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

  • 1,245
    Page views
  • 394
    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
  1. Further reading

Further reading

    1. Chromosomes and Gene Expression
    2. Immunology and Inflammation
    Allison R Wagner, Chi G Weindel ... Kristin L Patrick
    Research Article Updated

    To mount a protective response to infection while preventing hyperinflammation, gene expression in innate immune cells must be tightly regulated. Despite the importance of pre-mRNA splicing in shaping the proteome, its role in balancing immune outcomes remains understudied. Transcriptomic analysis of murine macrophage cell lines identified Serine/Arginine Rich Splicing factor 6 (SRSF6) as a gatekeeper of mitochondrial homeostasis. SRSF6-dependent orchestration of mitochondrial health is directed in large part by alternative splicing of the pro-apoptosis pore-forming protein BAX. Loss of SRSF6 promotes accumulation of BAX-κ, a variant that sensitizes macrophages to undergo cell death and triggers upregulation of interferon stimulated genes through cGAS sensing of cytosolic mitochondrial DNA. Upon pathogen sensing, macrophages regulate SRSF6 expression to control the liberation of immunogenic mtDNA and adjust the threshold for entry into programmed cell death. This work defines BAX alternative splicing by SRSF6 as a critical node not only in mitochondrial homeostasis but also in the macrophage’s response to pathogens.

    1. Chromosomes and Gene Expression
    2. Genetics and Genomics
    Evgeniya N Andreyeva, Alexander V Emelyanov ... Dmitry V Fyodorov
    Research Article

    Asynchronous replication of chromosome domains during S phase is essential for eukaryotic genome function, but the mechanisms establishing which domains replicate early versus late in different cell types remain incompletely understood. Intercalary heterochromatin domains replicate very late in both diploid chromosomes of dividing cells and in endoreplicating polytene chromosomes where they are also underrelicated. Drosophila SNF2-related factor SUUR imparts locus-specific underreplication of polytene chromosomes. SUUR negatively regulates DNA replication fork progression; however, its mechanism of action remains obscure. Here we developed a novel method termed MS-Enabled Rapid protein Complex Identification (MERCI) to isolate a stable stoichiometric native complex SUMM4 that comprises SUUR and a chromatin boundary protein Mod(Mdg4)-67.2. Mod(Mdg4) stimulates SUUR ATPase activity and is required for a normal spatiotemporal distribution of SUUR in vivo. SUUR and Mod(Mdg4)-67.2 together mediate the activities of gypsy insulator that prevent certain enhancer-promoter interactions and establish euchromatin-heterochromatin barriers in the genome. Furthermore, SuUR or mod(mdg4) mutations reverse underreplication of intercalary heterochromatin. Thus, SUMM4 can impart late replication of intercalary heterochromatin by attenuating the progression of replication forks through euchromatin/heterochromatin boundaries. Our findings implicate a SNF2 family ATP-dependent motor protein SUUR in the insulator function, reveal that DNA replication can be delayed by a chromatin barrier and uncover a critical role for architectural proteins in replication control. They suggest a mechanism for the establishment of late replication that does not depend on an asynchronous firing of late replication origins.