1. Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA
2. These authors contributed equally to this work.

Correspondence to: Xiao-Fan Wang Correspondence and requests for materials should be addressed to X.-F.W. (e-mail: Email: wang0011@mc.duke.edu).

Abstract

Genotoxic stress triggers the activation of checkpoints that delay cell-cycle progression to allow for DNA repair¹. 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^{2, 3, 4, 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 genotoxic agents induced ATM/ATR-dependent phosphorylation of hRad17 at Ser 635 and Ser 645. Overexpression of a hRad17 mutant (hRad17^AA) 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 hRad17^AA 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.