Abstract
The tumour-suppressor gene ATM, mutations in which cause the human genetic disease ataxia telangiectasia (A-T), encodes a key protein kinase that controls the cellular response to DNA double-strand breaks (DSBs)1,2. DNA DSBs caused by ionizing radiation or chemicals result in rapid ATM autophosphorylation, leading to checkpoint activation and phosphorylation of substrates that regulate cell-cycle progression, DNA repair, transcription and cell death3. However, the precise mechanism by which damaged DNA induces ATM and checkpoint activation remains unclear. Here, we demonstrate that linear DNA fragments added to Xenopus egg extracts mimic DSBs in genomic DNA and provide a platform for ATM autophosphorylation and activation. ATM autophosphorylation and phosphorylation of its substrate NBS1 are dependent on DNA fragment length and the concentration of DNA ends. The minimal DNA length required for efficient ATM autophosphorylation is ∼200 base pairs, with cooperative autophosphorylation induced by DNA fragments of at least 400 base pairs. Importantly, full ATM activation requires it to bind to DNA regions flanking DSB ends. These findings reveal a direct role for DNA flanking DSB ends in ATM activation.
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Acknowledgements
This article is dedicated to the memory of John Newport. We thank Matthew Weitzman, Walter Eckhart, Paul Russell and Robert Abraham for critical discussions, James Kadonaga, Dominique Ray-Gallet, Geneviève Almouzni, Paul Labhart, Takeo Kiskimoto, Graeme Smith, Mark O'Connor, Aaron Straight, Andrew Murray and Beth Baber for providing valuable reagents, Ramiro Verdun for technical assistance in dot-blotting, and Andrew Dillin for use of his microscopes. Z.Y. was supported by a Pioneer Fund Postdoctoral Fellowship. T.H. is a Frank and Else Schilling American Cancer Society Research Professor. This work was supported by Public Health Service Grants CA14195 and CA80100 from the National Cancer Institute (T.H.).
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Z.Y. designed and performed the majority of the experiments with contributions from T.H., J.B. and S.J. S.J. provided the Chk2 substrate used in Fig. 2a, and J.B. performed the chromatin fibre immunostaining experiments in Fig. 3c. S.D. provided critical reagents. Z.Y., J.B. and T.H. wrote the paper.
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Supplementary figures S1, S2, S3, S4, S5, S6 and S7 (PDF 670 kb)
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You, Z., Bailis, J., Johnson, S. et al. Rapid activation of ATM on DNA flanking double-strand breaks. Nat Cell Biol 9, 1311–1318 (2007). https://doi.org/10.1038/ncb1651
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DOI: https://doi.org/10.1038/ncb1651
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