Origin and direction of mini-R1 plasmid DNA replication in cell extracts of Escherichia coli

Replication of the mini-R1 plasmids pKN177 and pKN182 can be carried out efficiently in cell extracts of E. coli and depends on both transcription and translation. Heteroduplex and restriction analyses indicate that both plasmids are derived from the R1 copy mutant pKN104 by IS1-mediated recombination events without involving structural alterations in the replication region. To ascertain whether the in vitro replication of these miniplasmids corresponds to R1 replication in vivo, the origin and direction of replication were analyzed by electron microscopy of replicative intermediates. It was found that replication starts at a unique origin located within the RepA region at R1 coordinate at 82.4 kilobases and proceeds unidirectionally toward the IS1b sequence. The specification of the origin and the direction of in vitro replication are therefore in full agreement with the pattern observed previously for the in vivo replication of the closely related plasmids R100 and R6-5. This agreement provides additional evidence that R1 DNA synthesis in vitro employs the same replication mechanism as it does in vivo.