Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

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.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

References

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

    Article  ADS  CAS  PubMed  Google Scholar 

  2. Rouse, J. & Jackson, S. P. Interfaces between the detection, signaling, and repair of DNA damage. Science 297, 547–551 (2002)

    Article  ADS  CAS  PubMed  Google Scholar 

  3. Melo, J. & Toczyski, D. A unified view of the DNA-damage checkpoint. Curr. Opin. Cell Biol. 14, 237–245 (2002)

    Article  CAS  PubMed  Google Scholar 

  4. Shiloh, Y. ATM and related protein kinases: safeguarding genome integrity. Nature Rev. Cancer 3, 155–168 (2003)

    Article  CAS  Google Scholar 

  5. Meek, K., Gupta, S., Ramsden, D. A. & Lees-Miller, S. P. The DNA-dependent protein kinase: the director at the end. Immunol. Rev. 200, 132–141 (2004)

    Article  CAS  PubMed  Google Scholar 

  6. Hammarsten, O., DeFazio, L. G. & Chu, G. Activation of DNA-dependent protein kinase by single-stranded DNA ends. J. Biol. Chem. 275, 1541–1550 (2000)

    Article  CAS  PubMed  Google Scholar 

  7. Smith, G. C. M. et al. Purification and DNA binding properties of the ataxia-telangiectasia gene product ATM. Proc. Natl Acad. Sci. USA 96, 11134–11139 (1999)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  8. Ünsal-Kaçmaz, K., Makhov, A. M., Griffith, J. D. & Sancar, A. Preferential binding of ATR protein to UV-damaged DNA. Proc. Natl Acad. Sci. USA 99, 6673–6678 (2002)

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  9. Unsal-Kacmaz, K. & Sancar, A. Quaternary structure of ATR and effects of ATRIP and replication protein A on its DNA binding and kinase activities. Mol. Cell. Biol. 24, 1292–1300 (2004)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Zou, L. & Elledge, S. J. Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes. Science 300, 1542–1548 (2003)

    Article  ADS  CAS  PubMed  Google Scholar 

  11. Gottlieb, T. M. & Jackson, S. P. The DNA-dependent protein kinase: requirement for DNA ends and association with Ku antigen. Cell 72, 131–142 (1993)

    Article  CAS  PubMed  Google Scholar 

  12. Dvir, A., Peterson, S. R., Knuth, M. W., Lu, H. & Dynan, W. S. Ku autoantigen is the regulatory component of a template-associated protein kinase that phosphorylates RNA polymerase II. Proc. Natl Acad. Sci. USA 89, 11920–11924 (1992)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  13. Andegeko, Y. et al. Nuclear retention of ATM at sites of DNA double strand breaks. J. Biol. Chem. 276, 38224–38230 (2001)

    CAS  PubMed  Google Scholar 

  14. Carson, C. T. et al. The Mre11 complex is required for ATM activation and the G2/M checkpoint. EMBO J. 22, 6610–6620 (2003)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Kitagawa, R., Bakkenist, C. J., McKinnon, P. J. & Kastan, M. B. Phosphorylation of SMC1 is a critical downstream event in the ATM-NBS1-BRCA1 pathway. Genes Dev. 18, 1423–1438 (2004)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. D'Amours, D. & Jackson, S. P. The Mre11 complex: at the crossroads of DNA repair and checkpoint signalling. Nature Rev. Mol. Cell Biol. 3, 317–327 (2002)

    Article  CAS  Google Scholar 

  17. Bakkenist, C. J. & Kastan, M. B. DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation. Nature 421, 499–506 (2003)

    Article  ADS  CAS  PubMed  Google Scholar 

  18. Lukas, C., Falck, J., Bartkova, J., Bartek, J. & Lukas, J. Distinct spatiotemporal dynamics of mammalian checkpoint regulators induced by DNA damage. Nature Cell Biol. 5, 255–260 (2003)

    Article  CAS  PubMed  Google Scholar 

  19. Uziel, T. et al. Requirement of the MRN complex for ATM activation by DNA damage. EMBO J. 22, 5612–5621 (2003)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Horejsi, Z. et al. Distinct functional domains of Nbs1 modulate the timing and magnitude of ATM activation after low doses of ionizing radiation. Oncogene 23, 3122–3127 (2004)

    Article  CAS  PubMed  Google Scholar 

  21. Hickson, I. et al. Identification and characterization of a novel and specific inhibitor of the ataxia-telangiectasia mutated kinase ATM. Cancer Res. 64, 9152–9159 (2004)

    Article  CAS  PubMed  Google Scholar 

  22. Veuger, S. J., Curtin, N. J., Smith, G. C. & Durkacz, B. W. Effects of novel inhibitors of poly(ADP-ribose) polymerase-1 and the DNA-dependent protein kinase on enzyme activities and DNA repair. Oncogene 23, 7322–7329 (2004)

    Article  CAS  PubMed  Google Scholar 

  23. Stiff, T. et al. ATM and DNA-PK function redundantly to phosphorylate H2AX after exposure to ionizing radiation. Cancer Res. 64, 2390–2396 (2004)

    Article  CAS  PubMed  Google Scholar 

  24. Gell, D. & Jackson, S. P. Mapping of protein–protein interactions within the DNA-dependent protein kinase complex. Nucleic Acids Res. 27, 3494–3502 (1999)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Singleton, B. K., Taccioli, G. E., Rottinghaus, S. & Jeggo, P. A. Interaction of the C-terminus of Ku80 with the DNA-dependent protein kinase catalytic subunit. Mol. Cell. Biol. 19, 3267–3277 (1999)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Chan, D. W. et al. Autophosphorylation of the DNA-dependent protein kinase catalytic subunit is required for rejoining of DNA double-strand breaks. Genes Dev. 16, 2333–2338 (2002)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Douglas, P. et al. Identification of in vitro and in vivo phosphorylation sites in the catalytic subunit of the DNA-dependent protein kinase. Biochem. J. 368, 243–251 (2002)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Rothkamm, K. & Lobrich, M. Evidence for a lack of DNA double-strand break repair in human cells exposed to very low x-ray doses. Proc. Natl Acad. Sci. USA 100, 5057–5062 (2003)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  29. Zou, L., Cortez, D. & Elledge, S. J. Regulation of ATR substrate selection by Rad17-dependent loading of Rad9 complexes onto chromatin. Genes Dev. 16, 198–208 (2002)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Rouse, J. & Jackson, S. P. Lcd1p recruits Mec1p to DNA lesions in vitro and in vivo . Mol. Cell 9, 857–869 (2002)

    Article  CAS  PubMed  Google Scholar 

  31. Costanzo, V., Paull, T., Gottesman, M. & Gautier, J. Mre11 assembles linear DNA fragments into DNA damage signaling complexes. PLoS Biol. 2, E110 (2004)

    Article  PubMed  PubMed Central  Google Scholar 

  32. Nakada, D., Matsumoto, K. & Sugimoto, K. ATM-related Tel1 associates with double-strand breaks through an Xrs2-dependent mechanism. Genes Dev. 17, 1957–1962 (2003)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Brodsky, M. H., Sekelsky, J. J., Tsang, G., Hawley, R. S. & Rubin, G. M. mus304 encodes a novel DNA damage checkpoint protein required during Drosophila development. Genes Dev. 14, 666–678 (2000)

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Wakayama, T., Kondo, T., Ando, S., Matsumoto, K. & Sugimoto, K. Pie1, a protein interacting with Mec1, controls cell growth and checkpoint responses in Saccharomyces cerevisiae . Mol. Cell. Biol. 21, 755–764 (2001)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Wolkow, T. D. & Enoch, T. Fission yeast Rad26 is a regulatory subunit of the Rad3 checkpoint kinase. Mol. Biol. Cell 13, 480–492 (2002)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Lee, J. H. & Paull, T. T. Direct activation of the ATM protein kinase by the Mre11/Rad50/Nbs1 complex. Science 304, 93–96 (2004)

    Article  ADS  CAS  PubMed  Google Scholar 

  37. Brewerton, S. C., Dore, A. S., Drake, A. C., Leuther, K. K. & Blundell, T. L. Structural analysis of DNA-PKcs: modelling of the repeat units and insights into the detailed molecular architecture. J. Struct. Biol. 145, 295–306 (2004)

    Article  CAS  PubMed  Google Scholar 

  38. Perry, J. & Kleckner, N. The ATRs, ATMs, and TORs are giant HEAT repeat proteins. Cell 112, 151–155 (2003)

    Article  CAS  PubMed  Google Scholar 

  39. Singleton, B. K., Torres-Arzayus, M. I., Rottinghaus, S. T., Taccioli, G. E. & Jeggo, P. A. The C terminus of Ku80 activates the DNA-dependent protein kinase catalytic subunit. Mol. Cell. Biol. 19, 3267–3277 (1999)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Goldberg, M. et al. MDC1 is required for the intra-S-phase DNA damage checkpoint. Nature 421, 952–956 (2003)

    Article  ADS  CAS  PubMed  Google Scholar 

  41. Falck, J., Petrini, J. H. J., Williams, B. R., Lukas, J. & Bartek, J. The DNA damage-dependent intra-S phase checkpoint is regulated by parallel pathways. Nature Genet. 30, 290–294 (2002)

    Article  PubMed  Google Scholar 

  42. Xu, B., Kim, S.-T. & Kastan, M. B. Involvement of Brca1 in S-phase and G2-phase checkpoints after ionizing irradiation. Mol. Cell. Biol. 21, 3445–3450 (2001)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Tsao, C. C., Geisen, C. & Abraham, R. T. Interaction between human MCM7 and Rad17 proteins is required for replication checkpoint signaling. EMBO J. 23, 4660–4669 (2004)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

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.).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Stephen P. Jackson.

Ethics declarations

Competing interests

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)

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature03442

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing