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Conserved modes of recruitment of ATM, ATR and DNA-PKcs to sites of DNA damage

Abstract

Ataxia-telangiectasia mutated (ATM), ataxia-telangiectasia and Rad3-related (ATR) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) are members of the phosphoinositide-3-kinase-related protein kinase (PIKK) family, and are rapidly activated in response to DNA damage. ATM and DNA-PKcs respond mainly to DNA double-strand breaks, whereas ATR is activated by single-stranded DNA and stalled DNA replication forks. In all cases, activation involves their recruitment to the sites of damage. Here we identify related, conserved carboxy-terminal motifs in human Nbs1, ATRIP and Ku80 proteins that are required for their interaction with ATM, ATR and DNA-PKcs, respectively. These motifs are essential not only for efficient recruitment of ATM, ATR and DNA-PKcs to sites of damage, but are also critical for ATM-, ATR- and DNA-PKcs-mediated signalling events that trigger cell cycle checkpoints and DNA repair. Our findings reveal that recruitment of these PIKKs to DNA lesions occurs by common mechanisms through an evolutionarily conserved motif, and provide direct evidence that PIKK recruitment is required for PIKK-dependent DNA-damage signalling.

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Figure 1: The extreme C terminus of Nbs1 constitutes a conserved ATM interaction motif.
Figure 2: ATM association with sites of DNA damage requires its interaction with Nbs1.
Figure 3: A functional Nbs1–ATM interaction is required for ATM phosphorylation events and checkpoint functions.
Figure 4: The PIKK interaction motif of Ku80 is required for DNA-PKcs activation.
Figure 5: ATR-mediated signalling depends on the C-terminal PIKK interaction motif of ATRIP.

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Acknowledgements

We are grateful to K.-P. Hopfner and members of the Jackson laboratory for their suggestions, V. Smits for help with the G2/M checkpoint assay, and P. Jeggo, R. T. Abraham, G. Smith, D. J. Chen, H. H. Chun and R. A. Gatti for providing reagents. We thank M. Stucki and P. M. Reaper for critical reading of the manuscript. This study was supported by Cancer Research UK and by the Danish Cancer Society (to J.F.).

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Correspondence to Stephen P. Jackson.

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S.P.J. is Chief Scientific Officer at KuDOS Pharmaceuticals Ltd.

Supplementary information

Supplementary Figure S1

Nbs1 C-terminal peptide pulldowns from nuclear extracts (coomassie stained) and with purified recombinant ATM (silver stained). (PDF 126 kb)

Supplementary Figure S2

Immunoblots and immunostainings from complemented NBS cells showing expression levels and subcellular localization of Nbs1 and Mre11. (PDF 1591 kb)

Supplementary Figure S3

Immunoblots from cells transfected with ATRIP siRNA and siRNA-resistant HA-ATRIP. (PDF 91 kb)

Supplementary Figure S4

Peptide pulldowns and immunoprecipitations from nuclear extracts using Nbs1, ATRIP and Ku80 C-terminal peptides and antibodies, respectively. (PDF 682 kb)

Supplementary Figure S5

Pulldowns from nuclear extracts using GST-ATM fragments. (PDF 107 kb)

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Falck, J., Coates, J. & Jackson, S. Conserved modes of recruitment of ATM, ATR and DNA-PKcs to sites of DNA damage. Nature 434, 605–611 (2005). https://doi.org/10.1038/nature03442

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