FIG. 5.

Rep endonuclease activity on RBE′ substitution mutants. (A) The wt AAV TR is depicted after extrusion of trs stem-loop structure. The RBE is indicated with a box, the RBE′ is indicated with a dashed oval, the minimal trs is indicated with small circles, and the actual nicking site is indicated with a small arrow. Additionally, the position of the SmaI endonuclease site is indicated with a line. The terminal hairpins of flop and RBE′ substitution mutants are also depicted. Mutated sequences are indicated in boldface. (B) Rep68 endonuclease reactions were performed on wt and substitution substrates in the presence of 0.5 mM ATP as described in Materials and Methods. Products were resolved on a 10% denaturing polyacrylamide gel. Numbers above lanes indicate the total amount of Rep68 in the reactions expressed in femtomoles. The positions of substrates and products are indicated. (C) The gel from panel B and a second gel containing reaction products from wt and LINEAR substrates were phosphorimaged, and the amounts of substrate and product were determined. The relative Rep specific activity for each mutant was expressed as the fraction of mutant substrate nicked divided by the fraction of wt substrate nicked at the same Rep concentration. Ratios obtained at different Rep concentrations were then averaged and graphed for each mutant (n = 2 for all mutants except for LINEAR, where n = 4). Bars indicate ranges between the different Rep concentrations.