title: Slow unloading leads to DNA-bound β2-sliding clamp accumulation in live Escherichia coli cells.
abstract: The ubiquitous sliding clamp facilitates processivity of the replicative polymerase and acts as a platform to recruit proteins involved in replication, recombination and repair. While the dynamics of the E. coli β2-sliding clamp have been characterized in vitro, its in vivo stoichiometry and dynamics remain unclear. To probe both β2-clamp dynamics and stoichiometry in live E. coli cells, we use custom-built microfluidics in combination with single-molecule fluorescence microscopy and photoactivated fluorescence microscopy. We quantify the recruitment, binding and turnover of β2-sliding clamps on DNA during replication. These quantitative in vivo results demonstrate that numerous β2-clamps in E. coli remain on the DNA behind the replication fork for a protracted period of time, allowing them to form a docking platform for other enzymes involved in DNA metabolism.
authors.affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience, Faculty of Applied Sciences, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands.
authors.forename: M Charl
authors.initials: MC
authors.lastname: Moolman
authors.affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience, Faculty of Applied Sciences, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands.
authors.forename: Sriram Tiruvadi
authors.initials: ST
authors.lastname: Krishnan
authors.affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience, Faculty of Applied Sciences, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands.
authors.forename: Jacob W J
authors.initials: JW
authors.lastname: Kerssemakers
authors.affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience, Faculty of Applied Sciences, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands.
authors.forename: Aafke
authors.initials: A
authors.lastname: van den Berg
authors.affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience, Faculty of Applied Sciences, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands.
authors.forename: Pawel
authors.initials: P
authors.lastname: Tulinski
authors.affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience, Faculty of Applied Sciences, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands.
authors.forename: Martin
authors.initials: M
authors.lastname: Depken
authors.affiliation: Department of Biology, McGill University, Montreal, Quebec, Canada H3G 0B1.
authors.forename: Rodrigo
authors.initials: R
authors.lastname: Reyes-Lamothe
authors.affiliation: Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK.
authors.forename: David J
authors.initials: DJ
authors.lastname: Sherratt
authors.affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience, Faculty of Applied Sciences, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands.
authors.forename: Nynke H
authors.initials: NH
authors.lastname: Dekker
mesh_terms: D004258:DNA Polymerase III / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair; D004261:DNA Replication; D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D046210:Microfluidic Analytical Techniques; D008856:Microscopy, Fluorescence; D011995:Recombination, Genetic; D059008:Time-Lapse Imaging
chemical_list: D004269:DNA, Bacterial; D004258:DNA Polymerase III