Endonucleolytic Activity from Micrococcus luteus That Acts on γ-Ray-Induced Damage in Plasmid DNA of Escherichia coli Minicells

Abstract

A partially purified extract from Micrococcus luteus contains endonucleolytic activity toward ultraviolet (UV)-irradiated deoxyribonucleic acid (DNA). We found that the same extract also acts on superhelical, covalently-closed circular lambdadv DNA isolated from gamma-irradiated minicells of E. coli. The introduction of nicks in isolated covalently-closed circular DNA by an endonuclease in the extract results in relaxed circles, and these two circular DNA species are easily distinguishable by their sedimentation properties in alkaline sucrose. The frequency with which the endonuclease-susceptible lesions are produced in superhelical DNA is only marginally enhanced when (60)Co gamma-rays are administered to an aerobic rather than an anoxic minicell suspension. The ratio of endonuclease-sensitive defects to single-strand scissions, induced by gamma-irradiation in air, is about 1:3. The nuclease-sensitive lesions disappear from gamma-irradiated minicells during incubation after radiation presumably as a consequence of excision repair. Since the addition of UV-irradiated calf-thymus DNA depresses the ability of the M. luteus extract to attack not only UV-damaged E. coli DNA (a known substrate for the so-called UV-specific endonuclease that catalyzes the initial single-strand incision adjacent to the structural defect) but, also lambdadv DNA injured by gamma-rays, we conclude that physicochemical alterations induced by both types of radiation are recognized by one and the same endonuclease.