Regulation of ATR activity via the RNA polymerase II associated factors CDC73 and PNUTS-PP1

Nucleic Acids Res. 2019 Feb 28;47(4):1797-1813. doi: 10.1093/nar/gky1233.

Abstract

Ataxia telangiectasia mutated and Rad3-related (ATR) kinase is a key factor activated by DNA damage and replication stress. An alternative pathway for ATR activation has been proposed to occur via stalled RNA polymerase II (RNAPII). However, how RNAPII might signal to activate ATR remains unknown. Here, we show that ATR signaling is increased after depletion of the RNAPII phosphatase PNUTS-PP1, which dephosphorylates RNAPII in its carboxy-terminal domain (CTD). High ATR signaling was observed in the absence and presence of ionizing radiation, replication stress and even in G1, but did not correlate with DNA damage or RPA chromatin loading. R-loops were enhanced, but overexpression of EGFP-RNaseH1 only slightly reduced ATR signaling after PNUTS depletion. However, CDC73, which interacted with RNAPII in a phospho-CTD dependent manner, was required for the high ATR signaling, R-loop formation and for activation of the endogenous G2 checkpoint after depletion of PNUTS. In addition, ATR, RNAPII and CDC73 co-immunoprecipitated. Our results suggest a novel pathway involving RNAPII, CDC73 and PNUTS-PP1 in ATR signaling and give new insight into the diverse functions of ATR.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Ataxia Telangiectasia Mutated Proteins / genetics*
  • Chromatin / genetics
  • DNA Damage / radiation effects
  • DNA-Binding Proteins / genetics
  • Gene Expression Regulation / radiation effects
  • Green Fluorescent Proteins / genetics
  • Humans
  • Mice
  • Nuclear Proteins / genetics
  • Phosphorylation / radiation effects
  • RNA Polymerase II / genetics*
  • RNA-Binding Proteins / genetics
  • Radiation, Ionizing
  • Receptors, Neuropeptide Y / genetics
  • Ribonuclease H / genetics
  • Signal Transduction / radiation effects
  • Stress, Physiological / genetics*
  • Stress, Physiological / radiation effects
  • Tumor Suppressor Proteins / genetics*

Substances

  • CDC73 protein, human
  • Chromatin
  • DNA-Binding Proteins
  • Nuclear Proteins
  • PPP1R10 protein, human
  • RNA-Binding Proteins
  • Receptors, Neuropeptide Y
  • Tumor Suppressor Proteins
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins
  • neuropeptide Y4 receptor
  • ATR protein, human
  • Ataxia Telangiectasia Mutated Proteins
  • RNA Polymerase II
  • Ribonuclease H
  • ribonuclease HI