Visualizing protein-DNA interactions in live bacterial cells using photoactivated single-molecule tracking

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Abstract

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. © JoVE 2006-2014. All Rights Reserved.

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Uphoff, S., Sherratt, D. J., & Kapanidis, A. N. (2014). Visualizing protein-DNA interactions in live bacterial cells using photoactivated single-molecule tracking. Journal of Visualized Experiments, (85). https://doi.org/10.3791/51177

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