ATR/ATM-mediated phosphorylation of human Rad17 is required for genotoxic stress responses.
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.
Bao
S
S
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Tibbetts
R S
RS
Brumbaugh
K M
KM
Fang
Y
Y
Richardson
D A
DA
Ali
A
A
Chen
S M
SM
Abraham
R T
RT
Wang
X F
XF
D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002460:Cell Line; D004249:DNA Damage; D004268:DNA-Binding Proteins; D004318:Doxycycline / Q000494:pharmacology; D006801:Humans; D051379:Mice; D009153:Mutagens / Q000633:toxicity*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D009153:Mutagens; C113575:Rad17 protein, human; D025521:Tumor Suppressor Proteins; D012694:Serine; C576897:Atr protein, mouse; C576324:ATM protein, human; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C576325:Atm protein, mouse; D017346:Protein Serine-Threonine Kinases; D004318:Doxycycline