Initiation of bacteriophage lambda DNA replication in vitro with purified lambda replication proteins.

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Abstract
A soluble enzyme system that replicates exogenously added plasmid DNA (.lambda.dv) bearing the replication origin of the bacteriophage .lambda. chromosome was developed. The system contains pure phage .lambda. O and P replication proteins and a partially purified mixture of Escherichia coli replication proteins. The features of .lambda.dv replication in this system closely resemble the known characteristics of phage .lambda. DNA replication in vivo. The system depends completely on exogenously supplied DNA, specifically replicates supercoiled plasmid DNA that contains a .lambda. replicaton origin, depends on the .lambda. O protein and the .lambda. P protein, depends on RNA polymerase, depends on host replication proteins (e.g., primase, dnaB protein and several others that function in the priming of DNA synthesis in E. coli) as judged by antibody inhibitions and replicates as much as 32% of added .lambda.dv plasmid DNA through a single complete round to generate catenated daughter molecules. Replication of .lambda.dv DNA in vitro requires DNA gyrase and an ATP-regenerating system. It is notable that addition of .lambda. O and P proteins to the mixture of E. coli replication proteins inhiits replication of plasmids bearing the origin of the E. coli chromosome. Exploitation of this enzyme system should allow a detailed investigation of the biochemical mechanisms involved in bacteriophage .lambda. DNA replication and its regulation.