Solution conformation and mutagenic specificity of 1,N6-ethenoadenine.

Site-specific studies in several laboratories established that each of the three etheno adducts, 1,N6-ethenoadenine (epsilon A), 3,N4-ethenocytosine (epsilon C) and N2,3-ethenoguanine (N2,3-epsilon G), is mutagenic. In Escherichia coli, epsilon A is only weakly mutagenic in single-stranded DNA (mutation frequency, 0.1%), and epsilon C is at least 20 times more mutagenic than epsilon A. Prior treatment of host cells with ultraviolet irradiation enhances the mutagenic frequency of epsilon C by 30-60%, even when the E. coli is recA. Likewise, enhanced mutagenicity was observed when the host cells lacked 3'-->5' exonuclease activity of DNA polymerase III. epsilon A induces all three base substitutions, but A-->G predominates. epsilon C induces epsilon C-->T and epsilon C-->A substitutions, but only the latter was enhanced after ultraviolet irradiation of host cells. In contrast to the results in bacteria, both epsilon A and epsilon C are potent mutagenic lesions in simian kidney cells, inducing 70 and 81% base substitutions, respectively. In simian kidney cells, epsilon A exclusively induces epsilon A-->G transitions, whereas epsilon C-->A transversions are the major type of mutation induced by epsilon C. Nuclear magnetic resonance (NMR) spectrometry of the four possible pairs containing epsilon C indicated that both epsilon C:G and epsilon C:T pairs are stabilized by hydrogen bonds. Even though the latter forms the most stable pair containing epsilon C, the etheno adduct is in syn alignment. DNA polymerase appears to continue DNA synthesis with a syn-orientated base only in the absence of proofreading exonuclease activity or when ultraviolet irradiation-inducible proteins are present. For epsilon A, only epsilon A:T and epsilon A:G pairs have been studied by NMR, which showed that the former has no hydrogen bond whereas the latter maintains two hydrogen bonds with the etheno base in syn orientation. Determination of the relationship between a particular conformation of epsilon A and its mutagenic activity must await further studies. In a site-specific study of epsilon A with human cell extracts, an 11-mer oligonuclotide with a single epsilon A was inserted into an M13 bacteriophage containing an SV40 origin of replication. This vector was replicated in vitro with human fibroblast cell extracts, and the replicated products were analysed. In this experiment, epsilon A induced predominantly epsilon A-->G transitions but at a mutation frequency of 0.14%.