A quantitative study of ethyl methanesulfonate-induced alkylation of Vicia faba DNA

Abstract

In an earlier cytological study of the effect of the alkylating chemical ethyl methanesulfonate (EMS) on root tip chromosomes of Vicia faba, it was inferred that the localization of breaks along the chromosomes resulted from the affinity of EMS for guanine-rich areas. Here we report the results of a qualitative and quantitative investigation of the alkylation products formed by the in vitro treatment of Vicia faba root tip DNA with EMS. Reaction mixtures containing isolated Vicia faba DNA (44 mg and 22 mg) were combined with buffered solutions of EMS (0.5 M and 0.5 M respectively) and were incubated 18 h at 37°. After ethanol precipitation of the DNA, both the supernatant and the acid hydrolyzed DNA pellet were assayed and the percent of alkylation was determined. Investigation of the alkylation products by ascending paper chromatography indicated EMS is specific for guanine and that 7-ethylguanine was the only detectable product. Therefore, the effect of in vitro EMS alkylation of Vicia faba DNA is predominantly a guanine ethylation as predicted by current concepts of the molecular mechanisms of action of alkylating agents. On a mole per mole basis, about 46% of the total guanine present in the DNA reacted with EMS to produce 7-ethylguanine. One-third of the total 7-ethylguanine produced was found to have been released from the DNA by depurination. It was suggested that these depurinations would be responsible for base deletions, transitions and/or transversions in an in vivo system while the remaining 7-ethylguanines would be responsible for mispairing with thymine instead of cytosine.