Single-molecule visualization of fast polymerase turnover in the bacterial replisome

  1. Jacob S Lewis
  2. Lisanne M Spenkelink
  3. Slobodan Jergic
  4. Elizabeth A Wood
  5. Enrico Monachino
  6. Nicholas P Horan
  7. Karl E Duderstadt
  8. Michael M Cox
  9. Andrew Robinson
  10. Nicholas E Dixon  Is a corresponding author
  11. Antoine M van Oijen  Is a corresponding author
  1. University of Wollongong, Australia
  2. University of Wisconsin-Madison, United States
  3. University of Groningen, Netherlands

Abstract

The Escherichia coli DNA replication machinery has been used as a road map to uncover design rules that enable DNA duplication with high efficiency and fidelity. Although the enzymatic activities of the replicative DNA Pol III are well understood, its dynamics within the replisome are not. Here we test the accepted view that the Pol III holoenzyme remains stably associated within the replisome. We use in vitro single-molecule assays with fluorescently labeled polymerases to demonstrate that the Pol III* complex (holoenzyme lacking the β2 sliding clamp), is rapidly exchanged during processive DNA replication. Nevertheless, the replisome is highly resistant to dilution in the absence of Pol III* in solution. We further show similar exchange in live cells containing labeled clamp loader and polymerase. These observations suggest a concentration-dependent exchange mechanism providing a balance between stability and plasticity, facilitating replacement of replisomal components dependent on their availability in the environment.

Article and author information

Author details

  1. Jacob S Lewis

    Centre for Medical and Molecular Bioscience, University of Wollongong, Wollongong, Australia
    Competing interests
    No competing interests declared.
  2. Lisanne M Spenkelink

    Centre for Medical and Molecular Bioscience, University of Wollongong, Wollongong, Australia
    Competing interests
    No competing interests declared.
  3. Slobodan Jergic

    Centre for Medical and Molecular Bioscience, University of Wollongong, Wollongong, Australia
    Competing interests
    No competing interests declared.
  4. Elizabeth A Wood

    Department of Biochemistry, University of Wisconsin-Madison, Wisconsin, United States
    Competing interests
    No competing interests declared.
  5. Enrico Monachino

    Centre for Medical and Molecular Bioscience, University of Wollongong, Wollongong, Australia
    Competing interests
    No competing interests declared.
  6. Nicholas P Horan

    Centre for Medical and Molecular Bioscience, University of Wollongong, Wollongong, Australia
    Competing interests
    No competing interests declared.
  7. Karl E Duderstadt

    Zernike Institute for Advanced Materials, University of Groningen, Groningen, Netherlands
    Competing interests
    No competing interests declared.
  8. Michael M Cox

    Department of Biochemistry, University of Wisconsin-Madison, Madison, United States
    Competing interests
    No competing interests declared.
  9. Andrew Robinson

    Centre for Medical and Molecular Bioscience, University of Wollongong, Wollongong, Australia
    Competing interests
    No competing interests declared.
  10. Nicholas E Dixon

    Centre for Medical and Molecular Bioscience, University of Wollongong, Wollongong, Australia
    For correspondence
    nickd@uow.edu.au
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5958-6945
  11. Antoine M van Oijen

    Centre for Medical and Molecular Bioscience, University of Wollongong, Wollongong, Australia
    For correspondence
    vanoijen@uow.edu.au
    Competing interests
    Antoine M van Oijen, Reviewing editor, <i>eLife</i>.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1794-5161

Funding

Australian Research Council (DP150100956)

  • Nicholas E Dixon
  • Antoine M van Oijen

Australian Research Council (FL140100027)

  • Antoine M van Oijen

National Institute of Health (GM32335)

  • Michael M Cox

Fundamenteel onderzoek der materie (12CMCE03)

  • Lisanne M Spenkelink

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Taekjip Ha, Johns Hopkins University School of Medicine, United States

Publication history

  1. Received: December 6, 2016
  2. Accepted: April 20, 2017
  3. Accepted Manuscript published: April 22, 2017 (version 1)
  4. Version of Record published: May 5, 2017 (version 2)

Copyright

© 2017, Lewis et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Jacob S Lewis
  2. Lisanne M Spenkelink
  3. Slobodan Jergic
  4. Elizabeth A Wood
  5. Enrico Monachino
  6. Nicholas P Horan
  7. Karl E Duderstadt
  8. Michael M Cox
  9. Andrew Robinson
  10. Nicholas E Dixon
  11. Antoine M van Oijen
(2017)
Single-molecule visualization of fast polymerase turnover in the bacterial replisome
eLife 6:e23932.
https://doi.org/10.7554/eLife.23932

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