Binding of DnaA protein to a replication enhancer counteracts the inhibition of plasmid R6K g origin replication mediated by elevated levels of R6K p protein

Replication of the-y origin ofEscherichia coli plasmid R6K requires i protein, encoded by the R6Kpir gene, and many host factors, including DnaA protein. zr has dual roles, activating replication at low levels and inhibiting replication at high levels. The inhibitory function ofw is counteracted by integration host factor and a specific sequence of the origin called the enhancer. This 106-bp DNA segment contains a binding site for DnaA protein (DnaA box 1). In this study, we mutated this site to determine if it was required for the enhancer's function. Using-y origin derivative plasmids with the DnaA box 1 altered or deleted, we show that this site is necessary to protect the origin against levels of wild-type wF protein that would otherwise inhibit replication. To show that the base substitutions in DnaA box 1 weakened the binding of DnaA, we developed a new application of the agarose gel retardation assay. This quick and easy assay has broad applicability, as shown in binding studies with DNA fragments carrying a different segment of the R6K origin, the chromosomal origin (oriC), or the pUC origin. The gel retardation assay suggests a stoichiometry of DnaA binding different from that deduced from other assays. Many replication origins have a bipartite organization, in-cluding an essential segment and an auxiliary segment which augments replication. Such enhancers have been discovered i