Abstract

The essential Smc5/6 complex is required in response to replication stress and is best known for ensuring the fidelity of homologous recombination. Using single-molecule tracking in live fission yeast to investigate Smc5/6 chromatin association, we show that Smc5/6 is chromatin associated in unchallenged cells and this depends on the non-SMC protein Nse6. We define a minimum of two Nse6-dependent sub-pathways, one of which requires the BRCT-domain protein Brc1. Using defined mutants in genes encoding the core Smc5/6 complex subunits we show that the Nse3 double-stranded DNA binding activity and the arginine fingers of the two Smc5/6 ATPase binding sites are critical for chromatin association. Interestingly, disrupting the ssDNA binding activity at the hinge region does not prevent chromatin association but leads to elevated levels of gross chromosomal rearrangements during replication restart. This is consistent with a downstream function for ssDNA binding in regulating homologous recombination.

Data availability

Single molecule traces exported from GDSC SMLM plugin and used for analysis in SpotOn software are available via the Open Science Framework (osf.io/myxtr).

The following data sets were generated

Article and author information

Author details

  1. Thomas J Etheridge

    Genome Damage and Stability Centre, University of Sussex, Brighton, United Kingdom
    For correspondence
    t.etheridge@sussex.ac.uk
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8144-6917
  2. Desiree Villahermosa

    Genome Damage and Stability Centre, University of Sussex, Brighton, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  3. Eduard Campillo-Funollet

    Genome Damage and Stability Centre, University of Sussex, Brighton, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7021-1610
  4. Alex David Herbert

    Genome Damage and Stability Centre, University of Sussex, Brighton, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9843-9980
  5. Anja Irmisch

    Genome Damage and Stability Centre, University of Sussex, Brighton, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  6. Adam T Watson

    Genome Damage and Stability Centre, University of Sussex, Brighton, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  7. Hung Q Dang

    Genome Damage and Stability Centre, University of Sussex, Brighton, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1226-0235
  8. Mark A Osborne

    Department of Chemistry, University of Sussex, Brighton, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  9. Antony W Oliver

    Genome Damage and Stability Centre, University of Sussex, Brighton, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2912-8273
  10. Antony M Carr

    Genome Damage and Stability Centre, University of Sussex, Brighton, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2028-2389
  11. Johanne M Murray

    Genome Damage and Stability Centre, University of Sussex, Brighton, United Kingdom
    For correspondence
    j.m.murray@sussex.ac.uk
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9225-6289

Funding

Wellcome Trust (110047/Z/15/Z)

  • Antony M Carr

Medical Research Council (MR/P018955/1)

  • Antony W Oliver
  • Johanne M Murray

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

Reviewing Editor

  1. Wolf-Dietrich Heyer, University of California, Davis, United States

Version history

  1. Received: March 19, 2021
  2. Accepted: April 15, 2021
  3. Accepted Manuscript published: April 16, 2021 (version 1)
  4. Version of Record published: April 26, 2021 (version 2)

Copyright

© 2021, Etheridge 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. Thomas J Etheridge
  2. Desiree Villahermosa
  3. Eduard Campillo-Funollet
  4. Alex David Herbert
  5. Anja Irmisch
  6. Adam T Watson
  7. Hung Q Dang
  8. Mark A Osborne
  9. Antony W Oliver
  10. Antony M Carr
  11. Johanne M Murray
(2021)
Live-cell single-molecule tracking highlights requirements for stable Smc5/6 chromatin association in vivo
eLife 10:e68579.
https://doi.org/10.7554/eLife.68579

Share this article

https://doi.org/10.7554/eLife.68579

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