Effect of DNA cytosine methylation upon deamination-induced mutagenesis in a natural target sequence in duplex DNA

Are 5-methylcytosine residues in DNA hot spots for transition mutagenesis? Numerous studies identify 1) structural changes induced by DNA methylation, 2) high percentages of human mutations that result from GC to AT transition pathways, and 3) differences between G.C and G.mC base pairs in susceptibility to nonenzymatic deamination. However, investigations of chemical stability necessarily involve non-physiological conditions for chemical analysis of deamination. Here we describe an experiment that compares rates of deamination-induced mutagenesis between a G.C and G.mC base pair, when both are present in duplex DNA, incubated at 37 degrees C and pH 7.4, within identical sequence contexts, in a natural mutational target (the Escherichia coli lacZ alpha gene) that selects for mutagenesis at the specific site under investigation. Under these conditions the rate of spontaneous deamination at G.mC exceeds that at G.C by more than 21-fold. Our data implicate differences in chemical stability toward deamination as a major causal factor releasing DNA cytosine methylation to spontaneous mutagenesis.