Effects of alkylation by dimethyl sulfate, nitrogen mustard, and mitomycin C on DNA structure as studied by the ethidium binding assay.
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 identifiions of the ethidium assay. The distinctly different behavior of mitomycin C confirms previous conclusions that its alkylation, preferentially on guanine, does not take part at the N-7 position.
D000477:Alkylating Agents; D001665:Binding Sites; D004247:DNA; D003851:Deoxyribonucleases; D003853:Deoxyribonucleosides / Q000032:analysis; D004996:Ethidium; D006863:Hydrogen-Ion Concentration; D008466:Mechlorethamine; D008937:Mitomycins; D010727:Phosphoric Diester Hydrolases; D010744:Phosphoric Monoester Hydrolases; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013463:Sulfuric Acid Esters; D013997:Time Factors
D000477:Alkylating Agents; D003853:Deoxyribonucleosides; D008937:Mitomycins; D013463:Sulfuric Acid Esters; D008466:Mechlorethamine; D004247:DNA; D003851:Deoxyribonucleases; D010744:Phosphoric Monoester Hydrolases; D010727:Phosphoric Diester Hydrolases; D004996:Ethidium