Slow unloading leads to DNA-bound β2-sliding clamp accumulation in live Escherichia coli cells

M. Charl Moolman, Sriram Tiruvadi Krishnan, Jacob W. J. Kerssemakers, Aafke van den Berg, Pawel Tulinski, Martin Depken, Rodrigo Reyes-Lamothe, David J. Sherratt and Nynke H. Dekker ()
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M. Charl Moolman: Kavli Institute of Nanoscience, Faculty of Applied Sciences, Delft University of Technology
Sriram Tiruvadi Krishnan: Kavli Institute of Nanoscience, Faculty of Applied Sciences, Delft University of Technology
Jacob W. J. Kerssemakers: Kavli Institute of Nanoscience, Faculty of Applied Sciences, Delft University of Technology
Aafke van den Berg: Kavli Institute of Nanoscience, Faculty of Applied Sciences, Delft University of Technology
Pawel Tulinski: Kavli Institute of Nanoscience, Faculty of Applied Sciences, Delft University of Technology
Martin Depken: Kavli Institute of Nanoscience, Faculty of Applied Sciences, Delft University of Technology
Rodrigo Reyes-Lamothe: McGill University
David J. Sherratt: University of Oxford
Nynke H. Dekker: Kavli Institute of Nanoscience, Faculty of Applied Sciences, Delft University of Technology

Nature Communications, 2014, vol. 5, issue 1, 1-11

Abstract: 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.

Date: 2014
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DOI: 10.1038/ncomms6820

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