Figure 5.

DnaA(H136Q) is defective in opening the strands of oriC. Single-strand specific endonuclease P1 was used to measure ability of WT and mutant DnaA proteins to open the DNA duplex. The indicated amounts (0, 10, 20, 30 nM) of ADP-DnaA (•), ATP-DnaA (○), ATP-DnaA(H136Q) (▪), and ATP-DnaA(H136A) (□) were incubated with 60 fmol of supercoiled pAL70 (poriC) plasmid for 5 min at 38°C. Samples were treated with P1 endonuclease (0.6 units) for 10–15 s, and reactions were stopped by adding a stop solution and immediate transfer of the samples to ice. Different forms of plasmid DNA were separated by electrophoresis followed by ethidium bromide staining. The relative percentage of linear form was quantified using Image Quant software. Data represents the mean (± SD) from three independent sets of experiments.