Termination point of replication of colicin E1 plasmid DNA in cell extracts.

Closed-circular monomeric molecules were one of the major products of replication of colicin E1 plasmid DNA in cell extracts. However, when the plasmid DNA synthesized in the reaction mixture was labeled for 3 min after 27 min of incubation, most of the label was found in open-circular molecules. The open-circular molecules were converted to closed-circular molecules upon further incubation. The newly replicated open-circular molecules had a nick or small gap in their newly synthesized strand. The interruption was located at approximately 20% of the molecular length from the single site of cleavage by restriction endonuclease EcoR1 and at or very close to the origin of replication. The addition of 10 mM nicotinamide mononucleotide instead of nicotinamide adenine dinucleotide to extracts did not significantly affect the kinetics of the colicin E1 plasmid DNA synthesis. However, in the presence of nicotinamide mononucleotide the formation of completely replicated closed-circular molecules was suppressed and, instead, open-circular molecules accumulated with an interruption in the newly synthesized strand at the termination point of replication, which was located at or very close to the origin of replication.