4174 Nucleic Acids Research, 2012, Vol. 40, No. 9 the region of PARP1, which is not in direct contact with Ku. This is consistent with the biochemical observation that the pull-down of Ku is mediated by the PARP1 BRCT domain (22), since the BRCT domain is located N-terminally to the catalytic site, with the latter being at the C-terminus of the enzyme. As a control experiment, we also aligned the monomeric particles to our cryo-EM reconstruction of DNA-PKcs (29). We then classiﬁed them using a local MSA mask centered on the PARP1 density. The particles belonging to classes, which contained the density, were extracted to one subset. The particles belonging to classes, which did not contain the density, were extracted to a second subset. Particles belonging to classes representing top views, which are very similar for both complexes, were combined to both sub-data sets. We then processed these two subsets (DNA-PK/PARP = 5163 particles; DNA-PK = 4903 particles) separately, using the cryoEM reconstruction for DNA-PKcs as a starting model and an Eman-based projection matching reﬁnement. As illustrated in Supplementary Figure S1, these reconstruc- tions are in agreement with the overall features of the two complexes. In particular, the additional density for the PARP1-containing complex is shown in Supplementary Figure S1B, and the comparison between this DNA-PK/ PARP1 reconstruction and the more detailed one obtained with a more stringent choice of particles is shown in Supplementary Figure S1C. Synaptic DNA-PK/PARP1 dimers show orientation changes compared to DNA-PK dimers A total of 3125 dimeric appearances of the DNA-PK/ PARP1 complex were manually extracted to a sub-data set. They were then analysed in 2D by alignment and clas- siﬁcation, revealing a ‘T-shaped’ architecture. Each DNA-PK/PARP1 assembly within class averages of this dimer-of-heterotetramers retains the general features of the smaller DNA-PK/PARP1 particles, with a clear added density attached to the Ku end of the DNA-PK complex. Importantly, in contrast to DNA-PK dimers loaded on Y-shaped DNA (Figure 4A) and similarly to DNA-PK dimers loaded on hairpin DNA, one DNA-PKcs head domain is located in close contact with the DNA-PKcs arm domain within the DNA-PKcs opposite (Figure 4B). The orientation of the two halves of the complex is however different from the one of DNA-PK dimers on hairpin DNA (Figure 4C). This orientation is compatible with the ability to auto- phosphorylate in trans (47), since the two main DNA-PKcs autophosphorylation clusters have been mapped in the HEAT repeats region, forming the arm and palm domain of DNA-PKcs (64). The dimers-of- tetramers were analysed as a single stack of particles, which all seemed to contain the PARP1 density, since all the class averages calculated present a strong signal at the same position of the PARP1 density in the tetramer (white arrowhead in Figure 4B, while the black arrowhead points to the DNA-PKcs head domain). Figure 4. The dynamic nature of DNA-PKcs in the formation of dimers, as assessed by electron microscopy and single particle analysis. (A) DNA-PK dimers on Y-shaped DNA, with head domains facing opposite. (B) DNA-PK/PARP1 dimers on Y-shaped DNA, with head domains in close contact. (C) Schematics of the relative arrangement of DNA-PK head domains in DNA-PK dimers and DNA-PK/PARP1 dimers, both loaded on Y-shaped DNA. DNA-PKcs is coloured yellow, apart from the head domain, which is coloured red. Ku is represented as a blue ring and PARP1 as a green oval. DISCUSSION The interplay between the PARP1 and DNA-PK enzymes seems to be quite intricate. On one hand, an interaction between the two enzymes has been reported by several studies (12–22). On the other hand, other work implies that they have separate roles within the NHEJ DNA repair pathway (23,24). However, it may well be that different roles for PARP1 and DNA-PK coexist in NHEJ, since these molecules respond to a number of macromolecular interactions and post-translational modiﬁcations (36,65), which can seriously affect their conformation and functional properties. Here, we describe in vivo data suggesting that DNA-PK and PARP1 lack additivity in DSB repair. We puriﬁed a DNA-PK/PARP1 complex, which was tested for both kinase and PARP activities. This complex is organized in both ‘monomer-of-heterotetramers’ and ‘dimer-of- heterotetramers’ assemblies. In both cases, tight direct interactions of PARP1 with the Ku DNA recognition module are apparent. This is consistent with independent biochemical characterizations of a PARP1-Ku interaction, and with the observation that Ku is needed for PARP activation by 50-overhang DNA DSBs (19). By analogy with the DNA-PK heterotrimer and dimer-of- heterotrimers (6), these complexes do not form in the absence of DNA. This supports a mechanism of