Sae2/CtIP prevents R-loop accumulation in eukaryotic cells

  1. Nodar Makharashvili
  2. Sucheta Arora
  3. Yizhi Yin
  4. Qiong Fu
  5. Xuemei Wen
  6. Ji-Hoon Lee
  7. Chung-Hsuan Kao
  8. Justin WC Leung
  9. Kyle M Miller
  10. Tanya T Paull  Is a corresponding author
  1. The University of Texas at Austin, United States
  2. National Institutes of Health, United States
  3. University of Arkansas for Medical Sciences, United States

Abstract

The Sae2/CtIP protein is required for efficient processing of DNA double-strand breaks that initiate homologous recombination in eukaryotic cells. Sae2/CtIP is also important for survival of single-stranded Top1-induced lesions and CtIP is known to associate directly with transcription-associated complexes in mammalian cells. Here we investigate the role of Sae2/CtIP at single-strand lesions in budding yeast and in human cells and find that depletion of Sae2/CtIP promotes the accumulation of stalled RNA polymerase and RNA-DNA hybrids at sites of highly expressed genes. Overexpression of the RNA-DNA helicase Senataxin suppresses DNA damage sensitivity and R-loop accumulation in Sae2/CtIP-deficient cells, and a catalytic mutant of CtIP fails to complement this sensitivity, indicating a role for CtIP nuclease activity in the repair process. Based on this evidence, we propose that R-loop processing by 5' flap endonucleases is a necessary step in the stabilization and removal of nascent R-loop initiating structures in eukaryotic cells.

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting files. Sequencing data has been deposited in GEO (accession number GSE122782).

The following data sets were generated

Article and author information

Author details

  1. Nodar Makharashvili

    Department of Molecular Biosciences, The University of Texas at Austin, Austin, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Sucheta Arora

    Department of Molecular Biosciences, The University of Texas at Austin, Austin, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Yizhi Yin

    Department of Molecular Biosciences, The University of Texas at Austin, Austin, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Qiong Fu

    Gastrointestinal Malignancy Section, Thoracic and Gastrointestinal Oncology Branch, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Xuemei Wen

    Department of Molecular Biosciences, The University of Texas at Austin, Austin, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Ji-Hoon Lee

    Department of Molecular Biosciences, The University of Texas at Austin, Austin, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7387-935X
  7. Chung-Hsuan Kao

    Department of Molecular Biosciences, The University of Texas at Austin, Austin, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Justin WC Leung

    Department of Radiation Oncology, University of Arkansas for Medical Sciences, Little Rock, United States
    Competing interests
    The authors declare that no competing interests exist.
  9. Kyle M Miller

    Department of Molecular Biosciences, The University of Texas at Austin, Austin, United States
    Competing interests
    The authors declare that no competing interests exist.
  10. Tanya T Paull

    Department of Molecular Biosciences, The University of Texas at Austin, Austin, United States
    For correspondence
    tpaull@utexas.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2991-651X

Funding

Howard Hughes Medical Institute

  • Sucheta Arora
  • Qiong Fu
  • Xuemei Wen
  • Ji-Hoon Lee
  • Chung-Hsuan Kao
  • Tanya T Paull

Cancer Prevention and Research Institute of Texas

  • Nodar Makharashvili
  • Yizhi Yin

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

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.

Metrics

  • 3,809
    views
  • 604
    downloads
  • 64
    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. Nodar Makharashvili
  2. Sucheta Arora
  3. Yizhi Yin
  4. Qiong Fu
  5. Xuemei Wen
  6. Ji-Hoon Lee
  7. Chung-Hsuan Kao
  8. Justin WC Leung
  9. Kyle M Miller
  10. Tanya T Paull
(2018)
Sae2/CtIP prevents R-loop accumulation in eukaryotic cells
eLife 7:e42733.
https://doi.org/10.7554/eLife.42733

Share this article

https://doi.org/10.7554/eLife.42733

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.