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Phage PBS-2 DNA, which contains uracil in place of thymine, was selectively damaged and then used as substrate for purified Bacillus subtilis uracil-DNA glycosylase. This enzyme releases uracil from DNA in a limited processive manner. Irradiation by ultraviolet light (greater than 305 nm) in the presence of isopropanol and a free radical photoinitiator introduced covalently bound 8-(2-hydroxy-2-propyl)purines into DNA. Methylation by dimethylsulfate yielded 7-methylguanine. Apurinic sites were produced by gentle heating of methylated DNA. Rates of enzymic release of uracil from DNA varied among these three substrates. The Vmax was markedly decreased for DNA containing 8-(2-hydroxy-2-propyl)purines and apurinic sites but was unaffected by the presence of larger quantities of 7-methylguanine. This suggests that certain types of damaged DNA moieties may decrease the capacity for uracil excision. Therefore, interference with enzymic excision of this potentially mutagenic base may constitute a common mechanism of action of the reaction products of several unrelated DNA damaging agents.

Perturbations of enzymic uracil excision due to purine damage in DNA.