Miscoding Properties of 3,N4-Etheno-2‘-deoxycytidine in Reactions Catalyzed by Mammalian DNA Polymerases

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Abstract
We have investigated the miscoding properties of the exocyclic DNA adduct, 3,N4-etheno-2'-deoxycytidine (epsilon dC), using an experimental system designed to detect and quantify base substitutions and deletions generated by primer extension in reactions catalyzed by DNA polymerases alpha, beta, and delta. Oligodeoxynucleotides modified site-specifically with epsilon dC were used as DNA templates for this study. Pol alpha catalyzed incorporation of dTMP and dAMP opposite epsilon dC, accompanied by lesser amounts of dCMP and dGMP and some two-base deletions. Pol beta promoted incorporation of dCMP and dAMP, along with small amounts of one-base and two-base deletions. Pol delta catalyzed incorporation of dTMP and lesser amounts of dAMP and dGMP. The frequency of nucleotide insertion opposite epsilon dC and of chain extension from the 3'-primer terminus in reactions catalyzed by pol alpha and pol beta was established by steady-state kinetic analysis. Results of this study were consistent with those obtained in primer extension experiments. The miscoding properties of epsilon dC determined in vitro are consistent with observations of epsilon dC-->A transversions and epsilon dC-->T transitions in site-specific mutagenesis experiments in mammalian cells (Moriya et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 11899). We conclude from this study that DNA polymerases may differ significantly in their miscoding potential and that in vitro analysis can be used to predict mutagenic specificity of exocyclic DNA adducts in mammalian cells.