Escherichia coli host factor required specifically for the phi X174 stage III reaction: in vitro identification and partial purification.

Abstract

A cell-free extract prepared, from X174-infected Escherichia coli cells sustained in vitro synthesis of viral DNA (stage 11 reaction).when supplemented with fraction II from uninfected cells. The reaction was dependent upon deoxyribonucleoside triphosphate,.ATP, added 4X174 replicative form I DNA template, and the fraction II from uninfected cells. This reaction differed from the stage II reaction (semiconservative replication of duplex replicative form DNA) by the production of stable viral protein-DNA complexes sensitive to anti-+X174 antiserum. Three types ofprotein-DNA complexes were identified, '50S, 92S, and a 114S complex that cobanded in CsCl and cosedimented in neutral sucrose gradients with a 4X174 phage marker. The sensitivity of these complexes to anti-4X174.antiserum and Staphylococcus aureus provided a relatively rapid biochemical assay for direct measurement of the amount of DNA synthesized by -the stage m reaction. With this assay, an E. coli factor .(SI,,) required specifically for the synthesis of viral protein-DNA complexes was identified and purified 200-fold from uninfected E. coli cells.' The partially purified Sm factor was required for the synthesis ofDNA and viral protein-DNA complexes in the +X174-infected cell extracts and could not be replaced by rep The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

DNA replication of single-stranded Escherichia coli DNA phages

“…Experiments in crude in vitro systems [128a] have indicated a requirement for DNA gyrase subunit A [129] and another unidentified host factor [130]. These proteins are not necessary in the purified in vitro system [126].…”