Effects of alkylation by dimethyl sulfate, nitrogen mustard, and mitomycin C on DNA structure as studied by the ethidium binding assay

Abstract

The extent of alkylation of DNA by dimethyl sulfate, nitrogen mustard, and the antibiotic mitomycin C is related to the resulting decrease in the fluorescence of intercalated ethidium. The fluorescence losses due to the first two types of reagents show a marked pH dependence, with greater losses of fluorescence being observed at alkaline pH values. At pH 11.6 the fluorescence shows a slow recovery, so that with low levels of methylation (~ 4% deoxyguanosine residues modified) one observes complete return of fluorescence. We postulate that these phenomena are due to conversion of 7-methyldeoxyguanosine to the zwitterionic form, and partial denaturation of the DNA duplex with loss of ethidium binding sites. Hydroxide-ion-catalyzed imidazole ring opening, and the removal of the positive charge permits reannealing with concomitant return of the ethidium intercalation sites. This conclusion is substantiated by enzymatic hydrolysis of 14C-labelled methylated DNA and identification of the two types of deoxyguanosine residues formed under the different conditions of the ethidium assay. The distinctly different behavior of mitomycin C confirms previous conclusions that its alkylation, preferentially on guanine, does not take place at the N-7 position.