FIGURE 1.

Loss of ATM does not exacerbate HR deficiency in mouse H2AX^−/− ES cells. A, the HR reporter. Repair of an I-SceI-induced DSB by HR between sister chromatids generates WT GFP. B, relative HR of multiple isogenic ES clones containing the HR reporter. Relative HR of one A⁺H⁺ clone serves as control and is normalized to 100%. Symbols represent the mean of at least two independent experiments, each with triplicates. Error bars indicate S.E. Statistical significance was determined by Student's two-tailed unpaired t test (unknown variance): p < 0.0002 between A⁺H⁺ and others and p < 0.04 between A⁺H⁻ and A⁻H⁻. C and D, effect of KU55933 and KU60019 on HR in isogenic A⁺H⁺, A⁺H⁻, and A⁻H⁻ reporter ES cells. I-SceI-induced HR assays were performed on A⁺H⁺, A⁺H⁻, and A⁻H⁻ reporter cells transiently transfected with the I-SceI plasmid. Transfected cells were treated with 10 μm KU55933 (C) or 5 and 10 μm KU60019 (D) with DMSO as the mock control for 42 h starting at 5 h post-transfection, and I-SceI-induced GFP⁺ cells were quantified by flow cytometry 96 h post-transfection. Relative HR of A⁺H⁺ cells treated with DMSO serves as control and is set to 100%. Bars represent the mean of three independent experiments, each with triplicates. Error bars indicate S.E. Statistical significance was determined by paired t test between DMSO and ATM inhibitors: with KU55933 (C), p < 0.0004 in A⁺H⁺, not significant (p > 0.05) in A⁺H⁻, and p < 0.03 in A⁻H⁻, and with 5 μm KU60019 (D), p < 0.0006 in A⁺H⁺, p < 0.04 in A⁺H⁻, and p < 0.01 in A⁻H⁻ and with 10 μm KU60019, p < 0.0008 in A⁺H⁺, p < 0.04 in A⁺H⁻, and p < 0.02 in A⁻H⁻.