Fig. 5
From: PKA prevents the resection of DNA double-strand breaks and favours nonhomologous end-joining
Effect of PKA on NHEJ. (A) Strategy and reporter substrate used. Here, the substrate is chromosomally integrated into the genome of SV40-transformed human fibroblasts16. Before cleavage, the cells express the reporter H2Kd. The reporter CD4 is not expressed because it is too far from the promoter (prom). Two cleavage sites for the meganuclease I-SceI (18 nt recognition) are inserted, one between the promoter and the H2Kd sequence and the other between the H2Ks and CD4. The expression of I-SceI results in targeted cleavage of the two I-SceI sites and excision of the H2Kd intervening fragment. The end-joining of the DSBs that bear no sequence homology put the CD4 reporter under the control of the promoter, leading to its expression. The frequency of CD4-positive cells thus provides an estimation of NHEJ efficiency. This substrate has been largely validated and characterized11,12,16,17,33,34,35. (B) Effect of silencing PKA with siRNA. The values are shown normalized to those of the control siRNA and represent the average ± SEM of 3 independent experiments. (C) Effect of stimulating PKA (8-Bromo-cAMP) and inhibiting PKA (H89) on NHEJ. Upper panel: scheme of the experiment. Lower left panel: expression of I-SceI under different conditions. Uncropped gels are shown in supplementary data. Lower right panel: NHEJ efficiency under the different conditions. The values are shown normalized to those of the control (DMSO) and represent the average ± SEM of at least 3 independent experiments.
