proteins (e.g. histones). It recognizes DNA strand breaks and binds to them, both in vivo and in vitro (11). PARP1 is a 113 kDa enzyme with three functional domains: an N-terminal DNA-binding domain, a central automodiﬁcation domain, and a C-terminal catalytic domain. Genetic interaction between DNA-PK and PARP was initially related to recombinational events (12). The cross-talk between DNA-PK or its component Ku70:80 and PARP1 within NHEJ and V(D)J recombination has been described in various independent studies (13–21). A recent study, focusing on the role of PARP1 in V(D)J recombination, reports that the immunoprecipitation of the BRCT domain of PARP1 pulls down Ku70 and the DNA-PK complex in a DNA-independent manner (22). This ﬁnding suggests that PARP1 modulates DNA-PK in gene conversion, and that this is mediated through BRCT domain-mediated interactions. Alternatively, an import- ant and DNA-PK-independent role of PARP1 in NHEJ has recently emerged. PARP1 is proposed to operate in an alternative NHEJ pathway, which backs up the classical pathway and is particularly active in microhomology-facilitated NHEJ (23,24). Recent data indicate that PARP1 can modulate competition between HR and NHEJ -PARP inhibition alone causes cell death in HR-defective, e.g. BRCA1 mutant cells. However it now appears that co-inactivation of NHEJ (by inactiva- tion of DNA-PKcs or 53BP1) rescues BRCA1 mutant cells from PARP inhibitor cytotoxicity (25,26). These roles of PARP1 are not mutually exclusive, however they need a much ﬁner characterization to fully under- stand the interplay of PARP1 and other repair factors. DNA-PKcs and DNA-PK have been at the centre of numerous structural studies in the last 15 years. X-ray crystallography, NMR, electron microscopy (EM) and SAXS have all contributed to our understanding of these proteins, either in isolation or in complex (6,27–37). We were the ﬁrst in visualizing DNA-PK synaptic dimers by electron microscopy and single particle analysis (6). Our ﬁndings were later supported by a SAXS study, where DNA-PK synaptic dimers loaded on a Y-shaped DNA were shown to arrange in the same way in solution as in our single particle analysis (37). DNA-PK dimers loaded on hairpin DNA produced a different architecture in solution, with DNA-PKcs heads (and therefore kinase domains) in close proximity. This shows the plasticity of DNA-PK, which responds to different macromolecular interactions with substantial architectural rearrangements. Recently, we showed how autophosphorylation has a dramatic structural effect on DNA-PK dimers, causing disassembly and structural heterogeneity (8). The structural data available for PARP1 are so far limited to its isolated domains, probably due to its size and ﬂexibility. The human PARP1 enzyme is a modular protein of 113 kDa. Three zinc ﬁnger domains, Zn1–Zn3, are located at its extreme N-terminus. An internal automodiﬁcation region contains a BRCT domain involved in mediating protein–protein interactions and three lysines that are targeted for automodiﬁcation. The catalytic domain is located at the extreme C-terminus of Nucleic Acids Research, 2012, Vol. 40, No. 9 4169 the enzyme. The crystal structure of the catalytic domain (38) led to the development of a major breakthrough in structure-based drug design: PARP inhibitors are current- ly in clinical trials for the treatment of breast cancer (39). A crystal structure has recently been solved for the Zn3 domain (40). Early DNA binding studies using a Zn1–Zn2 domain fragment suggested that PARP1 binds to DNA as a dimer (41), and kinetic analysis of PARP1 activity indicated that PARP1 is a catalytic dimer (42). However, more recent data suggest that the Zn1–Zn2 fragment recognizes DNA single-strand breaks as a monomer (43). A direct interaction between Ku and PARP1 has been described (22), however the stoichiom- etry of the Ku/PARP1 assembly has not been investigated to date. Here, we present in vivo data on the effect of DNA-PK and PARP1 inhibitors in repair competent cell lines, as well as in cells deﬁcient in either DNA-PK or PARP1. Our data highlight that, upon exposure to a clinically relevant dose of radiation (2Gy) (44), gH2AX foci, which mark DNA DSBs (45), were formed. We show lack of additivity of the two inhibitors in DNA-PK proﬁ- cient V3-YAC cells, and a PARP inhibitor KU-0058684 having no further impact on the repair of DSBs in DNA-PK deﬁcient V3 cells. The same trends were observed with PARP1 deﬁcient and proﬁcient cells treated with a DNA-PK inhibitor or combination of inhibitors. Although a DNA-PK/PARP complex has been described in biochemical studies, no structural data is currently available for it. To understand the nature of the DNA-PK/PARP1 interplay, we performed structural studies of the puriﬁed DNA-PK/PARP1 complex loaded on DNA, analysed by electron microscopy and single particle analysis. We identiﬁed and analysed the structure of DNA-PK/PARP1 heterotetramers and dimers-of-tetra- mers. A tight interaction surface between the Ku dimer and PARP1 within these complexes is apparent. Fitting of the PARP1 catalytic domain into the EM map is consist- ent with it not being in direct contact with Ku, supporting the fact that the BRCT domain may be its interactor. Our ﬁtting also supports a model where one PARP1 subunit is involved in the complex with DNA-PK, opposite to the homodimerization occurring in free PARP1. The architecture of DNA-PK/PARP1 dimers is strikingly different from the DNA-PK assemblies we analysed in the past (6,8,46) and from those reported in SAXS studies on Y-shaped DNA (37). In the presence of PARP1, the catalytic domain of one of the DNA-PKcs molecules involved in the complex is in direct contact with the arm domain of its DNA-PKcs counterpart, supporting the possibility of an autophosphorylation in trans (47). The DNA-PKcs catalytic domains are not in the same orientation as in the SAXS analysis of DNA-PK loaded on hairpin DNA. This would suggest a role for PARP1 in modulating DNA repair by eliciting a major architectural rearrangement of the DNA-PK-mediated synapsis, as well as supporting a high degree of conform- ational articulation of DNA-PK in response to diverse stimuli.