Uphoff Stephan, Sherratt David J, Kapanidis Achillefs N
Microbiology Unit, Department of Biochemistry, University of Oxford; Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford;
Microbiology Unit, Department of Biochemistry, University of Oxford.
J Vis Exp. 2014 Mar 10(85):51177. doi: 10.3791/51177.
Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome organization are governed by DNA-binding proteins that recognize specific DNA structures or sequences. In vitro experiments have helped to generate detailed models for the function of many types of DNA-binding proteins, yet, the exact mechanisms of these processes and their organization in the complex environment of the living cell remain far less understood. We recently introduced a method for quantifying DNA-repair activities in live Escherichia coli cells using Photoactivated Localization Microscopy (PALM) combined with single-molecule tracking. Our general approach identifies individual DNA-binding events by the change in the mobility of a single protein upon association with the chromosome. The fraction of bound molecules provides a direct quantitative measure for the protein activity and abundance of substrates or binding sites at the single-cell level. Here, we describe the concept of the method and demonstrate sample preparation, data acquisition, and data analysis procedures.
蛋白质与DNA的相互作用是许多基本细胞过程的核心。例如，DNA复制、转录、修复和染色体组织都由识别特定DNA结构或序列的DNA结合蛋白控制。体外实验有助于生成许多类型DNA结合蛋白功能的详细模型，然而，这些过程的确切机制及其在活细胞复杂环境中的组织方式仍知之甚少。我们最近引入了一种方法，使用光活化定位显微镜（PALM）结合单分子追踪来量化活的大肠杆菌细胞中的DNA修复活性。我们的一般方法通过单个蛋白质与染色体结合时迁移率的变化来识别单个DNA结合事件。结合分子的比例为单细胞水平上蛋白质活性以及底物或结合位点的丰度提供了直接的定量测量。在这里，我们描述了该方法的概念，并展示了样品制备、数据采集和数据分析程序。
