FIG. 2.

Rep nicking activity on RBE insertion 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. The position of insertions is indicated with a large arrow. The inserted sequences are given next to the mutant identifier. (B) Rep68 endonuclease reactions were performed on wt and insertion substrates in the presence of 0.5 mM ATP as described in Materials and Methods. Products were resolved on a 10% denaturing polyacrylamide gel. A representative gel is shown. Numbers above lanes indicate the total amount of Rep68 in the reactions expressed in femtomoles. The positions of substrates and products are indicated. (C) Nicking data were obtained from two independent trials. 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 in the two trials were then averaged and graphed for each mutant (n = 4 for all substrates except WT+10, where n = 7). Bars indicate the standard deviations from means.