title: In vivo single-molecule imaging of bacterial DNA replication, transcription, and repair.
abstract: In vivo single-molecule experiments offer new perspectives on the behaviour of DNA binding proteins, from the molecular level to the length scale of whole bacterial cells. With technological advances in instrumentation and data analysis, fluorescence microscopy can detect single molecules in live cells, opening the doors to directly follow individual proteins binding to DNA in real time. In this review, we describe key technical considerations for implementing in vivo single-molecule fluorescence microscopy. We discuss how single-molecule tracking and quantitative super-resolution microscopy can be adapted to extract DNA binding kinetics, spatial distributions, and copy numbers of proteins, as well as stoichiometries of protein complexes. We highlight experiments which have exploited these techniques to answer important questions in the field of bacterial gene regulation and transcription, as well as chromosome replication, organisation and repair. Together, these studies demonstrate how single-molecule imaging is transforming our understanding of DNA-binding proteins in cells.
authors.affiliation: Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford OX1 3PU, United Kingdom.
authors.forename: Mathew
authors.initials: M
authors.lastname: Stracy
authors.affiliation: Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom; Department of Systems Biology, Harvard Medical School, Boston, MA 02138, USA.
authors.forename: Stephan
authors.initials: S
authors.lastname: Uphoff
authors.affiliation: Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford OX1 3PU, United Kingdom.
authors.forename: Federico
authors.initials: F
authors.lastname: Garza de Leon
authors.affiliation: Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford OX1 3PU, United Kingdom. Electronic address: kapanidis@physics.ox.ac.uk.
authors.forename: Achillefs N
authors.initials: AN
authors.lastname: Kapanidis
mesh_terms: D002876:Chromosomes, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair; D004261:DNA Replication; D004269:DNA, Bacterial / Q000096:biosynthesis* / Q000235:genetics*; D057054:Molecular Imaging / Q000379:methods*; D014158:Transcription, Genetic
chemical_list: D004269:DNA, Bacterial
keywords: Bacteriology; Chromosome organisation; DNA repair; DNA replication; Fluorescence microscopy; Single-molecule fluorescence; Single-particle tracking; Super-resolution microscopy; Transcription