Functional origin of replication of pT181 plasmid DNA is contained within a 168-base-pair segment.

We have used a recently developed in vitro replication system from Staphylococcus aureus to determine the origin and direction of replication of pT181 plasmid DNA. The origin was located to within 168 base pairs by two methods: (i) sequential labeling of restriction endonuclease fragments after synchronous initiation in vitro in the presence of various amounts of dideoxy-TTP and (ii) by constructing in vitro deletions of pT181 DNA close to the origin of replication and testing for their ability to replicate in vitro pT181 plasmid was found to replicate unidirectionally and anticlockwise, as the map is conventionally drawn. The nucleotide sequence of the region containing the origin of replication has been determined and found to be partially or entirely contained within the coding sequence for the repC protein, which is uniquely required for pT181 plasmid replication. Preliminary evidence suggesting that pT181 replicates by a rolling circle mechanism is discussed.