The Molecular Basis of Mutagenesis by Methyl and Ethyl Methanesulfonates

Abstract

The molecular basis of the biological effect of alkylation, mutation induction and inactivation, was studied using transforming DNA.Comparative studies of mutation induction after methylation (by methyl methanesulfonate) and ethylation (by ethyl methanesulfonate) showed that methylation was five to ten times more effective than ethylation. This observation confirms chemical studies of the reactivities of the individual bases of DNA but conflicts sharply with observations that methyl methanesulfonate is much less mutagenic than ethyl methanesulfonate in bacteriophage and some higher organisms. In each case the number of mutational events caused in transforming DNA followed single‐hit kinetics. After termination of alkylation, the rates of decrease of mutants caused by methylation was twice that caused by ethylation, which fits chemical estimates of depurination rates.Reverse mutation studies with different mutagens and base analogs indicated that methyl methanesulfonate predominantly methylates guanine whereas ethyl methanesulfonate alkylates guanine and also adenine; in both cases the major product is an N7‐alkyl purine. Accordingly the cause of base‐pairing mistakes is unlikely to be related to steric effects of the alkyl group but rather due to ionization of the alkylated guanine.In addition, inactivation of transforming activity was observed to be linear with treatment time of DNA with methyl or ethyl methanesulfonate in a semilogarithmic plot. Since the observed inactivation rates cannot be solely explained as inactivating mutagenic events produced by base alkylation, it is likely that phosphate alkylation is inactivating.Lethal and mutagenic DNA alterations are shown to be dissociated processes by the following observations. (a) After termination of alkylation, the number of mutants caused by alkylation of the purine bases decreased linearly with time indicating that depurination is inactivating. (b) Under identical conditions, the number of total transformants decreased three times faster than the number of mutants, indicating that in addition to depurination triester breaks must be responsible for inactivation. Therefore, base alkylations are mutagenic and depurination and triester breaks are lethal DNA alterations.