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

Nature volume 411, pages 969–974 (2001)Cite this article

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 checkpoints2,3,4,5. 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 (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.

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Figure 1: Association of hRad17 with ATR/ATM induced by DNA damage.
Figure 2: Phosphorylation of hRad17 induced by DNA damage.
Figure 3: Role of hRad17 phosphorylation in activation of the G2 checkpoint.
Figure 4: Overexpression of Flag–hRad17AA suppresses phosphorylation of hRad17.
Figure 5: Association of hRad17 with hRad1 induced by DNA damage.

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Acknowledgements

We thank B. Vogelstein for the pAdTrack-CMV and pAdEasy adenoviral vectors; Y. Xu for the ATM+/+ and ATM-/- mouse embryo fibroblasts; C. Counter for the BJ fibroblasts; L. M. Karnitz for the anti-hRad17 antibody; L. Martinek and M. Cook for the flow cytometric analysis; Y. Yu for technical help; and members of the Wang and Abraham laboratories for scientific discussions. This work was supported by grants from the National Institutes of Health, the A-T Children's Project, and the Johnson and Johnson Foundation.

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Author notes

  1. Shideng Bao and Randal S. Tibbetts: These authors contributed equally to this work.

  2. Xiao-Fan Wang: Correspondence and requests for materials should be addressed to X.-F.W.

Authors and Affiliations

  1. Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, 27710, North Carolina, USA

    Shideng Bao, Randal S. Tibbetts, Kathryn M. Brumbaugh, Yanan Fang, D. Ashley Richardson, Ambereen Ali, Susan M. Chen, Robert T. Abraham & Xiao-Fan Wang

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  1. Shideng Bao
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Correspondence to Xiao-Fan Wang.

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Bao, S., Tibbetts, R., Brumbaugh, K. et al. ATR/ATM-mediated phosphorylation of human Rad17 is required for genotoxic stress responses. Nature 411, 969–974 (2001). https://doi.org/10.1038/35082110

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