FIG. 4.

Structural distortions in the SV40 origin are not catalyzed by the KH512/513AA mutant. T-ag (12 pmol) was incubated under replication conditions with pSV01ΔEP (0.27 pmol) in the presence of ATP (lane 2), AMP-PNP (lane 3), or ADP (lane 4). Additional reactions were conducted with 12 pmol of the KH512/513AA double mutant (lanes 5 to 7) in the presence of the identical nucleotides. As a control, the reaction in lane 1 was conducted in the absence of T-ag. After treatment with KMnO₄, the locations of the oxidized bases were determined via primer extension reactions with a ³²P-labeled oligonucleotide (see Materials and Methods). The products of the primer extension reactions were analyzed by electrophoresis on a 7% polyacrylamide gel containing 8 M urea. The locations of regions of the SV40 core origin, including the EP, site II, and the AT-rich region, are indicated to the right of the gel.