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Crystal structure of DNA-PKcs reveals a large open-ring cradle comprised of HEAT repeats

Nature volume 463, pages 118–121 (2010)Cite this article

Abstract

Broken chromosomes arising from DNA double-strand breaks result from endogenous events such as the production of reactive oxygen species during cellular metabolism, as well as from exogenous sources such as ionizing radiation1,2,3. Left unrepaired or incorrectly repaired they can lead to genomic changes that may result in cell death or cancer. DNA-dependent protein kinase (DNA-PK), a holoenzyme that comprises the DNA-PK catalytic subunit (DNA-PKcs)4,5 and the heterodimer Ku70/Ku80, has a major role in non-homologous end joining—the main pathway in mammals used to repair double-strand breaks6,7,8. DNA-PKcs is a serine/threonine protein kinase comprising a single polypeptide chain of 4,128 amino acids and belonging to the phosphatidylinositol-3-OH kinase (PI(3)K)-related protein family9. DNA-PKcs is involved in the sensing and transmission of DNA damage signals to proteins such as p53, setting off events that lead to cell cycle arrest10,11. It phosphorylates a wide range of substrates in vitro, including Ku70/Ku80, which is translocated along DNA12. Here we present the crystal structure of human DNA-PKcs at 6.6 Å resolution, in which the overall fold is clearly visible, to our knowledge, for the first time. The many α-helical HEAT repeats (helix–turn–helix motifs) facilitate bending and allow the polypeptide chain to fold into a hollow circular structure. The carboxy-terminal kinase domain is located on top of this structure, and a small HEAT repeat domain that probably binds DNA is inside. The structure provides a flexible cradle to promote DNA double-strand-break repair.

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Figure 1: Crystal structure of DNA-PKcs at 6.6 Å resolution.
Figure 2: Overall view of the DNA-PKcs structure.
Figure 3: Location of the DNA-PKcs kinase domain.

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Accession codes

Primary accessions

Protein Data Bank

Data deposits

The atomic coordinates and structure factors for the reported crystal structure have been deposited with the Protein Data Bank (PDB) under accession code 3kgv.

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Acknowledgements

We wish to thank G. Smith for providing pure DNA-PKcs that was used in the initial experiments as a marker and also for providing the DNA-PKcs and Ku70 antibodies, L. Pellegrini for help and advice at the beginning of the project, and S. Jackson for discussions. We are also grateful to R. Peat for preparing HeLa cells, L. Packman for help in identifying DNA-PKcs in polyacrylamide gels, N. Ban for providing Ta6Br122+ and C. Müller-Dieckmann at the ESRF for support during the diffraction data collection experiments. This work was funded by the Wellcome Trust and CR-UK.

Author Contributions T.L.B. and B.L.S. conceived the project. B.L.S. characterized, purified, crystallized and analysed the electron density for the DNA-PKcs–Ku80ct complex. D.Y.C. and B.L.S. carried out data collection and structure modelling. D.Y.C. carried out data processing, electron density calculations and refinement, with significant input from T.L.B. in the interpretation of the data. B.L.S. wrote the paper and all authors contributed and edited the manuscript.

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  1. Department of Biochemistry, University of Cambridge, Old Addenbrooke’s site, 80 Tennis Court Road, Cambridge CB2 1GA, UK,

    Bancinyane L. Sibanda, Dimitri Y. Chirgadze & Tom L. Blundell

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  1. Bancinyane L. Sibanda
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Correspondence to Bancinyane L. Sibanda or Dimitri Y. Chirgadze.

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Supplementary Information

This file contains Supplementary Table 1, Supplementary Figures 1-3 with Legends and a Legend for Supplementary Movie 1. (PDF 1711 kb)

Supplementary Information

This movie shows the 360 degrees view of a single DNA-PKcs molecule displayed as molecular surface (see Supplementary Information file for full Legend). (MOV 7455 kb)

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Sibanda, B., Chirgadze, D. & Blundell, T. Crystal structure of DNA-PKcs reveals a large open-ring cradle comprised of HEAT repeats. Nature 463, 118–121 (2010). https://doi.org/10.1038/nature08648

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