Rad51 recruitment and exclusion of non-homologous end joining during homologous recombination at a Tus/Ter mammalian replication fork barrier
Fig 2
Stable re-expression of wtXrcc4 does not affect Tus/Ter-induced HR in Xrcc4Δ/Δ cells.
A, RT qPCR analysis of Xrcc4 expression in stably transduced Xrcc4fl/fl or Xrcc4Δ/Δ clones. Xrcc4 expression normalized to GAPDH and displayed as fold difference from Xrcc4fl/fl parental reporter clone 8 of the same experiment (x = -2ΔΔCt, with ΔΔCt = [CtXrcc4-CtGapdh]-[CtXrcc4-CtGAPDH]). Error-bars represent standard deviation of the ΔCt value (SDEV = √[SDEVXrcc42 + SDEVGAPDH2]). B, Xrcc4 protein abundance by Western blot in extracts of parental Xrcc4fl/fl clone #8 and Xrcc4Δ/Δ clone #11 and derivative cultures stably transduced with empty lentiviral vector (pHIV-NAT-hCD52, “EV”) or HA-tagged mouse Xrcc4 lentiviral expression vector (“X4”). C, Fold enrichment of cultures transiently expressing exogenous GFP. Results represent fold enrichment of cultures transiently co-transfected with pcDNA3beta and GFP-expression plasmid 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. D, Frequencies of Tus/Ter-induced and I-SceI-induced repair in Xrcc4fl/fl clone #8 or Xrcc4Δ/Δ clone #11 6xTer-HR reporter cells lentivirally transduced with pHIV-NAT-hCD52-EV (empty vector control) or pHIV-NAT-hCD52-mXrcc4 (expressing HA-tagged mouse Xrcc4 expression vector) with selection of transduced cells in 100 μg/ml NTC. Cells were transiently transfected with empty, 3xMyc-NLS Tus or 3xMyc-NLS I-SceI expression vectors. Each plot represents the mean of duplicate samples from six independent experiments (n = 6). Error bars: s.e.m. Tus-induced Total HR, t-test: flox8 +Xrcc4 vs. flox8 +EV p = 0.0662; del11 +Xrcc4 vs. del11 +EV p = 0.4509; del11 +EV vs. flox8 +EV p = 0.6719; del11 +Xrcc4 vs. flox8 +Xrcc4 p = 0.0588; del11 +Xrcc4 vs. flox8 +EV p = 0.5025. Tus-induced STGC, t-test: flox8 +Xrcc4 vs. flox8 +EV p = 0.0836; del11 +Xrcc4 vs. del11 +EV p = 0.4126; del11 +EV vs. flox8 +EV p = 0.6144; del11 +Xrcc4 vs. flox8 +Xrcc4 p = 0.0595; del11 +Xrcc4 vs. flox8 +EV p = 0.7215. Tus-induced LTGC, t-test: flox8 +Xrcc4 vs. flox8 +EV p = 0.6686; del11 +Xrcc4 vs. del11 +EV p = 0.5972; del11 +EV vs. flox8 +EV p = 0.5313; del11 +Xrcc4 vs. flox8 +Xrcc4 p = 0.3007; del11 +Xrcc4 vs. flox8 +EV p = 0.7870. Tus-induced LTGC/Total HR ratio, t-test: flox8 +Xrcc4 vs. flox8 +EV p = 0.9182; del11 +Xrcc4 vs. del11 +EV p = 0.2133; del11 +EV vs. flox8 +EV p = 0.4686; del11 +Xrcc4 vs. flox8 +Xrcc4 p = 0.8360; del11 +Xrcc4 vs. flox8 +EV p = 0.5771. I-SceI-induced Total HR, t-test: flox8 +Xrcc4 vs. flox8 +EV: p = 0.1292; del11 +Xrcc4 vs. del11 +EV p<0.0001; del11 +EV vs. flox8 +EV p<0.0001; del11 +Xrcc4 vs. flox8 +Xrcc4 p = 0.1030; del11 +Xrcc4 vs. flox8 +EV p = 0.8690. I-SceI-induced STGC, t-test: flox8 +Xrcc4 vs. flox8 +EV p = 0.1353; del11 +Xrcc4 vs. del11 +EV p<0.0001; del11 +EV vs. flox8 +EV p<0.0001; del11 +Xrcc4 vs. flox8 +Xrcc4 p = 0.0939; del11 +Xrcc4 vs. flox39 +EV p = 0.0081. I-SceI-induced LTGC, t-test: flox8 +Xrcc4 vs. flox8 +EV p = 0.1840; del11 +Xrcc4 vs. del11 +EV p<0.0001; del13 +EV vs. flox39 +EV p<0.0001; del11 +Xrcc4 vs. flox8 +Xrcc4 p = 0.7589; del11 +Xrcc4 vs. flox39 +EV p = 0.1347. I-SceI-induced LTGC/Total HR ratio, t-test: flox8 +Xrcc4 vs. flox8 +EV p = 0.5908; del11 +Xrcc4 vs. del11 +EV p = 0.0001; del11 +EV vs. flox8 +EV p = 0.0001; del11 +Xrcc4 vs. flox8 +Xrcc4 p = 0.3729; del11 +Xrcc4 vs. flox39 +EV p = 0.4615.