Fig. 6
From: PKA prevents the resection of DNA double-strand breaks and favours nonhomologous end-joining
Effect of PKA stimulation on HR. (A) Strategy and reporter substrate used. Here, the DR-GFP substrate is chromosomally integrated into the genome of SV40-transformed human fibroblasts38. DR-GFP contains a tandem repeat of two inactive EGFP-encoding genes (upper panel). The 5’ repeat is interrupted by the I-SceI cleavage sequence (18 nt), and the 3’ repeat is truncated both at the 5’ and 3’ ends. None of these sequences were expressed and the cells were GFP negative. The expression of the I-SceI enzyme results in a cleavage target into the 5’ repeat (the SceGFP cassette); gene conversion (HR) with the 3’ repeat (iGFP) recreates a functional EGFP, leading to the fluorescence of cells37. Recombinant cells can be detected via FACS. (B) Cell cycle distribution in GC92 cells (human fibroblasts) after I-SceI transfection and PKA activation (8-Bromo-cAmp). The image shows 3 independent experiments. (C) Effect of stimulation of PKA by 8-Bromo-cAMP on HR after I-SceI transfection. Upper panel scheme of the experiment. Uncropped gels are shown in supplementary data. The values are shown normalized to those of the control (DMSO) and represent the average ± SEM of at least 3 independent experiments.
