4170 Nucleic Acids Research, 2012, Vol. 40, No. 9 MATERIALS AND METHODS Inhibitors The DNA-PK inhibitor NU7441 (48) and the PARP1 inhibitor KU-0058684 (49), synthesized by Newcastle University, UK, and KuDOS Pharmaceuticals (Cambridge, UK), respectively, were dissolved in 100% (v/v) dimethyl sulfoxide (DMSO) at stock concentrations of 2 mM and 200 mM, respectively, and stored at 20 C. Drugs were added in 1% (v/v) ﬁnal DMSO concentration, and control cells were exposed to 1% DMSO. NU7441 (IC50 for DNA-PK inhibition = 14 nM) was used at 1 mM as previously described (50). KU-0058684 (IC50 for PARP1 inhibition = 3.2 nM) was used at 100 nM. Cell lines and culture Primary PARP1+/+ and PARP1 / mouse embryonic ﬁbroblasts (MEFs) were a gift from Professor Gilbert de Murcia, France (51). Chinese hamster ovary cell lines V3 (mutated in DNA-PKcs) and V3 YAC (V3 transfected with yeast artiﬁcial chromosome containing human DNA-PKcs cDNA) were provided by Dr Penny Jeggo, UK (52). Cell lines were cultured in RPMI 1640+10% (v/v) fetal calf serum and 1% (v/v) penicillin– streptomycin. V3 YAC cells were maintained under anti- biotic selection with 500 mg/ml geneticin to ensure YAC retention. PARP and DNA-PK activities of these cell lines have been described previously (20,53). DNA double strand break determination by gH2AX immunoﬂuorescence DSBs were detected by gH2AX immunoﬂuorescence assays utilizing an anti-phospho H2AX antibody as previously published (anti ser139, clone JBW301, Upstate, now Millipore Corp., USA) (54). NU7441 and KU-0058684 were added to cells 1 h before 2 Gy X-irradiation (Gulmay D3300 X-Ray System, Gulmay Medical Ltd., UK) as previously described (50,54). Image capture was performed as previously described (54), recording 2 viewﬁelds each containing 20 nuclei. Mean focus number per nucleus was converted to foci/pg DNA present to account for different cellular DNA content (determined by picogreen DNA quantita- tion assay, Qiagen, UK). DNA repair was calculated as percent focus loss. Isolation of the DNA-bound DNA-PKcs/Ku70/Ku80/ PARP1 complexes The complex containing DNA-PKcs, Ku70, Ku80 and PARP1 was puriﬁed from HeLa nuclear extract (CilBiotech, Belgium) with a buffer system based on 20 mM HEPES (pH 7.5), 1 mM DTT, 0.5 mM EDTA, 0.001% b-octylglucoside and 10% glycerol, with NaCl concentrations ranging from 50 to 1000 mM in different chromatographic steps. After fractionation on heparin and dsDNA cellulose resins (GE Healthcare, Sweden), fractions containing DNA-PKcs, Ku70 and Ku80 were loaded onto a 1 ml HiTrapQ column (GE Healthcare, Sweden) and eluted with a linear NaCl gradient. This differs from our previous protocol for the puriﬁcation of DNA-PK heterotrimer (6), since we used a HiTrapQ column instead of a Resource Q column, therefore obtain- ing a less stringent fractionation of proteins interacting with Q resin. Fractions containing all three protein com- ponents were incubated with -phosphatase for 1 h at 4 C. The sample was incubated for an additional hour at 4 C with dsDNA iminobiotinylated at the 50-end of the longer oligo (IBA GmbH, Germany). The sequences of the oligos (with the linear duplex segments in uppercase) are shown in Table 1. The samples were then loaded on a 18–60% glycerol gradient, with the buffer system 20 mM HEPES (pH 7.5), NaCl 200 mM, 1 mM DTT, 0.5 mM EDTA, 0.001% b-octylglucoside. Further separation was achieved by using a Beckman SW28 rotor, spinning for 72 h at 25 000 rpm. Fractions were collected from the bottom and analysed by means of SDS–PAGE on 4–12% Bis-Tris NOVEX gradient gels (Invitrogen, UK). This analysis highlighted the co-migration of stoichio- metric quantities of an additional protein with the three protein components of DNA-PK (Figure 2B). The fourth protein species was identiﬁed as PARP1 by mass spectrometry and immunoblotting (data not shown). Electron microscopy and single-particle analysis of negatively stained DNA-PKcs/Ku70/Ku80/PARP1 complexes The DNA-PKcs/Ku70/Ku80/PARP1 sample was applied to carbon-coated grids, negatively stained with 1% uranyl acetate and observed in a JEOL 1200EX electron micro- scope operating at 100 kV. Micrographs were recorded at a calibrated magniﬁcation of 34 000 under low-dose conditions and digitized with a Nikon Coolscan scanner. A total of 21 000 molecular images were extracted with the Boxer program from the EMAN package, processed and reﬁned with a combination of EMAN (55), IMAGIC-5 (56) and Spider (57) routines. 3D ﬁtting experiments were performed using Chimera (58). RESULTS DNA-PK and PARP1 lack additivity in DSB repair We studied the role of PARP1 in the cellular response to clinically relevant IR doses by measuring DNA DSB repair in DNA-PK+/+ (V3-YAC), DNA-PK / (V3), Table 1. Oligos used to assemble the structured DNA on which the DNA-PK/PARP1 complex is loaded Oligo A Oligo B Biotin-50-CGCGCCCAgctttcccagctaATAAACTAAAAACTATTATTATGGCCGCACGCGTCCACCATGGGGTACAACT-30 50-AGTTGTACCCCATGGTGGACGCGTGCGGCCATAATAATAGTTTTTAGTTTATTGGGCGCG-30 Upper case: bases annealing to for dsDNA. Lower case: bases forming a loop.