pC221 belongs to a family of staphylococcal plasmids, including pT181, pS194, pC223, pUB112 and pCW7 . All possess open reading frames with 70-80% homology to the pC221 rapD gene. REP D has sequence specific topoisomerase activity at the pC221 origin (or iD) which is thought to be involved in replication initiation. DNase I footprinting has been carried out, showing that REP D binds to a region of oriD downstream of the nick site. The pattern of DNase I cleavage suggests that REP D contacts one face of the DNA helix, which may be bent around the protein. Extracts of S. aureus support incorporation of radioactive dNTPs into pC221 in the presence of REP D. Labelling with a[32P] dATP shows that replication initiates within the region containing oriD and proceeds in the direction expected for elongation of a 3' OH generated by nicking at oriD. With supercoiled DNA, REP D initiates replication of other members of this plasmid family in Vitro. However, with relaxed DNA, REP D is specific for oriD, suggesting that a change in the DNA, stabilised by supercoiling of the DNA or by binding of REP D, may be required for nicking. Of three inverted repeat sequences (ICRI, II & III) at the origin, ICRII has the greatest predicted hairpin stability and is almost totally conserved. Nicking takes place within the loop of this proposed hairpin. Disruption of base pairing within this hairpin has been investigated by mutagenesis of cloned oriD and using oligonucleotides based on the ICRII sequence. These experiments show that the 3' side of ICRII is more important for nicking than the 5' side. This is in agreement with footprinting data which shows that REP D binds the 3' side of ICRII, along with the whole of ICRIII. However, there is no evidence for hairpin formation at ICRII being required for nicking.