Protein-primed replication of bacteriophage PRD1 genome in vitro

A cell-free system has been developed from cells of an Escherichia coli strain, carrying cloned genes 1 and 8 of bacteriophage PRD1, that catalyzes protein-primed DNA synthesis. DNA synthesis in vitro is entirely dependent upon the addition of PRD1 DNA-protein complex as template, Mg2+, and four deoxyribonucleoside triphosphates. No in vitro DNA synthesis was observed when deproteinized PRD1 DNA was used as template. The origin and direction of PRD1 DNA replication in vitro was determined by restriction enzyme analysis of 32P-labeled PRD1 DNA synthesized in this system. Replication starts at both ends of the linear PRD1 DNA template. Alkaline sucrose gradient centrifugation and agarose gel electrophoresis showed that full-length PRD1 DNA is synthesized in vitro. DNA synthesis in this system is inhibited by the drug aphidicolin. We also observed that dimethyl sulfoxide (DMSO) stimulates in vitro DNA synthesis, although it inhibits bacterial DNA polymerase.