Synthesis of 8-carbon-14-labeled O6-methyldeoxyguanosine and its deoxynucleotide copolymers

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Abstract
To study the nature and repair of the promutagenic DNA lesion O6-methylguanine, 8-14C-labeled O6-methyldeoxyguanosine triphosphate [mGTP] was synthesized and the kinetics of its incorporation into the synthetic copolymers poly(dC [deoxycytidine],m6dG [deoxyguanosine]) and poly(dT [deoxyribosylthymidine], m6dG) was investigated. Deoxy[8-14C]guanosine was methylated with ethereal diazomethane and the products were separated by high-pressure liquid chromatography. O6-Methyldeoxy[14C]guanosine was converted to the 5''-monophosphate with carrot phosphotransferase and then to the 5''-triphosphate via the phosphorimidazolidate formed by the action of N,N''-carbonyldiimidazole. Although m6dGTP was a poor substrate for Escherichia coli DNA polymerase I, copolymers could be synthesized from dCTP or dTTP and m6dGTP with terminal deoxynucleotidyl transferase. The percent of m6dG in the polymer increased linearly as the percentage of m6dGTP in the polymerization mixture was increased to 20% of the total. The percent incorporation of m6dGTP into poly(dT,m6dG) was higher than into poly(dC,m6dG). Good yields of both polymers were readily obtained. The stability of O6-methyldeoxyguanosine in poly(dT,m6dG) was pH dependent and the half-life was measured at 4 pH values. [The relationship between DNA lesions and carcinogenesis is discussed.].