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. 2011 Oct 24;286(49):42470–42482. doi: 10.1074/jbc.M111.309252

FIGURE 1.

FIGURE 1.

Inhibitor of DNA-PKcs kinase activity causes a marked shift to incorrect end use during repair of tandem DSBs. A, a diagram for the end use assay based on the EJ5-GFP reporter, which contains two tandem recognition sites for the I-SceI endonuclease. Expression of I-SceI and the nonprocessive 3′-exonuclease Trex2 leads to a high frequency of I-SceI-resistant EJ products between proximal DSB ends (Proximal-EJ), which can be examined by PCR amplification (primers p1 and p2) and I-SceI digestion analysis. Such expression also leads to I-SceI-resistant EJ products between distal DSB ends that are marked by restoration of a GFP+ cassette (Distal-EJ). For comparison, a relative measure of distal end use is quantified by dividing the distal EJ value by the proximal EJ value for individual samples. B, expression of I-SceI and Trex2 as a fusion protein leads to a high frequency of I-SceI-resistant proximal EJ products. U2OS cells with an integrated copy of the EJ5-GFP reporter were left untransfected, or transfected with expression vectors for I-SceI fused with either Trex2-WT or Trex2-H188A (nuclease dead). Subsequently, proximal EJ was evaluated as described in A. Shown are amplification products that are uncut (U) or digested with the I-SceI endonuclease (S). C, treating cells with kinase inhibitors of either ATM or DNA-PKcs cause a substantial shift toward distal end use. The U2OS EJ5-GFP cell line was transfected with an expression vector for the I-SceI-Trex2 fusion, and treated with a kinase inhibitor of ATM (ATMi, KU55933, 10 μm), a kinase inhibitor of DNA-PKcs (PKi, NU7026, 20 μm), or vehicle (DMSO). Shown are the frequencies of distal EJ (%GFP+), proximal EJ (%I-SceI-resistant, p1/p2), and relative distal end use (distal EJ/proximal EJ, DMSO = 1) from these transfections. Asterisks denote statistical difference from DMSO samples, p < 0.0022. D, treating cells with a kinase inhibitor of DNA-PKcs causes elevated distal EJ in WT mES cells but not DNA-PKcs−/− mES cells. Using WT and DNA-PKcs−/− mES cell lines the EJ5-GFP reporter was integrated into the pim1 locus, I-SceI was co-expressed with Trex2, and transfections were treated with PKi or DMSO as in C. Shown are distal EJ values for these transfections. The asterisk denotes a statistical difference from DMSO samples (p = 0.001).