BINDING OF DNAA PROTEIN TO A REPLICATION ENHANCER COUNTERACTS THE INHIBITION OF PLASMID R6K GAMMA-ORIGIN REPLICATION MEDIATED BY ELEVATED LEVELS OF R6K PI-PROTEIN

Replication of the gamma origin of Escherichia coli plasmid R6K requir es pi protein, encoded by the R6K pir gene, and many host factors, inc luding DnaA protein. pi has dual roles, activating replication at low levels and inhibiting replication at high levels. The inhibitory funct ion of pi is counteracted by integration host factor and a specific se quence of the origin called the enhancer. This 106-bp DNA segment cont ains a binding site for DnaA protein (DnaA box 1). In this study, we m utated this site to determine if it was required for the enhancer's fu nction. Using gamma origin derivative plasmids with the DnaA box 1 alt ered or deleted, we show that this site is necessary to protect the or igin against levels of wild-type pi protein that would otherwise inhib it replication. To show that the base substitutions in DnaA box 1 weak ened the binding of DnaA, we developed a new application of the agaros e gel retardation assay. This quick and easy assay has broad applicabi lity, as shown in binding studies with DNA fragments carrying a differ ent segment of the R6K origin, the chromosomal origin (oriC), or the p UC origin. The gel retardation assay suggests a stoichiometry of DnaA binding different from that deduced from other assays.