Extent of formation of O4-methylthymidine in calf thymus DNA methylated by N-methyl-N-nitrosourea and lack of repair of this product by rat liver O6-alkylguanine-DNA-alkyltransferase

Calf thymus DNA was methylated by reaction with N-[3H]-methyl-N-nitrosourea and the content of O6-methyldeoxyguanosine, 3-methylthymidine and O4-methylthymidine was determined. It was found that O4-methylthymidine represented only 0.06 +/- 0.02% of the total methylation and that the ratio of O6-methyldeoxyguanosine:O4-methylthymidine was 126 +/- 31. 3-Methylthymidine represented only 0.05 +/- 0.01% of the total radioactivity and the ratio of O6-methyldeoxyguanosine:3-methylthymidine was 171 +/- 16. The ability of O6-alkylguanine-DNA-alkyltransferases from Escherichia coli and from rat liver to repair O4-methylthymidine was determined using this methylated DNA as a substrate. When the methylated DNA substrate was incubated with an excess of either of the O6-alkylguanine-DNA-alkyltransferases greater than 95% of the O6-methyldeoxyguanosine was removed. The E. coli O6-alkylguanine-DNA-alkyltransferase also removed 89% of the O4-methylthymidine but the rat liver alkyltransferase did not alter the content of O4-methylthymidine. These results indicate that the mammalian O6-alkylguanine-DNA-alkyltransferase is specific for O6-methylguanine and differs from the bacterial protein in that it does not demethylate O4-methylthymine at any significant rate. This shows that the rat O6-alkylguanine-DNA-alkyltransferase is not able to protect against the possible hazards of the promutagenic lesion, O4-methylthymidine, but the very low extent of formation of this product may limit its significance in carcinogenesis and mutagenesis.