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Dimers of DNA-PK create a stage for DNA double-strand break repair

Abstract

DNA double-strand breaks are the most dangerous type of DNA damage and, if not repaired correctly, can lead to cancer. In humans, Ku70/80 recognizes DNA broken ends and recruits the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to form DNA-dependent protein kinase holoenzyme (DNA-PK) in the process of non-homologous end joining (NHEJ). We present a 2.8-Å-resolution cryo-EM structure of DNA-PKcs, allowing precise amino acid sequence registration in regions uninterpreted in previous 4.3-Å X-ray maps. We also report a cryo-EM structure of DNA-PK at 3.5-Å resolution and reveal a dimer mediated by the Ku80 C terminus. Central to dimer formation is a domain swap of the conserved C-terminal helix of Ku80. Our results suggest a new mechanism for NHEJ utilizing a DNA-PK dimer to bring broken DNA ends together. Furthermore, drug inhibition of NHEJ in combination with chemo- and radiotherapy has proved successful, making these models central to structure-based drug targeting efforts.

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Fig. 1: The structure and conformational flexibility of DNA-PKcs.
Fig. 2: The structure of DNA-PK.
Fig. 3: The DNA-PK dimer.
Fig. 4: An overall model for NHEJ.

Data availability

Data supporting the findings of this paper are available from the corresponding author upon reasonable request. All data generated or analysed during this study are included in this published Article and its Supplementary Information files. Cryo-EM density maps have been deposited in the Electron Microscopy Data Bank under accession codes EMD-11211, 11185, 11213, 11217, 11216, 11219 and 11215. Atomic coordinates have been deposited in the RCSB Protein Data Bank under accession codes PDB 6ZH2, 6ZFP, 6ZH4, 6ZHA, 6ZH8, 6ZHE and 6ZH6. Source data are provided with this paper.

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Acknowledgements

Model coordinates have been deposited in the Protein Data Bank and maps deposited at the Electron Microscopy Data Bank under the accession numbers shown in Table 1. We thank T. Terwilliger for advice regarding the usage of the ResolveCryoEM program in Phenix. We also thank B. Luisi, L. Pellegrini and N. Rzechorzek for their critical reading and feedback on the manuscript. We are grateful to the Wellcome Trust for a Program Grant (O93167/Z/10/Z; 2011–2016) and Investigator Award (200814/Z/16/Z; 2016) to support this research.

Author information

Affiliations

Authors

Contributions

A.K.C. purified and prepared complexes for cryo-EM, modelled the protein structures and wrote the manuscript. S.W.H. collected and processed the cryo-EM data and assisted with modelling the protein structures and writing the manuscript. S.L. carried out the initial cryo-EM analysis of DNA-PKcs. A.K.S. assisted with Ku70/80 expression and purification. A.H. expressed and purified the Ku80 CTR. D.Y.C. collected cryo-EM data and provided expertise. L.R.C. assisted with grid preparation and collected cryo-EM data. T.M.D.O. helped with research design and advice. T.L.B. directed the study, provided advice and edited the manuscript.

Corresponding author

Correspondence to Tom L. Blundell.

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Competing interests

T.M.D.O. is employed at AstraZeneca. The authors declare no other competing interests.

Additional information

Peer review information Peer reviewer reports are available. Beth Moorefield was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 DNA-PK complex formation and optimisation of cryo-EM samples.

a, Gel filtration profile of DNA-PKcs, Ku70/80 and the DNA-PK complex with Y-shaped 42-55 bp DNA. Inset, SDS-PAGE gel for DNA-PKcs, Ku70/80 and DNA-PK with molecular weight marker (kDa) indicated. Uncropped SDS gel image is available as Source Data. b and c, Example of 2D classes of DNA-PK and the angular distribution calculated in cryoSPARC for particle projections shown as a heat map, where b is before addition of CHAPSO and c after CHAPSO has been added. Scale bar = 20nm.

Extended Data Fig. 2 Cryo-EM maps before and after using ResolveCryoEM.

a, DNA-PKcs (state 2), b, DNA-PK monomer and c, DNA-PK dimer before (as green sticks) and after ResolveCryoEM (yellow sticks) for residues 3897-391814.

Extended Data Fig. 3 Sequence of DNA-PKcs.

Coloured blocks below the sequence represent a traffic light comparison to the modelling of DNA-PKcs reported previously (PDB 5LUQ12). Green illustrates regions where the two structures align exactly, orange when the sequence is misaligned by 1-3 residues and red when there is a change of 4+ residues.

Extended Data Fig. 4 Structure of state 2 DNA-PKcs showing key areas of modelled density.

The most extensively remodeled region is part of the circular cradle and is coloured purple. 1 and 2 show two areas previously unmodelled between residues 2573-2594 and 2769-2785, larger side chains are labelled, and residues shown as green sticks. 3 shows a loop previously not modelled between residues 890-907. 4 and 5 illustrate two areas where there is a continuous loop that was previously assumed to be a much larger disordered region. 6. Shows residues 1798-1818 as a continuous loop which was previously modelled as an unconnected helix.

Extended Data Fig. 5 Particle distributions, FSC curves and local resolution maps.

Local resolution maps are coloured according to resolutions and colours displayed in the specific key chart for each map. a) DNA-PKcs state 1, b) DNA-PKcs state 2 and c) DNA-PKcs state 3.

Extended Data Fig. 6 Particle distributions, FSC curves and local resolution maps for DNA-PK monomer and dimer.

Local resolution maps are coloured according to resolutions and colours displayed in the specific key chart for each map. a, DNA-PK monomer and b, DNA-PK dimer.

Extended Data Fig. 7 DNA-PKcs structure with extra density and the location of the C-terminal α-helix of Ku80.

a, DNA-PKcs structure with additional density close to the N-terminal arm predicted to correspond to the CTD of Ku80 labelled and highlighted in yellow15 at 5.8 Å resolution (EMD-8752). b, DNA-PKcs structure with a fragment of DNA solved to 3.8 Å resolution following density modification14, displayed in two orientations. c, The structure of PDB entry 5LUQ modelled into our cryo-EM map of DNA-PKcs with Ku80ct194. This structure highlights where the predicted C-terminal α-helix of Ku80 was previously modelled and where two further helices were predicted to bind and belong to Ku80. d, Structure of apo-DNA-PKcs (state 2, showing no extra density for the C-terminal α-helix of Ku80 or the extra helices predicted in 5LUQ (c). e, Cryo-EM structure of DNA-PKcs with Ku80ct194 to 3.8 Å resolution following density modification14 highlighting the C-terminal α-helix of Ku80 and the lack of the extra helices predicted by 5LUQ (identical to d).

Extended Data Fig. 8 Particle distributions, FSC curves and local resolution maps of DNA-PKcs and complexes with DNA and Ku80CTR.

Local resolution maps are coloured according to resolutions and colours displayed in the specific key chart for each map. a, DNA-PKcs with a fragment of DNA b, DNA-PKcs + Ku80CTR.

Supplementary information

Reporting Summary

Peer Review Information

Supplementary Video 1

Side view of the movement between DNA-PKcs states 1–3.

Supplementary Video 2

Back view of the movement between DNA-PKcs states 1–3.

Supplementary Video 3

Back view of the movement between DNA-PKcs states 2 and DNA-PK.

Supplementary Video 4

Side view of the movement between DNA-PKcs states 2 and DNA-PK.

Source data

Source Data Fig. 1

Uncropped SDS-PAGE gel image for Extended Data Fig. 1a.

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Chaplin, A.K., Hardwick, S.W., Liang, S. et al. Dimers of DNA-PK create a stage for DNA double-strand break repair. Nat Struct Mol Biol 28, 13–19 (2021). https://doi.org/10.1038/s41594-020-00517-x

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