Endonuclease Activities in Extracts of Micrococcus Luteus that Act on γ-irradiated DNA

Summary

Several protein fractions containing endonuclease activity against γ-irradiated DNA (γ-endonuclease) were isolated from M. luteus. The crude extract was eluted on a phosphocellulose column and chromatographed on TEAE cellulose and subsequently on hydroxyapatite. Five peaks of γ-endonuclease were obtained from each preparation. Repeated experiments showed comparable chromatographic behaviour of the fractions. There was no detectable activity of U.V.-endonuclease in the fractions with γ-endonuclease but a small contamination of endonuclease against unirradiated DNA and against DNA with apurinic sites. The γ-endonuclease is stimulated by, but is not dependent on, magnesium. Several tests for endonuclease activity have been used: the analysis of strand breaks in calf-thymus DNA or in PM2 DNA, and the determination of end-groups formed by endonuclease, either 3′OH end-groups or phosphomonoester end groups. From the results obtained it can be assumed that the strand breaks induced by the γ-endonuclease carry 3′OH and 5′ phosphate end groups.