A C B D Nucleic Acids Research, 2012, Vol. 40, No. 9 4171 Figure 1. The effect of DNA-PK and PARP inhibitors on DSB repair. The recovery from IR-induced DSBs in the presence, or absence, of the DNA-PK inhibitor NU7441 or the PARP1 inhibitor KU-0058684, or both, was measured with g2HAX assays. (A) The effect on NU7441 on DNA-PK proﬁcient and deﬁcient cells. (B) The effect of KU-0058648 on PARP1 proﬁcient and deﬁcient cells. (C) The effect of NU7441 on PARP1 deﬁcient cells and KU-0058684 on DNA-PK deﬁcient cells. (D) The combinatorial effect of DNA-PK and PARP1 inhibitors on DNA-PK proﬁcient cells. Cells were exposed to 2 Gy ionizing radiation and allowed to recover. Data are the mean ± standard error of three independent experiments over a 24-h time course. PARP1+/+ and PARP1 / cells. After irradiation of PARP1 proﬁcient cells, DNA DSBs were rapidly induced as determined by gH2AX focus formation after 2 Gy IR (Figure 1). In repair-competent cell lines and con- sistent with our previous data with these cells (19) and independent data (59), DSB resolution followed a biphasic pattern. There was a rapid resolution of DSBs during the ﬁrst 2 h with 80 ± 24% and 71 ± 10% being repaired in PARP1+/+ and V3-YAC cells, respectively. However, PARP1 / MEFs and DNA-PKcs deﬁcient V3 cells followed a different repair kinetics, with a much slower rate such that only 27 ± 21% and 25 ± 9%, respectively, had been repaired. We have previously shown that the DNA-PK inhibitor, NU7441, at 1 mM radiosensitized V3-YAC cells but not V3 cells and inhibited DSB repair in SW620 cells (50). Investigation of the speciﬁcity of the inhibitors for their target at the DNA repair level revealed that NU7441 in- hibited repair in the V3-YAC cells (20.6 ± 20.3% repair) but not V3 cells, such that there was no difference in repair between V3-YAC+NU7441 and V3 cells (± NU7441) at 2 h (Figure 1 A). Similarly, we previously showed that PARP inhibition radiosensitized PARP+/+ cells but not PARP1 / cells (53). In the current study, the PARP inhibitor, KU-0058684, at 100 nM, a concentration that completely inhibited PARP activity in permeabilized cells (data not shown) retarded repair in the PARP1+/+ cells but not the PARP1 / cells (Figure 1B). We found that inhibiting PARP in cells lacking DNA-PK, or inhibit- ing DNA-PK in cells lacking PARP1 had no further impact on repair (Figure 1C). This ﬁnding, that inhibition of one enzyme does not further retard repair in a cell line already lacking the second enzyme, suggests that DNA-PK and PARP1 may function in the same epistatic pathway or in a co-operating manner in DSB repair. This was supported by a non-additive effect of KU-0058684 and NU7441 when utilized in combination in V3 YAC cells (Figure 1D). Similar effects were observed in PARP1+/+ cells (data not shown). Previous research utilizing PARP inhibitors in DNA-PK-defective V3 cells has been ambiguous—early research showed V3 cells were hypersensitive to the PARP inhibitor, 4-amino-1,8- naphthalimide (60) but these have not been replicated using the more potent and speciﬁc inhibitor, AG14361 (61), suggesting a compound-speciﬁc rather than class-effect. Electron microscopy analysis of the DNA-PK/PARP1 complex reveals additional density compared to the DNA-PK complex DNA-PK and PARP1 have been shown to interact in several studies in the past. We puriﬁed to homogeneity a human DNA-PKcs/Ku70/Ku80/PARP1 complex assembled on DNA from non-irradiated HeLa cell nuclear extracts by batch chromatography, column chromatography and glycerol gradient centrifugation (Figure 2A and B). Consistently with earlier independent data (17), our sample had PARylation and kinase activities (data not shown).