Figure 7.

QnrB enhances the formation of the DnaA‐oriC open complex. A. Overview of the pUC‐oriC plasmid and the oriC unwinding assays. DnaA unwinds oriC and forms ssDNA. Digestion of the plasmid with P1 nuclease and AlwNI produces 1.2 and 2.2 kb fragments. The blue box indicates the oriC cloned from E. coli BW25113 genomic DNA. The red box indicates the plasmid ori. B. QnrB promotes the formation of the DnaA‐oriC open complex. Upper panel: The indicated concentrations of QnrB (0‐0.08 nM) were incubated for 5 min at 37°C in buffer containing pUC‐oriC (0.5 μg), ATP (1 mM), HU protein (30 ng) and DnaA (0 or 0.04 nmol), and then incubated for 3 min in the presence of P1 nuclease. After digestion with AlwNI, the DNA fragments were analyzed by 1% agarose gel electrophoresis. Data are representative of three independent experiments. Lower panel: QnrB had no effect on oriC unwinding. The open complex formation was performed in the absence of DnaA. [Color figure can be viewed at wileyonlinelibrary.com]