assembly of the complex on its substrate, in the same way as a number of chromatin remodeling factors. Opposite to the architecture of PARP1 in isolation, our 3D ﬁtting experiments show that PARP1 interacts with DNA-PK in a monomeric form, with the catalytic domain distal to Ku. In fact, the density for the extra protein is really well deﬁned, accounting for the volume of only one PARP1 molecule (not smaller or bigger lumps) with no blurring which might suggest ﬂexibility in the PARP1 region. Importantly to our understanding of the functional mechanisms of this complex, and in contrast to our previous analysis of DNA-PK dimers-of-trimers loaded on Y-shaped DNA (6), the DNA-PKcs heads are close in space, suggesting that in this complex autophos- phorylation may take place in trans, as reported in earlier biochemical studies (47). Our study does not however answer the question of how DNA assembles in this complex. This might be addressed by future cryo-EM or small angle neutron scattering studies. This orientation of the DNA-PK/PARP1 dimer-of-tetramers has some analogies with the conformation of DNA-PK dimers loaded on hairpin DNA recently analysed by SAXS (37), since DNA-PKcs head domains are close in space. However, the overall architecture of the dimer-of- heterotetramers is different, as it is arranged in ‘T-shaped’ assemblies. This observation is crucial towards understanding the function of DNA-PK, since it shows that this dynamic enzyme can adopt substantially different conformations not only upon recognition of dif- ferent DNA structures (37) or upon autophosphorylation (8), but also upon interaction with other DNA repair factors. This further structural snapshot within the DNA-PK interacting network emphasizes the need to characterize the structures of the full range of DNA-PK assemblies such as the DNA-PK/XRCC4/LigaseIV complex in order to fully understand the role of a pivotal enzyme in the DNA damage response. SUPPLEMENTARY DATA Supplementary Data are available at Supplementary Figure S1. NAR Online: ACKNOWLEDGEMENTS We are grateful to Paula C.A. da Fonseca for her advice. FUNDING Cancer Research UK (Programme Grant to L.H.P.; Project Grant C17335/A10470 to L.S.); Scottish Universities Life Sciences Alliance (to The Electron Microscopy Facility at Edinburgh); Wellcome Trust (087658/Z/08/Z) (to The Electron Microscopy Facility at Edinburgh). Funding for open access charge: Wellcome Trust grant 087658/Z/08/Z. Conﬂict of interest statement. None declared. Nucleic Acids Research, 2012, Vol. 40, No. 9 4175 REFERENCES 1. Pardo,B., Gomez-Gonzalez,B. and Aguilera,A. (2009) DNA repair in mammalian cells: DNA double-strand break repair: how to ﬁx a broken relationship. Cell Mol. Life Sci., 66, 1039. 2. Jackson,S.P. (1997) DNA-dependent protein kinase. Int. J. Biochem. Cell Biol., 29, 935. 3. Downs,J.A. and Jackson,S.P. (2004) A means to a DNA end: the many roles of Ku. Nat. Rev. Mol. Cell Biol., 5, 367. 4. Hartley,K.O., Gell,D., Smith,G.C., Zhang,H., Divecha,N., Connelly,M.A., Admon,A., Lees-Miller,S.P., Anderson,C.W. and Jackson,S.P. (1995) DNA-dependent protein kinase catalytic subunit: a relative of phosphatidylinositol 3-kinase and the ataxia telangiectasia gene product. Cell, 82, 849. 5. Ochi,T., Sibanda,B.L., Wu,Q., Chirgadze,D.Y., Bolanos- Garcia,V.M. and Blundell,T.L. (2010) Structural biology of DNA repair: spatial organisation of the multicomponent complexes of nonhomologous end joining. J. Nucleic Acids, Article ID 621695. 6. Spagnolo,L., Rivera-Calzada,A., Pearl,L.H. and Llorca,O. (2006) Three-dimensional structure of the human DNA-PKcs/Ku70/ Ku80 complex assembled on DNA and its implications for DNA DSB repair, Mol. Cell, 22, 511. 7. Mahaney,B.L., Meek,K. and Lees-Miller,S.P. (2009) Repair of ionizing radiation-induced DNA double-strand breaks by non-homologous end-joining. Biochem. J., 417, 639. 8. Morris,E.P., Rivera-Calzada,A., da Fonseca,P.C., Llorca,O., Pearl,L.H. and Spagnolo,L. (2011) Evidence for a remodelling of DNA-PK upon autophosphorylation from electron microscopy studies. Nucleic Acids Res., 39, 5757. 9. Schreiber,V., Dantzer,F., Ame,J.C. and de Murcia,G. (2006) Poly(ADP-ribose): novel functions for an old molecule. Nat. Rev. Mol. Cell Biol., 7, 517. 10. Dantzer,F., Schreiber,V., Niedergang,C., Trucco,C., Flatter,E., De La Rubia,G., Oliver,J., Rolli,V., Menissier-de Murcia,J. and de Murcia,G. (1999) Involvement of poly(ADP-ribose) polymerase in base excision repair. Biochimie, 81, 69. 11. Malanga,M. and Althaus,F.R. (2005) The role of poly(ADP-ribose) in the DNA damage signaling network. Biochem. Cell Biol., 83, 354. 12. Morrison,C., Smith,G.C., Stingl,L., Jackson,S.P., Wagner,E.F. and Wang,Z.Q. (1997) Genetic interaction between PARP and DNA-PK in V(D)J recombination and tumorigenesis. Nat. Genet., 17, 479. 13. Henrie,M.S., Kurimasa,A., Burma,S., Menissier-de Murcia,J., de Murcia,G., Li,G.C. and Chen,D.J. (2003) Lethality in PARP-1/ Ku80 double mutant mice reveals physiological synergy during early embryogenesis. DNA Repair, 2, 151. 14. Pleschke,J.M., Kleczkowska,H.E., Strohm,M. and Althaus,F.R. (2000) Poly(ADP-ribose) binds to speciﬁc domains in DNA damage checkpoint proteins. J. Biol. Chem., 275, 40974. 15. Galande,S. and Kohwi-Shigematsu,T. (2000) Caught in the act: binding of Ku and PARP to MARs reveals novel aspects of their functional interaction. Crit. Rev. Eukaryot. Gene Expr., 10, 63. 16. Galande,S. and Kohwi-Shigematsu,T. (1999) Poly(ADP-ribose) polymerase and Ku autoantigen form a complex and synergistically bind to matrix attachment sequences. J. Biol. Chem., 274, 20521. 17. Ruscetti,T., Lehnert,B.E., Halbrook,J., Le Trong,H., Hoekstra,M.F., Chen,D.J. and Peterson,S.R. (1998) Stimulation of the DNA-dependent protein kinase by poly(ADP-ribose) polymerase. J. Biol. Chem., 273, 14461. 18. Hossain,M.B., Ji,P., Anish,R., Jacobson,R.H. and Takada,S. (2009) Poly(ADP-ribose) polymerase 1 interacts with nuclear respiratory factor 1 (NRF-1) and plays a role in NRF-1 transcriptional regulation. J. Biol. Chem., 284, 8621. 19. Mitchell,J., Smith,G.C. and Curtin,N.J. (2009) Poly(ADP-Ribose) polymerase-1 and DNA-dependent protein kinase have equivalent roles in double strand break repair following ionizing radiation. Int. J. Radiat. Oncol. Biol. Phys., 75, 1520. 20. Veuger,S.J., Curtin,N.J., Smith,G.C. and Durkacz,B.W. (2004) 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.