RepA and DnaA proteins are required for initiation of R1 plasmid replication in vitro and interact with the oriR sequence

Abstract

RepA, an initiation protein of R1 plasmid replication, was purified from an Escherichia coli strain overproducing the protein. The purified RepA protein specifically initiated replication in vitro of plasmid DNA bearing the replication origin of R1 plasmid (oriR). The replication, strictly dependent on added RepA protein, was independent of host RNA polymerase but required other host replication functions (DnaB and DnaC proteins, the single-stranded-DNA-binding protein SSB, and DNA gyrase). The replication was also completely dependent on the host DnaA function. In filter binding assays in high salt (0.5 M KCl) conditions, RepA specifically binds to both supercoiled and linear plasmid DNA containing the oriR sequence, whereas it binds to nonspecific DNA in low salt. DNase I-protection studies on a linearized DNA fragment revealed that DnaA protein specifically binds to a 9-base-pair DnaA-recognition sequence ("DnaA box") within oriR only when RepA is bound to the sequence immediately downstream of the DnaA box. These results indicate that initiation of R1 plasmid replication is triggered by interaction of RepA and DnaA proteins with the oriR sequence.