Characterization of DNA/Protein Interactions at the Single Molecule Level

单分子水平 DNA/蛋白质相互作用的表征

• 批准号: 0239986
• 负责人: Neal Woodbury
• 金额: $ 36万
• 依托单位: Arizona State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0239986&HistoricalAwards=false
• 关键词: Characterization; DNA; Protein; Interactions; Single

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.

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）通过在两个或多个不相邻位点结合的蛋白质复合物在 DNA 中生成环，3）通过将碱基拉出堆叠构型来破坏碱基堆叠的蛋白质相互作用 邻近的基地。 这些过程中的每一个都将在群体分布水平（平衡状态下DNA/蛋白质复合物的不同几何形状或构象的相对群体）和动力学（构象或几何形状之间相互转换所需的时间，同样主要在平衡状态）方面进行探索。 单分子荧光光谱非常适合此类测量。 主要重点将放在三个 DNA/蛋白质系统上。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 与 DNA 结合域的研究，重点是寻找结合位点和导致 DNA 环的蛋白质/DNA 相互作用。一些工作将在一个最终的、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 模板和构建体。 该项目将与大学现有的 IGERT 项目密切相关，本科生将大量参与该研究。 该项目由数学和物理科学局物理和化学司以及生物科学局分子和细胞生物科学司共同资助。