Mutagenic specificity of imidazole ring-opened 7-methylpurines in M13mp18 phage DNA

The most abundant lesion formed in DNA upon modification with methylating a gents 7-methylguanine, under alkaline conditions is converted into 2,6-diam ino-4-hydroxy-5N-methyl-formamidopyrimidine (Fapy-7MeGua). We have previous ly shown that treatment of dimethylsulfate methylated DNA with NaOH creates mutagenic base derivatives leading to a 60-fold increase in the frequency of A-->G transitions and a 2-3-fold increase of G-->T and G-->C transversio ns. We have analyzed which lesions lead to these mutations. We compared mut agenic spectra in the lacZ gene of M13mp18 phage DNA modified with dimethyl sulfate and NaOH after selective elimination of damaged bases from molecule s used for transfection into SOS-induced E. coli. Partial elimination of Fapy-7MeGua from phage DNA performed by its digestio n with formamidopyrimidine-DNA glycosylase resulted in a 2-3-fold decrease of G-->T and G-->C transversions. Selective depurination of methylated base s (9 h, 37 degrees C, pH 7.0) resulting in almost complete loss of 7MeAde a s demonstrated by HPLC analysis of [H-3]MNU alkylated phage DNA used as a p robe, caused a dramatic, 9-fold decrease of A-->G transitions. Alkali-catal ysed rearrangement of 7MeAde was followed by HPLC analysis of [H-3]MNU alky lated poly(A) and poly(dA). After incubation of these oligonucleotides in N aOH, 7MeAde disappeared from both chromatograms, but only in polyA, 2 new p eaks migrating with retention time different from that of 1MeAde, 3MeAde or 7MeAde were detected, suggesting formation of two rotameric forms of Fapy- 7MeAde as observed for Fapy-7MeGua. Thus the miscoding lesion, giving rise to A-->G transitions derived from 7MeAde was Fapy-7MeAde. Fapy-7MeGua was a t least an order of magnitude less mutagenic, but in SOS-induced cells it g ave rise to G-->T and G-->C transversions.