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

Author

Listed:
  • 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)

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.

Suggested Citation

  • M. Charl Moolman & Sriram Tiruvadi Krishnan & Jacob W. J. Kerssemakers & Aafke van den Berg & Pawel Tulinski & Martin Depken & Rodrigo Reyes-Lamothe & David J. Sherratt & Nynke H. Dekker, 2014. "Slow unloading leads to DNA-bound β2-sliding clamp accumulation in live Escherichia coli cells," Nature Communications, Nature, vol. 5(1), pages 1-11, December.
    • Handle: RePEc:nat:natcom:v:5:y:2014:i:1:d:10.1038_ncomms6820
    DOI: 10.1038/ncomms6820
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    Cited by:

    1. Chen Zhang & Asha Mary Joseph & Laurent Casini & Justine Collier & Anjana Badrinarayanan & Suliana Manley, 2024. "Chromosome organization shapes replisome dynamics in Caulobacter crescentus," Nature Communications, Nature, vol. 15(1), pages 1-12, December.
    2. Agathe Couturier & Chloé Virolle & Kelly Goldlust & Annick Berne-Dedieu & Audrey Reuter & Sophie Nolivos & Yoshiharu Yamaichi & Sarah Bigot & Christian Lesterlin, 2023. "Real-time visualisation of the intracellular dynamics of conjugative plasmid transfer," Nature Communications, Nature, vol. 14(1), pages 1-15, December.
    3. Mareike Berger & Pieter Rein ten Wolde, 2022. "Robust replication initiation from coupled homeostatic mechanisms," Nature Communications, Nature, vol. 13(1), pages 1-13, December.

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