AN 18-BASE-PAIR SEQUENCE IS SUFFICIENT FOR TERMINATION OF ROLLING-CIRCLE REPLICATION OF PLASMID PT181

pT181 and related plasmids of gram-positive bacteria replicate by a ro lling circle mechanism. The replication initiator protein of pT181, Re pC, has origin-specific nicking-closing activities. Replication of the plasmid pT181 leading strand initiates by covalent extension of the R epC-generated nick, and the origin of replication contains signals for both initiation and termination of DNA replication. We have investiga ted the sequence requirements for the initiation and termination steps by using plasmids containing two pT181 origins. In vitro replication experiments showed that 18- and 24-bp synthetic oligonucleotides conta ining the RepC nick site were active in the termination of replication , However, initiation of replication required a larger region which al so includes the RepC binding site. Plasmids containing the 18- and 24- bp region were also found to be nicked by the RepC protein. Our result s demonstrate that sequence requirements for initiation and terminatio n of pT181 replication overlap, but while the RepC binding site is req uired for initiation, it is dispensable for termination.