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 Col E` 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 Col E1 and Clo DF13 DNA. It differs, from the system employed by Sakakibara and Tomizawa in two important points: (1) Its replicative capacity for Col 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 on 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°C consists mostly of monomeric supercoils. If Col 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 semiconservative replication process.