• 批准号: 6422004
• 负责人: Stephen Charles Kowalczykowski
• 金额: $ 40.91万
• 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 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6422004
• 关键词: DNA replication; bioimaging /biomedical imaging; biomedical equipment development; fluorescence microscopy; genetic recombination; green fluorescent proteins; helicase; imaging /visualization /scanning; intermolecular interaction; intracellular transport; lasers; nanotechnology; protein biosynthesis; protein structure function; protein transport; recombinase

DESCRIPTION (provided by applicant): The major objective of this grant proposal is to develop a new experimental technique for the study of protein-DNA interactions at the single-molecule level. This proposal describes a novel instrument to study the dynamics and function of individual complexes of proteins and DNA. The design will permit the direct visualization by fluorescence microscopy, in real-time, of the assembly, disassembly, and movement of proteins on single, optically-trapped DNA molecules using a novel, multi-port, laminar-flow, micro machined flow-cell. This instrument will allow us to readily introduce an individual, optically-trapped DNA molecule (or protein-DNA complex) sequentially to other proteins and/or reaction conditions, and visualize the changes in structure/assembly of the molecules in real-time using multi-wavelength fluorescence microscopy. The instrument will also measure the forces generated by these protein-DNA interactions. Several different protein-DNA complexes will be examined; each is an essential component of genetic recombination. Two classes of proteins that will be examined are the DNA motor proteins known as helicases, and the DNA strand exchange proteins known as recombinases. The DNA helicases central to this process in Escherichia coli are the RecBCD enzyme and the RecQ helicase. The recombinases essential to this process are RecA (prokaryotes) and Rad51 (eukaryotes) proteins, and their ancillary protein components. This grant proposal has two specific aims. The first is to develop an instrument that will permit analysis of individual assemblies of protein-DNA complexes. The second broad objective is to examine the formation, dissociation, and translocation of the several specific DNA helicases (motor proteins) and DNA-enzyme complexes (protein machines) that are involved in genetic recombination and DNA repair. Development of this instrument will enable new types of single-molecule experiments, experiments that will uncover information about the dynamics and function of protein-DNA interactions that cannot be obtained from large ensembles of DNA molecules.

描述（申请人提供）：本赠款提案的主要目标 是开发一种新的实验技术来研究蛋白质-DNA 单分子级的相互作用。该提议描述了一本小说 研究单个复合物的动态和功能的仪器 蛋白质和DNA。该设计将允许通过 实时的荧光显微镜组装，拆卸和 使用小说 多端口，层流 - 流，微加工流线。该工具将允许 我们很容易介绍一个单独的，光学捕获的DNA分子（或 依次与其他蛋白质和/或反应条件依次 并可视化分子实时的结构/组装变化 使用多波长荧光显微镜。该仪器也将 测量这些蛋白-DNA相互作用产生的力。 将检查几种不同的蛋白质-DNA复合物。每个都是必不可少的 遗传重组的组成部分。两类蛋白质将是 检查的是DNA运动蛋白称为解旋酶，DNA链 交换蛋白质称为重物组织酶。 DNA解旋酶核心 大肠杆菌的过程是RECBCD酶和RECQ解旋酶。这 重点酶对此过程必不可少的是RECA（原核生物）和RAD51 （真核生物）蛋白质及其辅助蛋白成分。这笔赠款 提案有两个具体的目标。首先是开发一种将会 允许分析蛋白质-DNA复合物的单个组件。第二个 广泛的目标是检查的形成，解离和易位 几种特定的DNA解旋酶（运动蛋白）和DNA-酶复合物 （蛋白质机器）涉及遗传重组和DNA修复。 该仪器的开发将使新型的单分子类型 实验，将发现有关动力学和的信息 蛋白质-DNA相互作用的功能，无法从大型 DNA分子的集合。