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

  • ️Wang, Xiao-Fan
  • ️Thu Jun 21 2001

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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References

  1. Zhou, B. B. & Elledge, S. J. The DNA damage response: putting checkpoints in perspective. Nature 408, 433–439 (2000).

    Article  ADS  CAS  Google Scholar 

  2. O'Connell, M. J., Walworth, N. C. & Carr, A. M. The G2-phase DNA-damage checkpoint. Trends Cell Biol. 10, 296–303 (2000).

    Article  CAS  Google Scholar 

  3. Lowndes, N. F. & Murguia, J. R. Sensing and responding to DNA damage. Curr. Opin. Gen. Dev. 10, 17–25 (2000).

    Article  CAS  Google Scholar 

  4. Weinert, T. Yeast checkpoint controls and relevance to cancer. Cancer Surveys 29, 109–132 (1997).

    CAS  PubMed  Google Scholar 

  5. Caspari, T. & Carr, A. M. DNA structure checkpoint pathways in Schizosaccharomyces pombe. Biochimie 81, 173–181 (1999).

    Article  CAS  Google Scholar 

  6. Venclovas, C. & Thelen, M. P. Structure-based predictions of Rad1, Rad9, Hus1 and Rad17 participation in sliding clamp and clamp-loading complexes. Nucleic Acids Res. 28, 2481–2493 (2000).

    Article  CAS  Google Scholar 

  7. Bao, S., Shen, X., Shen, K., Liu, Y. & Wang, X. F. The mammalian Rad24 homologous to yeast Saccharomyces cerevisiae Rad24 and Schizosaccharomyces pombe Rad17 is involved in DNA damage checkpoint. Cell Growth Diff. 9, 961–967 (1998).

    CAS  PubMed  Google Scholar 

  8. Parker, A. E., Van, D. W., Laus, M. C., Verhasselt, P. & Luyten, W. H. M. L. Identification of a human homologue of the Schizosaccharomyces pombe rad17(+) checkpoint gene. J. Biol. Chem. 274, 24438–24439 (1999).

    CAS  Google Scholar 

  9. Thelen, M. P., Venclovas, C. & Fidelis, K. A sliding clamp model for the Rad1 family of cell cycle checkpoint proteins. Cell 96, 769–770 (1999).

    Article  CAS  Google Scholar 

  10. Rauen, M., Burtelow, M. A., Dufault, V. M. & Karnitz, L. M. The human checkpoint protein hRad17 interacts with the PCNA-like proteins hRad1, hHus1, and hRad9. J. Biol. Chem. 275, 29767–29771 (2000).

    Article  CAS  Google Scholar 

  11. Shiloh, Y. ATM and ATR: networking cellular responses to DNA damage. Curr. Opin. Gen. Dev. 11, 71–77 (2001).

    Article  CAS  Google Scholar 

  12. Kim, S. T., Lim, D. S., Canman, C. E. & Kastan, M. B. Substrate specificities and identification of putative substrates of ATM kinase family members. J. Biol. Chem. 274, 37538–37543 (1999).

    Article  CAS  Google Scholar 

  13. Tibbetts, R. S. et al. A role for ATR in the DNA damage-induced phosphorylation of p53. Genes Dev. 13, 152–157 (1999).

    Article  CAS  Google Scholar 

  14. Banin, S. et al. Enhanced phosphorylation of p53 by ATM in response to DNA damage. Science 281, 1674–1677 (1998).

    Article  ADS  CAS  Google Scholar 

  15. Cliby, W. A. et al. Overexpression of a kinase-inactive ATR protein causes sensitivity to DNA-damaging agents and defects in cell cycle checkpoints. EMBO J. 17, 159–169 (1998).

    Article  CAS  Google Scholar 

  16. Tibbetts, R. S. et al. Functional interactions between BRCA1 and the checkpoint kinase ATR during genotoxic stress. Genes Dev. 14, 2989–3002 (2000).

    Article  CAS  Google Scholar 

  17. Burtelow, M. A., Kaufmann, S. H. & Karnitz, L. M. Retention of the human Rad9 checkpoint complex in extraction-resistant nuclear complexes after DNA damage. J. Biol. Chem. 275, 26343–26348 (2000).

    Article  CAS  Google Scholar 

  18. Sekulic, A. et al. A direct linkage between the phosphoinositide 3-kinase-AKT signaling pathway and the mammalian target of rapamycin in mitogen-stimulated and transformed cells. Cancer Res. 60, 3504–3513 (2000).

    CAS  PubMed  Google Scholar 

  19. He, T. C. et al. A simplified system for generating recombinant adenoviruses. Proc. Natl Acad. Sci. USA 95, 2509–2514 (1998).

    Article  ADS  CAS  Google Scholar 

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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

Authors

  1. Shideng Bao

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  2. Randal S. Tibbetts

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  3. Kathryn M. Brumbaugh

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  4. Yanan Fang

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  5. D. Ashley Richardson

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  6. Ambereen Ali

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  7. Susan M. Chen

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  8. Robert T. Abraham

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  9. Xiao-Fan Wang

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Corresponding author

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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  • Received: 31 January 2001

  • Accepted: 23 May 2001

  • Issue Date: 21 June 2001

  • DOI: https://doi.org/10.1038/35082110