Use of a DNA Polymerase III Bypass Mutant ofEscherichia coli, pcbA1, to Isolate Potentially Useful Mutations of a Complex Plasmid Replicon

Abstract The essential replicon region of plasmid pCU1 has, within 1.2 kb, two origins of replication that can function in the absence of Escherichia coli DNA polymerase I and one that requires this polymerase. To isolate mutants in the replicon pathway that uses the PolI-dependent origin in the presence of the two other origins, we examined the feasibility of exploiting E. coli strains carrying a polymerase c bypass mutation ( pcb A) and which can survive and form colonies with the polymerase activity of pol C inactivated at 42°C. The selection scheme that is described was successful and resulted in the isolation of a mutant replicon that is not maintained at 42°C in a PcbA − PolC +ts PolA + strain. Nucleotide sequencing identified the mutated region to be within the origin ( Ori V) that was known to be pol A-dependent. Electron microscopy of mutant plasmid molecules replicating in a Pcb + strain confirmed that Ori V is inactivated.