title: Visualizing protein-DNA interactions in live bacterial cells using photoactivated single-molecule tracking.
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.
authors.affiliation: Microbiology Unit, Department of Biochemistry, University of Oxford; Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford; stephan.uphoff@bioch.ox.ac.uk.
authors.forename: Stephan
authors.initials: S
authors.lastname: Uphoff
authors.affiliation: Microbiology Unit, Department of Biochemistry, University of Oxford.
authors.forename: David J
authors.initials: DJ
authors.lastname: Sherratt
authors.affiliation: Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford.
authors.forename: Achillefs N
authors.initials: AN
authors.lastname: Kapanidis
mesh_terms: D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004256:DNA Polymerase I / Q000737:chemistry / Q000378:metabolism; D004260:DNA Repair; D004273:DNA, Neoplasm / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D008856:Microscopy, Fluorescence / Q000379:methods; D038761:Photobleaching; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D004273:DNA, Neoplasm; D005456:Fluorescent Dyes; D011506:Proteins; D004247:DNA; D004256:DNA Polymerase I