Development of an in vitro bacteriophage N4 DNA replication system

An in vitro DNA replication system from bacteriophage N4-infected Escherichia coli has been developed. It requires MgCl2, all four deoxyribonucleoside triphosphates, and exogenously added N4 phage DNA; other DNAs are used inefficiently or not at all. Ribonucleoside triphosphates are not required, although they stimulate DNA synthesis. In vitro replication starts at the ends of the N4 genome and moves progressively inward. Initiation occurs through hairpin priming at the 3' ends of the genome, but shows a strong preference for the right end. Three N4 gene products (dnp, dbp, and exo) required in vivo for N4 DNA synthesis are absolutely required in the in vitro system. These findings are discussed with respect to the mode of N4 DNA replication.