Purification and Characterization of an Endonuclease from Micrococcus luteus that Acts on Depurinated and Carcinogen‐Modified DNA

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Abstract
An endonuclease which is active with regard to depurinated, alkylated, arylated and arylamidated DNA was purified 500-fold from M. luteus. In this purification, separation from the pyrimidine-dimer-specific UV-endonuclease was achieved. The enzyme has a MW of 30,000 on the basis of gel filtration; its activity is not absolutely dependent upon the presence of Mg2+, but 5-30 mM Mg2+ produces a 5-fold stimulation. KCl concentrations of less than 100 mM are optimal, while concentrations exceeding 100 mM inhibit. The enzyme has no effect on native DNA, but introduces single-strand breaks into DNA containing apurinic/apyrimidinic sites produced by heating at an acidic pH. DNA treated with such carcinogens as N-alkyl-N-nitrosoureas, alkyl methanesulfonates, alkyl sulfates, nitrogen mustard, .beta.-propiolactone, 7-bromomethyl-benz[a]anthracene, N-acetoxy-2-acetylaminofluorene and 7,12-dimethyl-benz[a]anthracene-5,6-oxide also becomes susceptible to enzymic action. The activity of the enzyme was detected by making use of the difference in mobility between supercoiled closed-circular DNA of Pseudomonas phage PM2 and its nicked form in agarose gel electrophoresis. Even depurinated or carcinogen-modified supercoiled PM2 DNA migrated faster than the respective relaxed nicked forms. A comparison of the number of enzyme-catalyzed single-strand breaks with the number of alkali-labile (i.e., apurinic) sites in carcinogen-modified PM2 DNA showed that the enzyme preparation introduced approximately twice as many breaks into the substrates as the number of apurinic sites and DNA bases carrying carcinogenic residues or contains DNA glycosidase activity besides the endonuclease activity. Exposure of UV-irradiated PM2 DNA to the endonuclease preparation showed that pyrimidine dimers were not substrates. The yields of enzyme-catalyzed single-strand breaks found in UV-irradiated DNA was 5 times the number of alkali-labile sites present suggesting that minor photoproducts, possibly 5,6-saturated pyrimidine residues, were recognized besides apurinic sites.