Plasmids used for host cell reactivation assays.

Abstract

(A) pSF-tdTomato-END (for NHEJ, left) and pSF-tdTomato-HOM (for SSA, right) plasmids used for DSB repair assays. XhoI+ApaI double-digestion (= DSB) generated in vitro is repaired by either NHEJ or SSA after transfection in cells, thereby restoring EYFP expression. The common sequence between the two overlapping halves of EYFP in the SSA template (“YF” in grey) is 350bp long. Repair is measured by the percentage of EYFP+ cells among tdTomato+ cells. (B) pM1-Luc plasmid (left) and pRL-CMV (right) plasmids. Oxidative (8-oxoG) or UV damage (pyrimidine dimers) generated in vitro on pM1-Luc plasmid impairs expression of firefly luciferase (FL) that is then restored by BER or NER, respectively, once transfected in cells. Repair is measured by the FL activity after normalization to renilla luciferase (RL) activity (transfection control) and in comparison to the undamaged FL template (100% expression) in the same cells.</p