DNA Synthesis in Lysates of RecB⁻ and Rec⁺ E. coli Cells

Abstract

THE isolation by De Lucia and Cairns² of a mutant (pol⁻)¹ of E. coli that lacks DNA polymerase I has led to the demonstration of in vitro DNA synthesis that is independent of this enzyme. Moses and Richardson² have observed prolonged and extensive DNA synthesis after treatment of pol⁻ or pol⁺ cells with toluene. This synthesis is strongly dependent on ATP as well as on deoxynucleoside triphosphates. Several groups of workers have observed synthesis in lysates of penicillin or lysozyme-induced pol⁻ spheroplasts^3–5. The synthesis was short lived but was reported to proceed at a rate of chain growth comparable to the rate in vivo, and was interpreted as a limited continuation of real replication at the original growth point. An enzyme, DNA polymerase II, which may be responsible for DNA replication has been extracted and purified from both pol⁻ and pol⁺ cells^6–9. In purified form, this enzyme does not require ATP. It can repair gaps in DNA which has been made partly single stranded by treatment with exo-nuclease III, but unlike polymerase I it is not able to act on a nicked, endonuclease-treated DNA primer. Sulphydryl blocking agents inhibit polymerase II, but not polymerase I.