Fig. 4
From: PKA prevents the resection of DNA double-strand breaks and favours nonhomologous end-joining
Effect of PKA on resection. (A) Scheme of the experimental strategy. The DIvA system is a U2OS cell containing an inducible Asi-SI endonuclease31,32. Nuclear translocation of the restriction enzyme Asi-SI is induced by 4-hydroxy-tamoxifen (4-OH-TAM), which then cleaves at restriction sites and then DNA. Resection of the Asi-SI-induced DSB will generate single-stranded DNA, which is then resistant to subsequent in vitro cleavage by the restriction enzyme BanI. We monitored cleavage at different BanI cleavage restriction sites located at different distances from the Asi-SI site on chromosome 22, which has been mapped and used in previous studies31,32. With the use of specific primers, PCR allows the quantification of uncleaved DNA (resulting from resection) and thus the extent of resection. (B) Quantification of the different siRNAs used for resection indicated in the figure. The distances of BanI from the Asi-SI site are indicated. The quantification corresponds to the means (± SEM) of 5 independent experiments.
