A COMPARISON OF THE FIDELITY OF COPYING 5-METHYLCYTOSINE AND CYTOSINE AT A DEFINED DNA-TEMPLATE SITE

5-Methylcytosine has been postulated to be an endogenous mutagen in procaryotes and eucaryotes leading to base substitution hot spots, C --> T transitions, resulting from spontaneous deamination of (m)C to T. The possibility remains, however, that a second mechanism involving mispairing of (m)C with A might also contribute to base substitution mutagenesis via G --> A transitions. Stimulation of the G --> A mutational pathway could involve preferential misincorporation of dAMP opposite template (m)C compared to C. To investigate this possibility, we synthesized a sequence containing (m)C at a defined template location. We compared the fidelity of copying (m)C versus C and the efficiency of extending mismatched base pairs at the (m)C position using three DNA polymerases, AMV reverse transcriptase, Drosophila DNA polymerase alpha, and mutant Escherichia coli Klenow fragment containing no proofreading exonuclease activity. Significant differences in misinsertion and mismatch extension efficiencies were observed only for the case of AMV reverse transcriptase. AMV reverse transcriptase was observed to incorporate dAMP 4 to 5-fold more efficiently opposite (m)C than C. Favored extension of a 5-MeC . A over C . A mispair was also observed with a difference of about 3-fold. In contrast to AMV reverse transcriptase, Klenow fragment showed no significant difference when copying either (m)C or C sites or when extending mispairs involving (m)C and C. Incorporation of dAMP opposite either C or (m)C was barely detectable using pol alpha, although pol alpha has been observed to form A . C mismatches in other sequences. While we cannot completely exclude the possibility that dAMP might be incorporated opposite (m)C in preference to C, our results suggest that contributions of the G --> A pathway to (m)C mutagenic hot spots are likely to be minor, lending additional support to the model invoking deamination of (m)C.