Genotoxic stress triggers the activation of checkpoints that delay cell-cyc
le progression to allow for DNA repair(1). Studies in fission yeast implica
te members of the Rad family of checkpoint proteins, which includes Rad17,
Rad1, Rad9 and Hus1, as key early-response elements during the activation o
f both the DNA damage and replication checkpoints(2-5). 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 genotox
ic agents induced ATM/ATR-dependent phosphorylation of hRad17 at Ser 635 an
d Ser 645. Overexpression of a hRad17 mutant (hRad17(AA)) bearing Ala subst
itutions at both phosphorylation sites abrogated the DNA-damage-induced G(2
) checkpoint, and sensitized human fibroblasts to genotoxic stress. In cont
rast to wild-type hRad17, the hRad17(AA) mutant showed no ionizing-radiatio
n-inducible association with hRad1, a component of the hRad1-hRad9-hHus1 ch
eckpoint complex. These findings demonstrate that ATR/ATM-dependent phospho
rylation of hRad17 is a critical early event during checkpoint signalling i
n DNA-damaged cells.