Development of a 32P-postlabeling assay for 7-methylguanines in human DNA.

The application of a 32P-postlabeling assay for 7-methylguanines in DNA was studied either by labeling the imidazole ring-opened dinucleotide derivatives or by using strong-anion-exchange column chromatography for the adduct enrichment from normal nucleotides. Data showed that 7-methylguanines can be efficiently labeled as dinucleotides when in vitro methylated DNA was first imidazole ring-opened and then digested to the dinucleotide level with deoxyribonuclease I, snake venom phosphodiesterase, and prostatic acid phosphatase. When using ion exchange chromatography for the adduct enrichment, DNA was digested with micrococcal nuclease and spleen phosphodiesterase. Anion exchange chromatography was applied for 7-methylguanine measurements in white blood cell DNA of healthy nonsmokers (n = 17) and patients (n = 4) treated with the methylating drugs procarbazine and decarbazine. We found that the mean level of 7-methylguanine residues in nonsmokers was 2.5 per 10(7) nucleotides. The corresponding level in the patient samples immediately after the drug treatment was 57 per 10(7) nucleotides.