Fig 2: Transient KU70 expression does not affect Tus/Ter-induced HR in Ku70–/–cells.A, Frequencies of Tus/Ter-induced and I-SceI-induced repair in four independently derived Ku70–/– 6xTer-HR reporter clones (clones 27, 34, 41, and 47) with and without transient expression of exogenous hKU70. Cells transiently co-transfected with empty pcDNA3beta or pcDNA3beta-hKU70 expression vector and either empty, 3xMyc-NLS Tus or 3xMyc-NLS I-SceI expression vectors. Each column represents the mean of duplicate samples from eight independent experiments (n = 8), values are corrected for transfection efficiency. Error bars: s.e.m. Tus-induced total HR, t test: #27 +EV vs. +hKU70, p = 0.8355; #34 +EV vs. +hKU70, p = 0.3799; #41 +EV vs. +hKU70, p = 0.2710; #47 +EV vs. +hKU70, p = 0.4703; Tus-induced STGC, t test: #27 +EV vs. +hKU70, p = 0.7892; #34 +EV vs. +hKU70, p = 0.4223; #41 +EV vs. +hKU70, p = 0.2345; #47 +EV vs. +hKU70, p = 0.4426; Tus-induced LTGC, t test: #27 +EV vs. +hKU70, p = 0.4375; #34 +EV vs. +hKU70, p = 0.2413; #41 +EV vs. +hKU70, p = 0.8608; #47 +EV vs. +hKU70, p = 0.9872; Tus-induced LTGC/(Total HR), t test: #27 +EV vs. +hKU70, p = 0.2701; #34 +EV vs. +hKU70, p = 0.4964; #41 +EV vs. +hKU70, p = 0.2507; #47 +EV vs. +hKU70, p = 0.8222. I-SceI-induced total HR, t test: #27 +EV vs. +hKU70, p<0.0001; #34 +EV vs. +hKU70, p<0.0001; #41 +EV vs. +hKU70, p<0.0001; #47 +EV vs. +hKU70, p<0.0001; I-SceI-induced STGC, t test: #27 +EV vs. +hKU70, p<0.0001; #34 +EV vs. +hKU70, p<0.0001; #41 +EV vs. +hKU70, p<0.0001; #47 +EV vs. +hKU70, p<0.0001; I-SceI-induced LTGC, t test: #27 +EV vs. +hKU70, p = 0.0002; #34 +EV vs. +hKU70, p<0.0001; #41 +EV vs. +hKU70, p<0.0001; #47 +EV vs. +hKU70, p<0.0001; I-SceI-induced LTGC/(Total HR), t test: #27 +EV vs. +hKU70, p = 0.0004; #34 +EV vs. +hKU70, p<0.0001; #41 +EV vs. +hKU70, p<0.0001; #47 +EV vs. +hKU70, p = 0.0003. One-way ANOVA (Analysis of Variance) test comparing trend in HR: Tus-induced HR, total HR, p = 0.2388; STGC, p = 0.1923; LTGC, p = 0.9660; LTGC/(Total HR), p = 0.5923. I-SceI-induced HR, total HR, p<0.0001; STGC, p<0.0001; LTGC, p<0.0001; LTGC/(Total HR), p<0.0001. B, RT qPCR analysis of hKU70 in transfected Ku70–/–clones. hKU70 expression normalized to GAPDH and displayed as fold difference from Ku70–/–reporter clone 27 of the same experiment (x = -2??Ct, with ??Ct = [CtKU70-CtGAPDH]-[CtKU70-CtGAPDH]). Error-bars represent standard deviation of the ?Ct value (SDEV = v[SDEVKU702 + SDEVGAPDH2]). C, Western blot for abundance of hKU70 protein in Ku70–/–reporter clones transiently transfected with empty pcDNA3beta or pcDNA3beta-hKU70. D, Fold enrichment of cultures transiently expressing exogenous GFP. Results represent fold enrichment of cultures transiently co-transfected with GFP-expression plasmid and either pcDNA3beta or pcDNA3beta-hKU70 expression vector over co-cultured cells transiently transfected with pcDNA3beta alone. Each plot represents the mean of triplicate samples from three independent experiments (n = 3), fold enrichment GFP+ cells normalized to 0 µg/mL phleomycin control. Error bars: s.e.m.

Fig 3: Impact of CtIP depletion on repair frequencies in the presence or absence of hKU70.A, Frequencies of Tus/Ter-induced repair in three independently derived Ku70–/– 6xTer-HR reporter clones (clones 27, 41, and 47) transiently expressing exogenous hKU70 and transfected with siRNAs shown. Cells transiently co-transfected with empty pcDNA3beta or pcDNA3beta-hKU70 expression vector and empty or 3xMyc-NLS Tus expression vectors treated with either siLUC or siCtIP. Each column represents the mean of duplicate samples from eight independent experiments (n = 8), values are corrected for transfection efficiency. Error bars: s.e.m. Tus-induced total HR, siLUC vs. siCtIP t test: #27 +EV, p = 0.0030; #41 +EV, p = 0.0207; #47 +EV, p = 0.0070; #27 +hKU70, p = 0.0047; #41 + hKU70, p = 0.0281; #47 + hKU70, p = 0.0148; Tus-induced STGC, t test: #27 +EV, p = 0.0011; #41 +EV, p = 0.0104; #47 +EV, p = 0.0070; #27 +hKU70, p = 0.0030; #41 + hKU70, p = 0.0207; #47 + hKU70, p = 0.0148; Tus-induced LTGC, t test: #27 +EV, p = 0.2786; #41 +EV, p = 0.5737; #47 +EV, p = 0.1304; #27 +hKU70, p = 0.8785; #41 + hKU70, p = 0.5737; #47 + hKU70, p = 0.5737; Tus-induced LTGC/(Total HR), t test: #27 +EV, p = 0.0002; #41 +EV, p = 0.0006; #47 +EV, p = 0.0006; #27 +hKU70, p = 0.0019; #41 + hKU70, p = 0.0030; #47 + hKU70, p = 0.0650; One-way ANOVA (Analysis of Variance) test comparing trend in Tus-induced Total HR: siLUC vs siCtIP all, p<0.0001; siLUC vs siCtIP +EV, p<0.0001; siLUC vs siCtIP +hKU70, p = 0.0002; siLUC all, p = 0.8904; siCtIP all, p = 0.1322. Tus-induced STGC, one-way ANOVA test: siLUC vs siCtIP all, p<0.0001; siLUC vs siCtIP +EV, p<0.0001; siLUC vs siCtIP +hKU70, p<0.0001; siLUC all, p = 0.9108; siCtIP all, p = 0.1155. Tus-induced LTGC, one-way ANOVA test: siLUC vs siCtIP all, p = 0.4334; siLUC vs siCtIP +EV, p = 0.3194; siLUC vs siCtIP +hKU70, p = 0.4144; siLUC all, p = 0.6254; siCtIP all, p = 0.2231. Tus-induced LTGC/(Total HR), one-way ANOVA test: siLUC vs siCtIP all, p<0.0001; siLUC vs siCtIP +EV, p = 0.0004; siLUC vs siCtIP +hKU70, p = 0.0012; siLUC all, p = 0.9449; siCtIP all, p = 0.2989. B, Frequencies of I-SceI-induced repair in three independently derived KU70?/? 6xTer-HR reporter clones. Cells transiently co-transfected with empty pcDNA3beta or pcDNA3beta-hKU70 expression vector and empty or 3xMyc-NLS I-SceI expression vectors treated with either siLUC or siCtIP. Each column represents the mean of duplicate samples from eight independent experiments (n = 8), values are corrected for transfection efficiency. Error bars: s.e.m. I-SceI-induced total HR, siLUC vs. siCtIP t test: #27 +EV, p = 0.0379; #41 +EV, p = 0.0281; #47 +EV, p = 0.0499; #27 +hKU70, p = 0.0003; #41 + hKU70, p = 0.0019; #47 + hKU70, p = 0.0011; I-SceI-induced STGC, siLUC vs. siCtIP t test: #27 +EV, p = 0.0379; #41 +EV, p = 0.0281; #47 +EV, p = 0.0379; #27 +hKU70, p = 0.0002; #41 + hKU70, p = 0.0019; #47 + hKU70, p = 0.0011; I-SceI-induced LTGC, siLUC vs. siCtIP t test: #27 +EV, p = 0.1104; #41 +EV, p = 0.7984; #47 +EV, p = 0.3282; #27 +hKU70, p = 0.3282; #41 + hKU70, p = 0.1949; #47 + hKU70, p = 0.1949; I-SceI-induced LTGC/(Total HR), siLUC vs. siCtIP t test: #27 +EV, p = 0.0011; #41 +EV, p = 0.0379; #47 +EV, p = 0.0070; #27 +hKU70, p = 0.0006; #41 + hKU70, p = 0.0650; #47 + hKU70, p = 0.0070; I-SceI-induced Total HR, one-way ANOVA test: siLUC vs siCtIP all, p<0.0001; siLUC vs siCtIP +EV, p = 0.0139; siLUC vs siCtIP +hKU70, p<0.0001; siLUC all, p<0.0001; siCtIP all, p<0.0001. I-SceI-induced STGC, one-way ANOVA test: siLUC vs siCtIP all, p<0.0001; siLUC vs siCtIP +EV, p = 0.0106; siLUC vs siCtIP +hKU70, p<0.0001; siLUC all, p<0.0001; siCtIP all, p<0.0001. I-SceI-induced LTGC, one-way ANOVA test: siLUC vs siCtIP all, p<0.0001; siLUC vs siCtIP +EV, p = 0.1503; siLUC vs siCtIP +hKU70, p = 0.1010; siLUC all, p = 0.0010; siCtIP all, p<0.0001. I-SceI-induced LTGC/(Total HR), one-way ANOVA test: siLUC vs siCtIP all, p<0.0001; siLUC vs siCtIP +EV, p<0.0001; siLUC vs siCtIP +hKU70, p<0.0001; siLUC all, p = 0.0147; siCtIP all, p<0.0001. C, Observed repair frequencies for Tus or I-SceI induced HR, STGC and LTGC expressed as the ratio of siCtIP frequency/siLUC frequency for data from clones 27, 41 and 47 shown in panels A and B. One-way ANOVA test: Tus-induced total HR, p = 0.0779; Tus-induced STGC, p = 0.0564; Tus-induced LTGC, p = 0.2067. One-way ANOVA test: I-SceI-induced total HR, p<0.0001; I-SceI-induced STGC, p<0.0001; I-SceI-induced LTGC, p = 0.0832. D, RT qPCR analysis of CtIP mRNA in siRNA-transfected Ku70–/–clones. Data normalized to GAPDH and expressed as fold difference from siLUC sample from the same experiment (x = -2??Ct, with ??Ct = [CtsiCtIP-CtGapdh]-[CtsiLUC-CtsiGAPDH]). Error-bars represent standard deviation of the ?Ct value (SDEV = v[SDEVCtIP2 + SDEVGAPDH2]).

Fig 4: Phosphorylation of S426 by CDK2 shifts WRN’s DNA repair activity toward homologous recombination. (a) Recruitment of mCherry-WRN to laser tracks. U2OS cells expressing mCherry-tagged WT/S426A/D-WRN were micro-irradiated with 435 nm laser to induce DSBs. Images were captured at 10 sec intervals for 5 min. Scale bar, 10 µm. n = 3 for each group. (b) Real-time recruitment of mCherry-WRN to DSB tracks in cells as in panel A. (c) U2OS cells were ?-irradiated (10 Gy), 30 min after irradiation, cells were lysed and mixed with FLAG-tagged purified WT/S426A/D-WRN protein for binding assay. Indicated WRN interacting proteins were probed by Western blot for the antigens shown. (d) HR efficiency in DR-GFP cells. (e) Total NHEJ efficiency in EJ5 cells. DR-GFP and EJ5 cells were induced by co-transfecting I-SceI and mCherry WT/S426A/D-WRN expression plasmid. Graph represents relative repair efficiency as measured by GFP positive cells normalized to mCherry expression. Immunoblots represent protein expression levels. (f) Schematic diagram of CDK2-mediated WRN phosphorylation on DSB pathway choice. When DSBs occur in the G1 phase of the cell cycle, the KU70/80 complex interacts directly with WRN and stimulates WRN’s exonuclease activity. Stimulated by KU70/80, DNA-PKcs also regulate WRN’s enzymatic activities. Finally, fully activated WRN utilizes its nuclease activity to create DNA ends suitable for XRCC4-DNA ligase IV complex-mediated ligation. When cells progress into S phase of the cell cycle, CDK2 becomes active via CycE, and phosphorylates WRN on S426. S426 phosphorylated WRN has increased affinity for RPA, thereby promoting end resection and stabilization of the resected single-stranded DNA. Also, S426 phosphorylation enhances WRN’s strand annealing activity to promote strand annealing activity to form D-loop formation for efficient HR. Error bars represent SD from four independent experiments. p-value, *, <0.05; **, <0.01; ***, <0.001