Methylation of DNA by incubation with methylamine and nitrite

DNA was incubated in septum-closed reaction vials with [14C]methylamine and nitrite. The DNA was purified, hydrolysed with hydrochloric acid, and the purines were analysed by h.p.l.c. 7-Methylguanine was detectable as a result of DNA methylation in experiments performed in 100 mM acetate at pH 4. Using different concentrations of amine and nitrite a first order reaction for total amine and a second order for total nitrite could be shown. A study on the pH dependence using 100 mM malonate buffer, pH 2.0-6.0, revealed a maximum rate at pH 3.5, with steep slopes above and below this pH value, in agreement with a mathematical analysis of the reaction equations. The data show that the alkylating agent formed spontaneously by nitrosation and deamination of a primary amine has a long enough lifetime to react with DNA in vitro. Using the reaction orders established here, an extrapolation to lower concentrations found in the stomach can now be performed. Future in vivo experiments on the methylation of gastro-intestinal DNA then would show to what extent DNA in a cell is protected from alkylation.