Replication of Colicin El Plasmid DNA In Vitro

Abstract

Several methods for in vitro synthesis of DNA have been developed in recent years (cf. Gefter, 1975; Geider, 1976). These methods have been applied to the synthesis of DNA fragments on double-stranded template DNA and to the synthesis of the strands complementary to the viral strands of single-stranded phages. However, complete in vitro replication of double-stranded DNA had not been successful at the time we started the work to be described. Considering the importance of circular double-stranded DNA and the inherent limitation of in vivo experiments as means to elucidate mechanisms of DNA replication, we attempted to construct an in vitro system to replicate circular double-stranded DNA. After selecting colicin El plasmid (Col El) DNA as a model material, an in vitro system capable of carrying out a complete cycle of replication of the plasmid DNA was established (Sakakibara and Tomizawa, 1974; Tomizawa et al., 1975).