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.

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.

Metrics

  • 1,881
    views
  • 298
    downloads
  • 24
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  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

Further reading

    1. Chromosomes and Gene Expression
    2. Genetics and Genomics
    Omid Gholamalamdari, Tom van Schaik ... Andrew S Belmont
    Research Article

    Models of nuclear genome organization often propose a binary division into active versus inactive compartments yet typically overlook nuclear bodies. Here, we integrated analysis of sequencing and image-based data to compare genome organization in four human cell types relative to three different nuclear locales: the nuclear lamina, nuclear speckles, and nucleoli. Although gene expression correlates mostly with nuclear speckle proximity, DNA replication timing correlates with proximity to multiple nuclear locales. Speckle attachment regions emerge as DNA replication initiation zones whose replication timing and gene composition vary with their attachment frequency. Most facultative LADs retain a partially repressed state as iLADs, despite their positioning in the nuclear interior. Knock out of two lamina proteins, Lamin A and LBR, causes a shift of H3K9me3-enriched LADs from lamina to nucleolus, and a reciprocal relocation of H3K27me3-enriched partially repressed iLADs from nucleolus to lamina. Thus, these partially repressed iLADs appear to compete with LADs for nuclear lamina attachment with consequences for replication timing. The nuclear organization in adherent cells is polarized with nuclear bodies and genomic regions segregating both radially and relative to the equatorial plane. Together, our results underscore the importance of considering genome organization relative to nuclear locales for a more complete understanding of the spatial and functional organization of the human genome.

    1. Chromosomes and Gene Expression
    Ashwin Govindan, Nicholas K Conrad
    Research Article

    O-GlcNAcylation is the reversible post-translational addition of β-N-acetylglucosamine to serine and threonine residues of nuclear and cytoplasmic proteins. It plays an important role in several cellular processes through the modification of thousands of protein substrates. O-GlcNAcylation in humans is mediated by a single essential enzyme, O-GlcNAc transferase (OGT). OGT, together with the sole O-GlcNAcase OGA, form an intricate feedback loop to maintain O-GlcNAc homeostasis in response to changes in cellular O-GlcNAc using a dynamic mechanism involving nuclear retention of its fourth intron. However, the molecular mechanism of this dynamic regulation remains unclear. Using an O-GlcNAc responsive GFP reporter cell line, we identify SFSWAP, a poorly characterized splicing factor, as a trans-acting factor regulating OGT intron detention. We show that SFSWAP is a global regulator of retained intron splicing and exon skipping that primarily acts as a negative regulator of splicing. In contrast, knockdown of SFSWAP leads to reduced inclusion of a ‘decoy exon’ present in the OGT retained intron which may mediate its role in OGT intron detention. Global analysis of decoy exon inclusion in SFSWAP and UPF1 double knockdown cells indicate altered patterns of decoy exon usage. Together, these data indicate a role for SFSWAP as a global negative regulator of pre-mRNA splicing and positive regulator of intron retention.