DNA-PK promotes DNA end resection at DNA double strand breaks in G0 cells

  1. Faith C Fowler
  2. Bo-Ruei Chen
  3. Nicholas Zolnerowich
  4. Wei Wu
  5. Raphael Pavani
  6. Jacob Paiano
  7. Chelsea Peart
  8. Zulong Chen
  9. André Nussenzweig
  10. Barry P Sleckman  Is a corresponding author
  11. Jessica K Tyler  Is a corresponding author
  1. Weill Cornell Medicine, United States
  2. University of Alabama at Birmingham, United States
  3. National Cancer Institute, United States

Abstract

DNA double-strand break (DSB) repair by homologous recombination is confined to the S and G2 phases of the cell cycle partly due to 53BP1 antagonizing DNA end resection in G1 phase and non-cycling quiescent (G0) cells where DSBs are predominately repaired by non-homologous end joining (NHEJ). Unexpectedly, we uncovered extensive MRE11- and CtIP-dependent DNA end resection at DSBs in G0 murine and human cells. A whole genome CRISPR/Cas9 screen revealed the DNA-dependent kinase (DNA-PK) complex as a key factor in promoting DNA end resection in G0 cells. In agreement, depletion of FBXL12, which promotes ubiquitylation and removal of the KU70/KU80 subunits of DNA-PK from DSBs, promotes even more extensive resection in G0 cells. In contrast, a requirement for DNA-PK in promoting DNA end resection in proliferating cells at the G1 or G2 phase of the cell cycle was not observed. Our findings establish that DNA-PK uniquely promotes DNA end resection in G0, but not in G1 or G2 phase cells, which has important implications for DNA DSB repair in quiescent cells.

Data availability

Sequencing data have been deposited in GEO under accession codesGSE186087

The following data sets were generated

Article and author information

Author details

  1. Faith C Fowler

    Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7180-8141
  2. Bo-Ruei Chen

    Department of Medicine, University of Alabama at Birmingham, Birmingham, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6404-2099
  3. Nicholas Zolnerowich

    Laboratory of Genome Integrity, National Cancer Institute, Bethesda, United States
    Competing interests
    No competing interests declared.
  4. Wei Wu

    Laboratory of Genome Integrity, National Cancer Institute, Bethesda, United States
    Competing interests
    No competing interests declared.
  5. Raphael Pavani

    Laboratory of Genome Integrity, National Cancer Institute, Bethesda, United States
    Competing interests
    No competing interests declared.
  6. Jacob Paiano

    Laboratory of Genome Integrity, National Cancer Institute, Bethesda, United States
    Competing interests
    No competing interests declared.
  7. Chelsea Peart

    Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York, United States
    Competing interests
    No competing interests declared.
  8. Zulong Chen

    Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York, United States
    Competing interests
    No competing interests declared.
  9. André Nussenzweig

    Laboratory of Genome Integrity, National Cancer Institute, Bethesda, United States
    Competing interests
    No competing interests declared.
  10. Barry P Sleckman

    Department of Medicine, University of Alabama at Birmingham, Birmingham, United States
    For correspondence
    bps@uab.edu
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8295-4462
  11. Jessica K Tyler

    Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York, United States
    For correspondence
    jet2021@med.cornell.edu
    Competing interests
    Jessica K Tyler, Senior editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9765-1659

Funding

NIH Office of the Director (R35 GM139816)

  • Jessica K Tyler

NIH Office of the Director (RO1 CA95641)

  • Jessica K Tyler

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

Publication history

  1. Received: October 14, 2021
  2. Preprint posted: October 21, 2021 (view preprint)
  3. Accepted: May 6, 2022
  4. Accepted Manuscript published: May 16, 2022 (version 1)
  5. Version of Record published: May 20, 2022 (version 2)

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

  • 1,727
    Page views
  • 462
    Downloads
  • 2
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

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. Faith C Fowler
  2. Bo-Ruei Chen
  3. Nicholas Zolnerowich
  4. Wei Wu
  5. Raphael Pavani
  6. Jacob Paiano
  7. Chelsea Peart
  8. Zulong Chen
  9. André Nussenzweig
  10. Barry P Sleckman
  11. Jessica K Tyler
(2022)
DNA-PK promotes DNA end resection at DNA double strand breaks in G0 cells
eLife 11:e74700.
https://doi.org/10.7554/eLife.74700

Further reading

    1. Chromosomes and Gene Expression
    2. Genetics and Genomics
    Anil Kumar Vijjamarri, Xiao Niu ... Alan G Hinnebusch
    Research Article

    Degradation of most yeast mRNAs involves decapping by Dcp1/Dcp2. DEAD-box protein Dhh1 has been implicated as an activator of decapping, in coupling codon non-optimality to enhanced degradation, and as a translational repressor, but its functions in cells are incompletely understood. RNA-Seq analyses coupled with CAGE sequencing of all capped mRNAs revealed increased abundance of hundreds of mRNAs in dcp2Δ cells that appears to result directly from impaired decapping rather than elevated transcription. Interestingly, only a subset of mRNAs requires Dhh1 for targeting by Dcp2, and also generally requires the other decapping activators Pat1, Edc3 or Scd6; whereas most of the remaining transcripts utilize NMD factors for Dcp2-mediated turnover. Neither inefficient translation initiation nor stalled elongation appears to be a major driver of Dhh1-enhanced mRNA degradation. Surprisingly, ribosome profiling revealed that dcp2Δ confers widespread changes in relative translational efficiencies (TEs) that generally favor well-translated mRNAs. Because ribosome biogenesis is reduced while capped mRNA abundance is increased by dcp2&Delta, we propose that an increased ratio of mRNA to ribosomes increases competition among mRNAs for limiting ribosomes to favor efficiently translated mRNAs in dcp2Δ cells. Interestingly, genes involved in respiration or utilization of alternative carbon or nitrogen sources are up-regulated, and both mitochondrial function and cell filamentation are elevated in dcp2Δ cells, suggesting that decapping sculpts gene expression post-transcriptionally to fine-tune metabolic pathways and morphological transitions according to nutrient availability.

    1. Chromosomes and Gene Expression
    2. Genetics and Genomics
    Isabella Horton, Conor J Kelly ... Edward B Chuong
    Research Article Updated

    Regulatory networks underlying innate immunity continually face selective pressures to adapt to new and evolving pathogens. Transposable elements (TEs) can affect immune gene expression as a source of inducible regulatory elements, but the significance of these elements in facilitating evolutionary diversification of innate immunity remains largely unexplored. Here, we investigated the mouse epigenomic response to type II interferon (IFN) signaling and discovered that elements from a subfamily of B2 SINE (B2_Mm2) contain STAT1 binding sites and function as IFN-inducible enhancers. CRISPR deletion experiments in mouse cells demonstrated that a B2_Mm2 element has been co-opted as an enhancer driving IFN-inducible expression of Dicer1. The rodent-specific B2 SINE family is highly abundant in the mouse genome and elements have been previously characterized to exhibit promoter, insulator, and non-coding RNA activity. Our work establishes a new role for B2 elements as inducible enhancer elements that influence mouse immunity, and exemplifies how lineage-specific TEs can facilitate evolutionary turnover and divergence of innate immune regulatory networks.