1. Biochemistry and Chemical Biology
  2. Structural Biology and Molecular Biophysics
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Synergy between RecBCD subunits is essential for efficient DNA unwinding

  1. Rani Zananiri
  2. Omri Malik
  3. Sergei Rudnizky
  4. Vera Gaydar
  5. Roman Kreiserman
  6. Arnon Henn
  7. Ariel Kaplan  Is a corresponding author
  1. Technion - Israel Institute of Technology, Israel
Research Article
  • Cited 4
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Cite this article as: eLife 2019;8:e40836 doi: 10.7554/eLife.40836

Abstract

The subunits of the bacterial RecBCD act in coordination, rapidly and processively unwinding DNA at the site of a double strand break. RecBCD is able to displace DNA binding proteins, suggesting that it generates high forces, but the specific role of each subunit in the force generation is unclear. Here, we present a novel optical tweezers assay that allows monitoring the activity of RecBCD's individual subunits, when they are part of an intact full complex. We show that RecBCD and its subunits are able to generate forces up to 25-40 pN without a significant effect on their velocity. Moreover, the isolated RecD translocates fast, but is a weak helicase with limited processivity. Experiments at a broad range of [ATP] and forces suggest that RecD unwinds DNA as a Brownian ratchet, rectified by ATP binding, and that the presence of the other subunits shifts the ratchet equilibrium towards the post-translocation state.

Data availability

All data generated during this study have been deposited in Dryad under accession code doi:10.5061/dryad.jb10510

The following data sets were generated

Article and author information

Author details

  1. Rani Zananiri

    Faculty of Biology, Technion - Israel Institute of Technology, Haifa, Israel
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0094-4197
  2. Omri Malik

    Faculty of Biology, Technion - Israel Institute of Technology, Haifa, Israel
    Competing interests
    The authors declare that no competing interests exist.
  3. Sergei Rudnizky

    Faculty of Biology, Technion - Israel Institute of Technology, Haifa, Israel
    Competing interests
    The authors declare that no competing interests exist.
  4. Vera Gaydar

    Faculty of Biology, Technion - Israel Institute of Technology, Haifa, Israel
    Competing interests
    The authors declare that no competing interests exist.
  5. Roman Kreiserman

    Faculty of Biology, Technion - Israel Institute of Technology, Haifa, Israel
    Competing interests
    The authors declare that no competing interests exist.
  6. Arnon Henn

    Faculty of Biology, Technion - Israel Institute of Technology, Haifa, Israel
    Competing interests
    The authors declare that no competing interests exist.
  7. Ariel Kaplan

    Faculty of Biology, Technion - Israel Institute of Technology, Haifa, Israel
    For correspondence
    akaplanz@technion.ac.il
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9731-6962

Funding

Israel Science Foundation (1782/17)

  • Ariel Kaplan

Israel Centers of Research Excellence (1902/12)

  • Ariel Kaplan

Elyhau Pen Research Fund

  • Ariel Kaplan

Israel Science Foundation (1403705/11)

  • Arnon Henn

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: August 6, 2018
  2. Accepted: January 1, 2019
  3. Accepted Manuscript published: January 2, 2019 (version 1)
  4. Version of Record published: January 18, 2019 (version 2)

Copyright

© 2019, Zananiri 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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