Phage PBS-2 DNA, which contains uracil in place of thymine, was selectively damaged and then used as substrate for purified Bacillu subtilis uracil-DNA glycosylase. This enzyme releases uracil from DNA in a limited processive manner. Irradiation by ultraviolet light (>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 Vm. 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.Uracil-DNA glycosylase [dUra(DNA) glycosylase] specifically removes uracil from DNA. Such uracil may result either from incorporation in place of thymine during DNA synthesis or deamination of DNA cytosine (1), which may occur at physiological conditions (2). Left unrepaired, deaminated cytosines would result in transition mutations (3). Escherichia coli mutants deficient in dUra(DNA) glycosylase (ung-) are mutators, and in vivo deamination of cytosines is the source of these mutations (4,5). This implies that dUra(DNA) glycosylase activity is necessary to preserve the integrity of DNA in the face ofcontinuous potentially mutagenic damage. The finding that this enzyme is apparently ubiquitous supports the suggestions that this is its prime function (1, 4,6).The presence of UV photoproducts results in alteration of dUra(DNA) glycosylase activity toward phage PBS-2 DNA, which contains uracil in place of thymine. The rate of enzymic release ofuracil from such UV-irradiated DNA decreases as the number of photodimers increases (7). This indicates that the presence of pyrimidine dimers in DNA may affect excision of uracil.We investigated whether this alteration occurs with other types of DNA modifications. Three different types of purine damage were introduced into PBS-2 DNA, which was then used as substrate for dUra(DNA) glycosylase. Photoalkylation produced the modified purines 8-(2-hydroxy-2-propyl)guanine (HPG) and 8-(2-hydroxy-2-propyl)adenine (HPA) in DNA. (9), except that the samples were not flushed with nitrogen before irradiation. Actinometry (10) showed the incident dose to be 6.6 x 10-5 einstein cm-2 min-1. Photoalkylated DNA was extensively dialyzed into 10 mM Tris HCl/1 mM EDTA, pH 8.0. DNA was methylated with 10 mM Me2SO4 for 1 hr (11) and purified by passage through a Sephadex G-50 column in 10 mM Tris HCI/ 1 mM EDTA, pH 8.0 (12). In some experiments, methylated PBS-2 DNA was partially depurinated by heat (13) followed by dialysis ...

The effects of photoalkylation on superhelical PM2 DNA were examined. The chief product was 8-(2-hydroxy-2-propyl)guanine, formed exclusively in sequences of alternating purines and pyrimidines. Other purine damages included 8-(2-hydroxy-2-propyl)adenine and smaller quantities of two uncharacterized adenine products. DNA strand breaks were formed with increasing irradiation. A small quantity of thymine-containing photodimers was formed. Photoalkylation of poly(dG-dC):poly(dG-dC) reduced the concentration of salt required to effect inversion of the circular dichroic spectrum. This suggests that photoalkylation induces the transition of poly(dG-dC):poly(dG-dC) from the right-handed B form of DNA to the left-handed Z form.

The effects of UV irradiation on double-stranded PBS-2 phage DNA, which contains uracil in place of thymine, were studied. Irradiation by 254nm light, or by 313nm light in the presence of the triplet sensitizer acetophenone, resulted in production of uracil-containing pyrimidine photodimers. DNA chain breaks were also produced under both conditions. Uracil-DNA glycosylase released free uracil, but no uracil-containing dimers from such irradiated DNA. The rate of enzymatic release of uracil from irradiated PBS-2 DNA was decreased as the number of photodimers per DNA molecule was increased.

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