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Distinct roles for DNA-PK, ATM and ATR in RPA phosphorylation and checkpoint activation in response to replication

Shengqin Liu1, Stephen O Opiyo, Karoline Manthey

  • 1Department of Oral Biology, University of Nebraska Medical Center, Omaha, NE 68583, USA.

Nucleic Acids Research
|September 15, 2012
PubMed

Insights

DNA-PKcs is crucial for preventing genome instability during replication stress. Its absence or mutation leads to cell cycle defects and mitotic entry with unrepaired DNA damage, increasing cancer risk.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Cancer Research

Background:

  • DNA damage at replication forks can lead to genome instability and cancer.
  • DNA damage checkpoint systems are critical for suppressing cancer by arresting the cell cycle, promoting repair, or inducing cell death.
  • Replication stress, caused by DNA damage or replication machinery perturbation, activates signaling pathways like ATR-Chk1, involving RPA phosphorylation.

Purpose of the Study:

  • To investigate the role of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in replication stress-induced RPA phosphorylation.
  • To understand the consequences of impaired DNA-PKcs function on cell cycle control and DNA damage response.

Main Methods:

  • Utilized DNA-PKcs mutant cells and cells with mutations in RPA32 phosphorylation sites.
  • Assessed cell cycle arrest, ATR signaling to Chk1, and H2AX phosphorylation.
  • Inhibited ATR and DNA-PK (but not ATM) to mimic observed defects.

Main Results:

  • DNA-PKcs mutant cells failed to arrest replication following stress.
  • Mutations in RPA32 phosphorylation sites impaired ATR signaling and caused a G2/M arrest defect.
  • Inhibition of ATR and DNA-PK mimicked these defects.
  • Cells with mutant RPA32 or DNA-PKcs showed sustained H2AX phosphorylation, indicating entry into mitosis with unrepaired damage.

Conclusions:

  • DNA-PKcs plays a significant role in replication stress response by regulating RPA phosphorylation.
  • Impaired DNA-PKcs function or RPA32 phosphorylation leads to defective cell cycle arrest and inappropriate mitotic entry with unrepaired DNA.
  • These findings highlight a novel mechanism by which DNA-PKcs contributes to genome stability and cancer suppression.

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