Gene 6 Exonuclease of Bacteriophage T7: I. PURIFICATION AND PROPERTIES OF THE ENZYME

Abstract

An exonuclease activity was detectable in crude extracts from Escherichia coli 1200 cells (Endo I-, su-) infected with T7 phage bearing amber mutations in gene 3 (T7 endonuclease I) and gene 5 (T7 DNA polymerase). The activity was not detectable if the infecting phage also contained an amber mutation in gene 6. That gene 6 is the structural gene for this enzyme was shown by the fact that phage bearing a temperature-sensitive mutation in gene 6 induced an exonuclease activity which was more heat labile than the wild type enzyme. The enzyme has been purified 500-fold, and the purified preparations are essentially free of ribonuclease, endonuclease, DNA polymerase, and exonuclease I activity although they do contain measurable 3′-phosphatase activity, likely the reflection of residual E. coli exonuclease III. The enzyme has an absolute requirement for divalent cations and a sulfhydryl reagent and is stimulated greatly by potassium ions. The enzyme shows a marked preference for duplex DNA and liberates 5′-mononucleotides as the sole acid-soluble product. The gene 6 exonuclease is involved in the degradation of cellular DNA to acid-soluble products after T7 phage infection.