title: Single-molecule DNA repair in live bacteria.
abstract: Cellular DNA damage is reversed by balanced repair pathways that avoid accumulation of toxic intermediates. Despite their importance, the organization of DNA repair pathways and the function of repair enzymes in vivo have remained unclear because of the inability to directly observe individual reactions in living cells. Here, we used photoactivation, localization, and tracking in live Escherichia coli to directly visualize single fluorescent labeled DNA polymerase I (Pol) and ligase (Lig) molecules searching for DNA gaps and nicks, performing transient reactions, and releasing their products. Our general approach provides enzymatic rates and copy numbers, substrate-search times, diffusion characteristics, and the spatial distribution of reaction sites, at the single-cell level, all in one measurement. Single repair events last 2.1 s (Pol) and 2.5 s (Lig), respectively. Pol and Lig activities increased fivefold over the basal level within minutes of DNA methylation damage; their rates were limited by upstream base excision repair pathway steps. Pol and Lig spent >80% of their time searching for free substrates, thereby minimizing both the number and lifetime of toxic repair intermediates. We integrated these single-molecule observations to generate a quantitative, systems-level description of a model repair pathway in vivo.
authors: Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford OX1 3PU, United Kingdom. s.uphoff1@physics.ox.ac.uk
authors: Stephan
authors: S
authors: Uphoff
authors: Rodrigo
authors: R
authors: Reyes-Lamothe
authors: Federico
authors: F
authors: Garza de Leon
authors: David J
authors: DJ
authors: Sherratt
authors: Achillefs N
authors: AN
authors: Kapanidis
mesh_terms: D001419:Bacteria / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004249:DNA Damage; D019175:DNA Methylation; D004260:DNA Repair; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004058:Diffusion; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D013379:Substrate Specificity
chemical_list: D004259:DNA-Directed DNA Polymerase
keywords: DNA damage response; cytosolic diffusion; protein-DNA interaction; single-molecule tracking; super-resolution microscopy
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