Characterization of DNA/Protein Interactions at the Single Molecule Level

这项工作的目标是在单分子水平上探索三种不同类型的DNA/蛋白质相互作用：1)蛋白质和DNA之间的结合相互作用，特别强调一维扩散的作用；2)结合在两个或多个非相邻位点的蛋白质复合物在DNA中产生环；3)通过将一个碱基从相邻碱基的堆叠配置中拉出而破坏碱基堆叠的蛋白质相互作用。这些过程中的每一个都将在种群分布水平（处于平衡状态的DNA/蛋白质复合物的不同几何形状或构象的相对种群）和动力学方面（构象或几何形状之间相互转换所需的时间，主要是在平衡状态下）进行探索。单分子荧光光谱非常适合这种测量。主要的重点将放在三个DNA/蛋白质系统。这些是嵌入染料与DNA的相互作用作为蛋白质/DNA相互作用的模型系统，以及各种DNA结合域与DNA的研究，重点是寻找结合位点和导致DNA环的蛋白质/DNA相互作用。一些工作将在最终和雄心勃勃的项目中进行，在单分子水平上监测DNA聚合酶的作用。这项工作将与阿尔伯塔大学的Reha-Krantz博士合作完成，他将提供实验所需的一些DNA模板和结构。该项目将与该大学现有的IGERT项目密切相关，本科生将大量参与研究。该项目由数学和物理科学理事会的物理和化学司以及生物科学理事会的分子和细胞生物科学司共同资助。

The goal of this work is to explore, at the single molecule level, three different types of DNA/protein interactions: 1) Binding interactions between proteins and DNA with a particular emphasis on the role of one-dimensional diffusion, 2) Generation of loops in DNA by protein complexes that bind at two or more nonadjacent sites, 3) Protein interactions which disrupt base stacking by pulling a base out of the stacking configuration with neighboring bases. Each of these processes will be explored at both the level of population distributions (relative populations of different geometries or conformations of the DNA/protein complex at equilibrium) and in terms of dynamics (times required for interconversion between conformations or geometries, again primarily at equilibrium). Single molecule fluorescence spectroscopy is well suited for such measurements. The primary emphasis will be placed on three DNA/Protein systems. These are the interaction of intercalating dyes with DNA as a model system for protein/DNA interaction and the study of various DNA binding domains with DNA with an emphasis on searching for binding sites and protein/DNA interactions which result in DNA looping. Some work will be performed on a final and ambitious project, monitoring the action of DNA polymerase at the single molecule level. This work will be done in collaboration with Dr. Reha-Krantz at the University of Alberta, who will provide some of the DNA templates and constructs required for the experiments. The project will be closely connected to an existing IGERT program at the university, and undergraduate students will be heavily involved in the research. This project is jointly funded by the Physics and Chemistry Divisions in the Directorate for Mathematical and Physical Sciences and the Division of Molecular and Cellular Biosciences in the Directorate for Biological Sciences.