Replication of small plasmids in extracts of Escherichia coli

Abstract

Sakakibara and Tomizawa (1974a) have described a soluble in vitro system that can carry the semi-conservative replication of the Co1 E1 plasmid. However, the usefulness of this system is restricted by its rapid inactivation during storage. This paper describes a stable soluble system prepared by freeze-thaw lysis of chloramphenicol-treated E. coli cells which replicates added Co1 E1 and C1o DF13 DNA. It differs from the system employed by Sakakibara and Tomizawa in two important points: (1) Its replicative capacity for Co1 E1 DNA is by an order of magnitude higher and (2) it can be stored in liquid nitrogen for several months without loss of activity Plasmid replication in vitro is dependent of DNA polymerase I and requires de novo RNA synthesis. It is completely inhibited by rifampicin, oxolinic acid, and novobiocin. The DNA synthesized during a 60 min incubation at 30 degrees C consists mostly of monomeric supercoils. If Co1 E1 DNA is used as template, a minor portion of the label is also found in closed dimeric catenanes. Density labelling experiments indicate that plasmid DNA synthesis occurs by a semi-conservative replication process.