Replication of the ampicillin resistance plasmid RSF1030 in extracts of Escherichia coli: Separation of the replication cycle into early and late stages

The replication cycle of the small resistance plasmid RSF1030 can be divided into two stages with different enzyme requirements: (1) Synthesis of early replicative intermediates containing 7S DNA catalyzed by DNA polymerase I in the absence of functional dnaZ protein, and (2) replication of early intermediates requiring DNA polymerase III holoenzyme (including the dnaZ protein). Early intermediate DNA synthesized in a dnaZ^- extract can be converted to fully replicated plasmid molecules upon addition to a replication enzyme fraction prepared by ammonium sulfate fractionation of polA1 extracts. The firststage reaction is sensitive to rifampicin, novobiocin, and oxolinic acid, but insensitive to arabinosylcytosine triphosphate (aCTP). Addition of aCTP interferes with the second-stage reaction resulting in the accumulation of late replicative intermediates.