ATR/ATM-mediated phosphorylation of human Rad17 is required for genotoxic stress responses

Nature. 2001 Jun 21;411(6840):969-74. doi: 10.1038/35082110.

Abstract

Genotoxic stress triggers the activation of checkpoints that delay cell-cycle progression to allow for DNA repair. Studies in fission yeast implicate members of the Rad family of checkpoint proteins, which includes Rad17, Rad1, Rad9 and Hus1, as key early-response elements during the activation of both the DNA damage and replication checkpoints. Here we demonstrate a direct regulatory linkage between the human Rad17 homologue (hRad17) and the checkpoint kinases, ATM and ATR. Treatment of human cells with genotoxic agents induced ATM/ATR-dependent phosphorylation of hRad17 at Ser 635 and Ser 645. Overexpression of a hRad17 mutant (hRad17AA) bearing Ala substitutions at both phosphorylation sites abrogated the DNA-damage-induced G2 checkpoint, and sensitized human fibroblasts to genotoxic stress. In contrast to wild-type hRad17, the hRad17AA mutant showed no ionizing-radiation-inducible association with hRad1, a component of the hRad1-hRad9-hHus1 checkpoint complex. These findings demonstrate that ATR/ATM-dependent phosphorylation of hRad17 is a critical early event during checkpoint signalling in DNA-damaged cells.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Ataxia Telangiectasia Mutated Proteins
  • Cell Cycle
  • Cell Cycle Proteins / metabolism*
  • Cell Line
  • DNA Damage
  • DNA-Binding Proteins
  • Doxycycline / pharmacology
  • Humans
  • Mice
  • Mutagens / toxicity*
  • Phosphorylation
  • Protein-Serine-Threonine Kinases / metabolism*
  • Serine / metabolism
  • Tumor Cells, Cultured
  • Tumor Suppressor Proteins

Substances

  • Cell Cycle Proteins
  • DNA-Binding Proteins
  • Mutagens
  • Rad17 protein, human
  • Tumor Suppressor Proteins
  • Serine
  • Atr protein, mouse
  • ATM protein, human
  • ATR protein, human
  • Ataxia Telangiectasia Mutated Proteins
  • Atm protein, mouse
  • Protein-Serine-Threonine Kinases
  • Doxycycline