FIG. 3.

Rep nicking activity on RBE insertion mutants in the NOSTEM background. (A) The two TR substrates containing preferentially extruded trs stem-loop structure are illustrated. NOSTEM and NOSTEM+10 TRs are depicted after formation 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 and sequence of the NOSTEM+10 insertion are indicated with boldface italics. (B) Rep68 endonuclease reactions were performed on wt and the WT+10 insertion substrates in the presence of 0.5 mM ATP as described in Materials and Methods. Rep68 endonuclease reactions were performed on NOSTEM and NOSTEM+10 insertion substrates in the absence of ATP. Products were resolved on a 10% denaturing polyacrylamide gel. Numbers above lanes indicate the total amount of Rep68 in the reactions expressed in femtomoles. (C) The gel from panel B was phosphorimaged, and the amounts of substrate and product were determined. The fraction of nicked substrate for wt and mutant TRs was then calculated at each Rep concentration and plotted. Closed squares, wt; closed triangles, NOSTEM; open squares, WT+10; open triangles, NOSTEM+10.