DNAA DEPENDENT REPLICATION OF PLASMID R1 OCCURS IN THE PRESENCE OF POINT MUTATIONS THAT DISRUPT THE DNAA BOX OF ORIR

We have found that DnaA dependent replication of R1 still occurred when 5 of the 9 bases in the dnaA box present in oriR were changed by site directed mutagenesis although the replication efficiency decreased to 20% and 70% of the wild-type origin in vitro and in vivo respectively. Additional mutation of a second dnaA box, 28 bp upstream oriR, that differs in only one base from the consensus sequence, did not affect the level of replication whereas polyclonal antibodies against DnaA totally abolished in vitro replication in the absence of the dnaA box. Wild-type RepA as well as a RepA mutant, RepA2623, that binds to oriR but that is inactive in promoting in vitro replication of plasmid R1, induce efficient binding of DnaA to the dnaA box. However, specific binding of DnaA to oriR was not detected by DNase I protection experiments in the absence of the dnaA box. These results suggest that the entrance of the DnaA protein in oriR is promoted initially by interactions with a RepA-oriR pre-initiation complex and that, in the absence of the dnaA box, these interactions can support, with reduced efficiency, DnaA dependent replication of plasmid R1.