Visualizing Protein-DNA Interactions in Live Bacterial Cells Using Photoactivated Single-molecule Tracking
Visualizing Protein-DNA Interactions in Live Bacterial Cells Using Photoactivated Single-molecule Tracking
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DOI：
10.3791/51177
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发表时间：
2014-03-01
影响因子：
1.2
通讯作者：
Kapanidis, Achillefs N.
中科院分区：
文献类型：
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作者：
Uphoff, Stephan;Sherratt, David J.;Kapanidis, Achillefs N.
蛋白质- dna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是由识别特定DNA结构或序列的DNA结合蛋白控制的。体外实验已经帮助生成了许多类型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仍然知之甚少。我们最近介绍了一种利用光激活定位显微镜（PALM）结合单分子跟踪来定量活大肠杆菌细胞dna修复活性的方法。我们的一般方法通过与染色体相关的单个蛋白质的流动性变化来识别单个dna结合事件。结合分子的比例在单细胞水平上为蛋白质活性和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，并演示了样品制备、数据采集和数据分析程序。
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.