FIG. 6.

Rep endonuclease activity on RBE′ substitution mutants in the NOSTEM background. (A) The NOSTEM substrate is depicted. 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 terminal hairpins of the RBE′ substitution mutants are also depicted. Mutated sequences are indicated in boldface. (B) Rep68 endonuclease reactions were performed on NOSTEM and NOSTEM RBE′ substitution substrates in the absence of ATP as described in Materials and Methods. Products were resolved on a 10% denaturing polyacrylamide gel. The gel was 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 = 4 for all mutants). Bars indicate standard deviations from the means.