Role of CIS in replication of an IncB plasmid

Replication of the IncB plasmid pMU720 requires the synthesis of the cis-ac ting RepA protein and the presence of two DNA elements, ori and CIS. CIS is the 166-bp sequence separating the RepA coding sequence from ori. To inves tigate how this organization of the pMU720 replicon contributes to the mech anism of initiation of replication, mutations in the sequence and/or the le ngth of CIS were introduced into the CIS region and their effects on the ef ficiency of replication of the pMU720 replicon in vivo was determined. The CIS region was found to be composed of two domains. The repA-proximal domai n, which shelved strong transcription termination activity, could be replac ed by equivalent sequences from I-complex and IncL/M plasmids, whose replic ons are organized in the same fashion as pMU720. Replacement by a trpA tran scription terminator afforded only partial replication activity. The repA-d istal domain was shown to be a spacer whose role was to position sequence(s ) within ori on the correct face of the DNA helix vis-g-vis the repA-proxim al portion of CIS. A model for the loading of RepA protein onto ori is disc ussed.