Determination of the binding sites of RepA, a replication initiator protein of the basic replicon of the IncN group plasmid pCU1.

A 2kb DNA region of the broad-host-range plasmid pCU1 carries all of the information essential for the stable maintenance of the plasmid and to express the same host-range specificity. It was predicted that the protein required to initiate replication from at least one of the three origins of the plasmid is encoded by the longest open-reading frame (ORF239) of the three overlapping in-frame ORFs located within the 2 kb region. The product of ORF239 has been named RepA. The initiator protein was overexpressed, purified and used for in vitro binding studies. Gel mobility shift experiments were performed to localize RepA binding sites. The DNA sequence protected by the bound RepA molecule(s) was determined by DNase I footprinting and 19 of a 20 bp long sequence that is part of the protected sequence were located in two clusters flanking the repA gene. A plasmid created by linking a 310 bp fragment (nucleotides 238 to 547) of the 2 kb region to the antibiotic resistance genes carried by the omega fragment, can be maintained stably if the RepA protein is supplied in trans. We conclude that this 310 bp DNA fragment, which consists of a short G+C and a long A+T rich region and the cluster of five RepA binding sites, carries a functional origin of the plasmid-protein dependent replication. The position of this origin indicates that it is oriB, one of the three origins previously identified by electron microscopy. The second cluster of RepA binding sites is downstream of the repA gene and consists of 14 sites that are in inverted orientation compared with the binding sites located in the oriB region. They are part of the region that was shown formerly to be involved in controlling the copy number of the plasmid. In contrast to oriB, binding of RepA to neither the oriS nor oriV region was detected.