1. Physics of Living Systems
  2. Structural Biology and Molecular Biophysics
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Spatial and temporal organization of RecA in the Escherichia coli DNA-damage response

  1. Harshad Ghodke
  2. Bishnu P Paudel
  3. Jacob S Lewis
  4. Slobodan Jergic
  5. Kamya Gopal
  6. Zachary J Romero
  7. Elizabeth A Wood
  8. Roger Woodgate
  9. Michael M Cox
  10. Antoine M van Oijen  Is a corresponding author
  1. University of Wollongong, Australia
  2. University of Wisconsin-Madison, United States
  3. National Institutes of Health, United States
Research Article
  • Cited 20
  • Views 3,350
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Cite this article as: eLife 2019;8:e42761 doi: 10.7554/eLife.42761

Abstract

The RecA protein orchestrates the cellular response to DNA damage via its multiple roles in the bacterial SOS response. Lack of tools that provide unambiguous access to the various RecA states within the cell have prevented understanding of the spatial and temporal changes in RecA structure/function that underlie control of the damage response. Here, we develop a monomeric C-terminal fragment of the l repressor as a novel fluorescent probe that specifically interacts with RecA filaments on single-stranded DNA (RecA*). Single-molecule imaging techniques in live cells demonstrate that RecA is largely sequestered in storage structures during normal metabolism. Upon DNA damage, the storage structures dissolve and the cytosolic pool of RecA rapidly nucleates to form early SOS-signaling complexes, maturing into DNA-bound RecA bundles at later time points. Both before and after SOS induction, RecA* largely appears at locations distal from replisomes. Upon completion of repair, RecA storage structures reform.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Codes used for analysis are publicly available (in GitHub as described in previous publications). Scripts using these codes are also now provided in this submission as Source Code files for the relevant figures.

Article and author information

Author details

  1. Harshad Ghodke

    School of Chemistry and Molecular Bioscience, University of Wollongong, Wollongong, Australia
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6628-876X
  2. Bishnu P Paudel

    School of Chemistry and Molecular Bioscience, University of Wollongong, Wollongong, Australia
    Competing interests
    The authors declare that no competing interests exist.
  3. Jacob S Lewis

    School of Chemistry and Molecular Bioscience, University of Wollongong, Wollongong, Australia
    Competing interests
    The authors declare that no competing interests exist.
  4. Slobodan Jergic

    School of Chemistry and Molecular Bioscience, University of Wollongong, Wollongong, Australia
    Competing interests
    The authors declare that no competing interests exist.
  5. Kamya Gopal

    Department of Biochemistry, University of Wisconsin-Madison, Madison, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Zachary J Romero

    Department of Biochemistry, University of Wisconsin-Madison, Madison, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Elizabeth A Wood

    Department of Biochemistry, University of Wisconsin-Madison, Madison, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Roger Woodgate

    Laboratory of Genomic Integrity, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United States
    Competing interests
    The authors declare that no competing interests exist.
  9. Michael M Cox

    Department of Biochemistry, University of Wisconsin-Madison, Madison, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3606-5722
  10. Antoine M van Oijen

    School of Chemistry and Molecular Bioscience, University of Wollongong, Wollongong, Australia
    For correspondence
    vanoijen@uow.edu.au
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1794-5161

Funding

Australian Research Council (DP150100956)

  • Antoine M van Oijen

National Institutes of Health (GM32335)

  • Michael M Cox

Australian Research Council (FL140100027)

  • Antoine M van Oijen

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, United States

Publication history

  1. Received: October 18, 2018
  2. Accepted: January 22, 2019
  3. Accepted Manuscript published: February 5, 2019 (version 1)
  4. Version of Record published: February 5, 2019 (version 2)

Copyright

This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

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