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Nucleic Acids Research, 1992, Vol. 20, No. 19 5119-5125
© 1992

MOLECULAR BIOLOGY

A comparison of the fidelity of copying 5-methylcytosine and cytosine at a difined DNA template site

Jiang-Cheng Shen, Steven Creighton1, Peter A. Jones and Myron F. Goodman1,*

Department of Biochemistry and Comprehensive Cancer Center, University of Southern California School of Medicine Los Angeles, CA 90033, USA 1Department of Biological Sciences, Molecular Biology Section, University Park Los Angeles, CA 90089-1340, USA

* To whom correspondence should be addressed

Received June 24, 1992. Revised August 24, 1992. Accepted August 24, 1992.

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 deamlnation of mC to T. The possibility remains, however, that a second mechanism involving mispairing of mC with A might also contribute to base substitution mutagenesls via G-A transitions. Stimulation of the G-A mutational pathway could involve preferential misincorporatlon of dAMP opposite template mC compared to C. To investigate this possibility, we synthesized a sequence containing mC at a defined template location. We compared the fidelity of copying mC versus C and the efficiency of extending mismatched base pairs at the mC position using three DNA polymerases, AMV reverse transcriptase, Drosophila DNA polymerase {alpha}, and mutant Escherichla 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 mC 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 mC or C sites or when extending mispairs involving mC and C. Incorporation of dAMP opposite either C or mC was barely detectable using pol {alpha}, although pol a has been observed to form A·C mismatches in other sequences. While we cannot completely exclude the possibility that dAMP might be incorporated opposite mC in preference to C, our results suggest that contributions of the G-A pathway to mC mutagenic hot spots are likely to be minor, lending additional support to the model invoking deamination of mC.


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