Figure 7: Activity of APOBEC3A (A3A) mutants in 293T transfectants

(a) A3A protein level in whole-cell lysates (20 μg protein) of cells transfected with an empty vector (Ctrl.) or expression constructs for wild-type (WT) A3A or its C101S, E72D or P134A variants. (b) Cytidine deamination activity of the transfectant lysates was examined in an in vitro reaction with a 5′ fluorescent dye-labelled ssDNA substrate of 40 bases (b). C>U deamination of the single cytidine residue of the substrate at position 23 followed by deglycosylation of the uridine and subsequent cleavage of the product at the abasic site was evaluated by electrophoresis of reactions of one hour duration on a polyacrylamide gel, whose fluorographic image is shown. (c) SDHB c.136C>U RNA editing in the transfectants. (d) Retrotransposition of a human LINE-1 element in a separate set of 293T transfectants. Retrotransposition, relative to the Ctrl. transfectant, was assessed with a luciferase reporter-based assay and is quantified as the ratio of firefly and Renilla luciferase activities. Mean and range (n=3) are shown for c and d.