Single-Molecule Assembly of Protein-DNA Complexes

描述(由申请人提供)：这项资助计划的主要目的 是开发一种新的研究蛋白质-DNA的实验技术 单分子水平上的相互作用。这份提案描述了一部小说 用于研究单个复合体的动力学和功能的仪器 蛋白质和DNA。该设计将允许通过以下方式直接可视化 实时荧光显微镜下的组装、拆卸和 利用一种新型的、光学捕获的DNA分子上的蛋白质运动， 多端口、层流、微机械加工的流量计。这台仪器将允许 美国容易地引入一种单独的、光学捕获的DNA分子(或 蛋白质-DNA复合体)顺次到其他蛋白质和/或反应条件， 并实时可视化分子的结构/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。 我们将研究几种不同的蛋白质-DNA复合体；每个复合体都是必不可少的 基因重组的组成部分。两类蛋白质将是 被检查的是被称为解旋酶的DNA马达蛋白，以及DNA链 交换蛋白称为重组酶。对此至关重要的DNA解旋酶 在大肠杆菌中的过程是RecBCD酶和RecQ解旋酶。这个 对这一过程至关重要的重组酶是RecA(原核生物)和RAD51 (真核生物)蛋白质及其辅助蛋白质组分。这笔赠款 提案有两个具体目标。首先是开发一种仪器，它将 允许分析蛋白质-DNA复合体的单个组装。第二 更广泛的目标是检查的形成、解离和移位 几种特异的DNA解旋酶(马达蛋白)和DNA-酶复合体 (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，实验将揭示关于动力学和 蛋白质-DNA相互作用的功能不能从大的 DNA分子的集合。

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.