Sequence context is an important determinant in the mutagenic potential of 1,N6-ethenodeoxyadenosine (epsilonA): formation of epsilonA basepairs and elongation in defined templates.

Abstract

Many laboratories have obtained data on mutagenicity of modified bases in naturally occurring DNA sequences. It has often been noted that mutation is favored in certain sequence contexts, sometimes termed 'hot spots'. This approach to the contribution of neighboring sequences does not permit a systematic study of both the qualitative and quantitative mutational frequencies. In the present experiments we have chosen to use the exocyclic adduct, 1,N6-etheno A (epsilonA), site-specifically placed in a defined 25-mer oligonucleotides in which epsilonA is flanked by differing 5' and 3' tandem bases. Mutation was assessed using an in vitro replication assay and five polymerases of varying fidelity. The relevant central sequences were 3' --> 5' -CC-epsilonA-CC-, -GG-epsilonA-GG-, -TT-epsilonA-TT-, -AA-epsilonA-AA-, -GG-epsilonA-TT-, -TT-epsilonA-AA-, -AT-epsilonA-TT- and -TA-epsilonA-TA-. Using the Klenow fragment (Kf) (exo+ or exo-) of E. coli Pol I, it was found the epsilonA is an ambiguous base and, with varying efficiencies, all four dNTPs could be inserted opposite epsilonA in all sequences. However, only 3' --> 5' -TT-epsilonA-TT-, -GG-epsilonA-TT- and -AT-epsilonA-TT- were fully extended to a significant extent. The only sequences essentially blocked at the position of epsilonA were -AA-epsilonA-AA- and -TT-epsilonA-AA-. The others were intermediate. When replication was performed with Sequenase, MMLV RT or HIV RT, different patterns were observed, in which replication terminated one base prior to epsilonA, at epsilonA, or one base after epsilonA without further extension. In favored sequences, using the Klenow fragment, an epsilonA x N pair could be extended to form normal basepairs. No extension could be demonstrated in sequences in which tandem adenines were 5' to epsilonA. Kinetic data showed that two of the epsilonA x N pairs, epsilonA x A and epsilonA x C, could form at 10 microM or less dNTP. Which bases were preferentially inserted opposite epsilonA was a function of the flanking bases. Under the kinetic conditions used, epsilonA x T did not form even at 1 mM dTTP. These results indicate that the chemical structure of an adduct is not the only determinant of mutagenic efficiency. It is likely that the effect of the adduct on replication is due to the changes in the structural environment conferred by the flanking bases.