title: Single-Molecule Observation of DNA Replication Repair Pathways in E. coli.
abstract: The method of action of many antibiotics is to interfere with DNA replication-quinolones trap DNA gyrase and topoisomerase proteins onto DNA while metronidazole causes single- and double-stranded breaks in DNA. To understand how bacteria respond to these drugs, it is important to understand the repair processes utilised when DNA replication is blocked. We have used tandem lac operators inserted into the chromosome bound by fluorescently labelled lac repressors as a model protein block to replication in E. coli. We have used dual-colour, alternating-laser, single-molecule narrowfield microscopy to quantify the amount of operator at the block and simultaneously image fluorescently labelled DNA polymerase. We anticipate use of this system as a quantitative platform to study replication stalling and repair proteins.
authors.affiliation: Department of Physics and Biology, Biological Physical Sciences Institute, University of York, York, YO10 5DD, UK. adam.wollman@york.ac.uk.
authors.forename: Adam J M
authors.initials: AJ
authors.lastname: Wollman
authors.affiliation: Department of Physics and Biology, Biological Physical Sciences Institute, University of York, York, YO10 5DD, UK.
authors.forename: Aisha H
authors.initials: AH
authors.lastname: Syeda
authors.affiliation: Department of Physics and Biology, Biological Physical Sciences Institute, University of York, York, YO10 5DD, UK.
authors.forename: Peter
authors.initials: P
authors.lastname: McGlynn
authors.affiliation: Department of Physics and Biology, Biological Physical Sciences Institute, University of York, York, YO10 5DD, UK.
authors.forename: Mark C
authors.initials: MC
authors.lastname: Leake
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D004261:DNA Replication / Q000187:drug effects*; D004269:DNA, Bacterial / Q000096:biosynthesis / Q000187:drug effects* / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D004927:Escherichia coli Infections / Q000188:drug therapy* / Q000276:immunology / Q000382:microbiology; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D017930:Genes, Reporter; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007763:Lac Operon; D056968:Lac Repressors / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D057054:Molecular Imaging / Q000379:methods*
chemical_list: D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D056968:Lac Repressors; C539064:LacI protein, E coli; D008164:Luminescent Proteins; D004259:DNA-Directed DNA Polymerase
keywords: DNA repair; Fluorescent protein; In vivo imaging; Single-molecule; Super-resolution