SPECIFIC NICKING-CLOSING ACTIVITY OF THE INITIATOR OF REPLICATION PROTEIN REPB OF PLASMID PMV158 ON SUPERCOILED OR SINGLE-STRANDED-DNA

Asymmetric rolling circle replication of the promiscuous replicon pMV1 58 is initiated by the plasmid-encoded RepB protein. In vitro, purifie d RepB protein introduces a nick within the leading strand origin of r eplication by a nucleophylic attack on the phosphodiester bond at the dinucleotide GpA. Some changes within and around this dinucleotide wer e recognized by the protein. RepB nicked and closed supercoiled pMV158 DNA, having an optimum activity at 60 degrees C. We have imitated, in vitro, a process of rolling circle replication, since RepB was able t o nick (initiation) and to covalently close (termination) single-stran ded oligonucleotides containing the protein cleavage sequence. Covalen t DNA-protein complexes were not found, indicating that RepB has uniqu e features among plasmid-encoded proteins involved in rolling circle r eplication or conjugative mobilization.