Fig 2: The influence of DNAPKcs and XLF on the sizes of deletions and insertions at EJ junctions, and on a +1 insertion EJ event likely caused by staggered Cas9 DSBs.a Shown is the frequency of deletion sizes for parental cells with or without M3814, PRKDC-KO, and XLF-KO cells for the deletion events shown in Fig. 3d. n = 3 biologically independent transfections. Statistics with unpaired two-tailed t test using Holm–Sidak correction. 1–5 nt deletion: ***P = 0.000475, **P = 0.00506, *P = 0.0447. 6–10nt deletion: Parental DMSO vs 500 nM M3814 ***P = 0.000937, Parental DMSO vs. PRKDC-KO ***P = 0.000596, *P = 0.0247. 11–15 nt deletion: **P = 0.00824. 16–20 nt deletion: ***P = 0.000189, *P = 0.0491. n.s.= not significant. b Shown is the frequency of deletion sizes for parental and XLF-KO cells with or without M3814 for the deletion events shown in Fig. 3e. n = 3 biologically independent transfections. Statistics as in (a). 1–5nt deletion: ****P < 0.0001, Parental EV DMSO vs. 500 nM M3814 ***P = 0.000473, XLF-KO XLF-WT DMSO vs. 500 nM M3814 ***P = 0.000473, **P = 0.00374. 6–10nt deletion: ****P < 0.0001, Parental EV DMSO vs. 500 nM M3814 **P = 0.00127, XLF-KO XLF-WT DMSO vs. 500 nM M3814 **P = 0.00127, *P = 0.041. 11–15nt deletion: n.s.= not significant. 16–20 nt deletion: Parental EV DMSO vs. 500 nM M3814 ***P = 0.000189, XLF-KO XLF-WT DMSO vs. 500 nM M3814 ***P = 0.000274, XLF-KO XLF-K160D DMSO vs. 500 nM M3814 ***P = 0.000121, *P = 0.0104. >20 nt deletion: †P = 0.0478 but not significant after correction (unadjusted P-value), n.s.= not significant. c Shown is the frequency of insertion sizes for parental cells with or without M3814, PRKDC-KO, and XLF-KO cells for the insertion events shown in Fig. 3d. n = 3 biologically independent transfections. Statistics as in (a). 1 nt insertion: **P = 0.00147. 2 nt insertion: **P = 0.00692. =3 nt insertion: *P = 0.0446, †P = 0.0257 but not significant after correction (unadjusted P-value). n.s.= not significant. d DNAPKcs is less important to promote EJ with 1 nt insertions (+1 EJ) compared to XLF. Shown is the EJ7+1-GFP reporter to measure +1 EJ, which is the same chromosomal reporter as in Fig. 1, but using a different 5' sgRNA. n = 6 biologically independent transfections. Statistics as in (a). ****P < 0.0001. Data are represented as mean values ± SD.

Fig 3: Olaparib treatment sensitizes cells to ionizing radiation. (A) Clonogenic assay. After trypsinization, 300 HeLa cells were plated. Once cells had attached, they were treated with olaparib for 1 h and then irradiated. The DMSO group was used as a control. The cells were cultured for two weeks, and the surviving colonies were observed and counted. *P<0.05 and **P<0.01. (B) The effect of combinational treatment with olaparib and the DNA-PKcs inhibitor NU7441 on the cell colony formation of irradiated HeLa cells. After trypsinization, HeLa cells were seeded at different cell concentrations as indicated (in DMSO controls 300 cells seeded/plate). After the cells were attached, the cells were treated with olaparib (10 µM), or olaparib (10 µM) and NU7441 (5 µM) for 1 h and then irradiated. The DMSO group was used as a control and cultured continuously for two weeks. Colonies containing 50 or more cells were scored. ***P<0.001, as indicated. (C) HeLa cells were prepared as cell suspensions of 5×104 cells/ml and seeded into 6-well cell culture plates (3 ml/dish, n=3). After the cells were attached, different inhibitors [DMSO, NU7441 (5 µM), olaparib (10 µM), and olaparib (10 µM) + NU7441 (5 µM)] were added to the culture. The number of cells was counted by flow cytometry to calculate the mean concentration of each group on each day. *P<0.05 and **P<0.01. DNA-PKcs, DNA-dependent protein kinase catalytic subunit.

Fig 4: DNA-PK is modified by PAR in response to DNA damage. (A) IP with an anti-DNA-PKcs antibody was performed to detect the PAR modification of DNA-PKcs in HeLa cells after treatment with different irradiation doses (0, 0.5, 1, 2, 4, 6 and 8 Gy). The cells were harvested 2 h after irradiation and then lysed. (B) Western blotting was used to detect DNA-PKcs in the IP product of the anti-PAR antibody from HeLa cells treated with or without irradiation. (C) The effect of olaparib on the PARylation of DNA-PKcs. HeLa cells were treated with different concentrations of olaparib. After 24 h, the cells were harvested and lysed, and IP was used to detected the PAR modification of DNA-PKcs. (D) The effect of olaparib on the chromatin binding of DNA-PKcs in irradiated HeLa cells. DNA-PKcs, DNA-dependent protein kinase catalytic subunit; PAR, poly(ADP-ribose); IP, immunoprecipitation; IR, irradiation.

Fig 5: Olaparib treatment promotes the autophosphorylation of DNA-PKcs. (A) The effect of olaparib on the phosphorylation of DNA-PKcs in response to IR. HeLa cells were treated with 10 µM olaparib for 24 h and irradiated with 8 Gy IR. After 1, 2, 4 and 8 h, the expression of phosphorylated DNA-PKcsSer2056 and DNA-PKcsThr2609 was detected by western blotting. (B) HeLa cells were treated with 1 or 10 µM olaparib for 24 h, and the cells were irradiated with 8 Gy IR. After 1 h, the expression of phosphorylated DNA-PKcs/S2056 was detected by western blotting. The effects of olaparib on the formation of ?H2AX and DNA-PKcs/S2056 foci were detected by immunofluorescence staining in 8 Gy-irradiated HeLa cells. (C) The dynamic changes in ?H2AX foci in 8 Gy-irradiated HeLa cells (magnification ×100). (D) The dynamic changes in ?H2AX foci in 8 Gy-irradiated HeLa cells. (E) The dynamic changes in DNA-PKcs/S2056 foci in 8 Gy-irradiated HeLa cells. *P<0.05 and **P<0.01 vs. DMSO treatment. DNA-PKcs, DNA-dependent protein kinase catalytic subunit; IR, irradiation; ?H2AX, ?-histone family member 2AX; PARP1, poly(ADP-ribose) polymerase 1.