Replication of mini RK2 plasmid in extracts of Escherichia coli requires plasmid-encoded protein TrfA and host-encoded proteins DnaA, B, G DNA gyrase and DNA polymerase III.

Soluble extracts of Escherichia coli capable of carrying out replication of the mini-RK2 derivative pCT461 have been prepared from cells carrying this plasmid or from plasmid-free bacteria. The latter are dependent upon exogenously added plasmid-encoded replication protein (TrfA) and require additional DnaA protein for optimum activity. This dependence upon DnaA was confirmed by the failure of DnaA-deficient cell extracts to support replication of pCT461 in the absence of added DnaA protein. Replication is unidirectional and begins at or near oriV, the vegetative replication origin of RK2. DNase I protection studies with purified TrfA indicate that this protein acts by binding to short (17 base-pairs) directly repeated DNA sequences present in oriV. The in vitro replication is resistant to rifampicin but can be abolished by antibodies against DnaG protein (E. coli primase) or DnaB protein (helicase) and by DNA gyrase inhibitors. Inhibition by arabinosyl-CTP suggests that DNA polymerase III is responsible for elongation of nascent DNA strands. These results are discussed in relation to the mechanism of RK2 replication and in the context of the host range of the plasmid.