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（由申请人提供）：本赠款提案的主要目标 是发展一种新的实验技术来研究蛋白质-DNA 在单分子水平上的相互作用。这篇文章描述了一部小说。 仪器研究的动态和功能的个别复合物 蛋白质和DNA。该设计将允许直接可视化， 荧光显微镜，实时，组装，拆卸， 使用一种新的， 多端口、层流、微加工流动池。该仪器将允许 我们很容易引入一个单独的，光学捕获的DNA分子（或 蛋白质-DNA复合物）依次与其它蛋白质和/或反应条件反应， 并实时可视化分子结构/组装的变化 使用多波长荧光显微镜。该仪器还将 测量这些蛋白质-DNA相互作用产生的力。 将检查几种不同的蛋白质-DNA复合物;每一种都是必不可少的。 基因重组的组成部分。两类蛋白质， 研究的是被称为解旋酶的DNA马达蛋白， 交换蛋白质称为重组酶。DNA解旋酶对此至关重要， 在大肠杆菌中的主要酶是RecBCD酶和RecQ解旋酶。的 该过程所必需的重组酶是RecA（原核生物）和Rad 51 （真核生物）蛋白质及其辅助蛋白质组分。这笔赠款 该提案有两个具体目标。第一是开发一种工具， 允许分析蛋白质-DNA复合物的单独组装。第二 一个广泛的目标是检查的形成，解离，和易位 几种特异性DNA解旋酶（马达蛋白）和DNA-酶复合物 （蛋白质机器）参与基因重组和DNA修复。 该仪器的开发将使新型单分子 实验，这些实验将揭示有关动力学的信息， 蛋白质-DNA相互作用的功能，不能从大的 DNA分子的集合。