CtIP forms a tetrameric dumbbell-shaped particle which bridges complex DNA end structures for double-strand break repair

  1. Oliver J Wilkinson
  2. Alejandro Martín-González
  3. Haejoo Kang
  4. Sarah J Northall
  5. Dale B Wigley
  6. Fernando Moreno-Herrero
  7. Mark Simon Dillingham  Is a corresponding author
  1. University of Bristol, United Kingdom
  2. Centro Nacional de Biotecnologia, Consejo Superior de Investigaciones Cientificas, Spain
  3. Imperial College London, United Kingdom

Abstract

CtIP is involved in the resection of broken DNA during the S and G2 phases of the cell cycle for repair by recombination. Acting with the MRN complex, it plays a particularly important role in handling complex DNA end structures by localised nucleolytic processing of DNA termini in preparation for longer range resection. Here we show that human CtIP is a tetrameric protein adopting a dumbbell architecture in which DNA binding domains are connected by long coiled-coils. The protein complex binds two short DNA duplexes with high affinity and bridges DNA molecules in trans. DNA binding is potentiated by dephosphorylation and is not specific for DNA end structures per se. However, the affinity for linear DNA molecules is increased if the DNA terminates with complex structures including forked ssDNA overhangs and nucleoprotein conjugates. This work provides a biochemical and structural basis for the function of CtIP at complex DNA breaks.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files.

Article and author information

Author details

  1. Oliver J Wilkinson

    School of Biochemistry, University of Bristol, Bristol, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  2. Alejandro Martín-González

    Department of Macromolecular Structures, Centro Nacional de Biotecnologia, Consejo Superior de Investigaciones Cientificas, Madrid, Spain
    Competing interests
    The authors declare that no competing interests exist.
  3. Haejoo Kang

    Section of Structural Biology, Department of Medicine, Imperial College London, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  4. Sarah J Northall

    School of Biochemistry, University of Bristol, Bristol, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  5. Dale B Wigley

    Section of Structural Biology, Department of Medicine, Imperial College London, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0786-6726
  6. Fernando Moreno-Herrero

    Department of Macromolecular Structures, Centro Nacional de Biotecnologia, Consejo Superior de Investigaciones Cientificas, Madrid, Spain
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4083-1709
  7. Mark Simon Dillingham

    School of Biochemistry, University of Bristol, Bristol, United Kingdom
    For correspondence
    mark.dillingham@bristol.ac.uk
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4612-7141

Funding

Wellcome (100401/Z/12/Z)

  • Oliver J Wilkinson
  • Sarah J Northall
  • Mark Simon Dillingham

Cancer Research UK (C6913/A21608)

  • Haejoo Kang
  • Dale B Wigley

Spanish Ministry of Science (BFU2017-83794-P)

  • Fernando Moreno-Herrero

European Research Council (681299)

  • Fernando Moreno-Herrero

Spanish MINECO (BES-2015-071244)

  • Alejandro Martín-González

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

Copyright

© 2019, Wilkinson 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

  • 4,275
    views
  • 577
    downloads
  • 26
    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. Oliver J Wilkinson
  2. Alejandro Martín-González
  3. Haejoo Kang
  4. Sarah J Northall
  5. Dale B Wigley
  6. Fernando Moreno-Herrero
  7. Mark Simon Dillingham
(2019)
CtIP forms a tetrameric dumbbell-shaped particle which bridges complex DNA end structures for double-strand break repair
eLife 8:e42129.
https://doi.org/10.7554/eLife.42129

Share this article

https://doi.org/10.7554/eLife.42129

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.