title: A dynamic DNA-repair complex observed by correlative single-molecule nanomanipulation and fluorescence.
abstract: We characterize in real time the composition and catalytic state of the initial Escherichia coli transcription-coupled repair (TCR) machinery by using correlative single-molecule methods. TCR initiates when RNA polymerase (RNAP) stalled by a lesion is displaced by the Mfd DNA translocase, thus giving repair components access to the damage. We previously used DNA nanomanipulation to obtain a nanomechanical readout of protein-DNA interactions during TCR initiation. Here we correlate this signal with simultaneous single-molecule fluorescence imaging of labeled components (RNAP, Mfd or RNA) to monitor the composition and localization of the complex. Displacement of stalled RNAP by Mfd results in loss of nascent RNA but not of RNAP, which remains associated with Mfd as a long-lived complex on the DNA. This complex translocates at ∼4 bp/s along the DNA, in a manner determined by the orientation of the stalled RNAP on the DNA.
authors.affiliation: Institut Jacques Monod, Centre National de la Recherche Scientifique, University of Paris Diderot and Sorbonne Paris Cité, Paris, France.
authors.forename: Evan T
authors.initials: ET
authors.lastname: Graves
authors.affiliation: Institut Jacques Monod, Centre National de la Recherche Scientifique, University of Paris Diderot and Sorbonne Paris Cité, Paris, France.
authors.forename: Camille
authors.initials: C
authors.lastname: Duboc
authors.affiliation: Institut Jacques Monod, Centre National de la Recherche Scientifique, University of Paris Diderot and Sorbonne Paris Cité, Paris, France.
authors.forename: Jun
authors.initials: J
authors.lastname: Fan
authors.affiliation: Institut Jacques Monod, Centre National de la Recherche Scientifique, University of Paris Diderot and Sorbonne Paris Cité, Paris, France.
authors.forename: François
authors.initials: F
authors.lastname: Stransky
authors.affiliation: Institut Jacques Monod, Centre National de la Recherche Scientifique, University of Paris Diderot and Sorbonne Paris Cité, Paris, France.
authors.forename: Mathieu
authors.initials: M
authors.lastname: Leroux-Coyau
authors.affiliation: Institut Jacques Monod, Centre National de la Recherche Scientifique, University of Paris Diderot and Sorbonne Paris Cité, Paris, France.
authors.forename: Terence R
authors.initials: TR
authors.lastname: Strick
mesh_terms: D004247:DNA / Q000378:metabolism; D004260:DNA Repair; D045643:DNA Repair Enzymes / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000502:physiology*; D007700:Kinetics; D008856:Microscopy, Fluorescence / Q000379:methods; D009097:Multienzyme Complexes / Q000378:metabolism; D061848:Optical Imaging / Q000379:methods; D011485:Protein Binding; D055503:Protein Multimerization
chemical_list: D009097:Multienzyme Complexes; D004247:DNA; D045643:DNA Repair Enzymes