Double-stranded origin nicking and replication initiation are coupled in the replication of a rolling circle plasmid, pT181.

The Staphylococcus aureus rolling circle plasmid pT181 initiator RepC is modified by the addition of an oligodeoxynucleotide, giving rise to a new form, RepC*. RepC/RepC* heterodimer is an inhibitor of replication. However, in order to act effectively, the initiator/inhibitor protein must be stable. We show here that RepC is stable for at least 90 min, which enables it to function effectively as an inhibitor of replication. This finding also allowed us to carry out the two stages in pT181 replication sequentially: first, binding/nicking of the double-strand origin (DSO) by the pT181-encoded RepC, followed by initiation/elongation by the host cell's DNA replication apparatus. The results demonstrate that these two stages in pT181 replication are functionally coupled and that interruptions in this continuous process generate relaxed pT181 DNA that cannot be used as a template for replication.