Utilization of 1, N6-Etheno-2'-deoxyadenosine 5'-triphosphate during DNA synthesis on natural templates, catalyzed by DNA polymerase I of Escherichia coli

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Abstract
To test whether vinyl chloride-induced mutagenesis might involve ambiguous base pairing of 1,N6-etheno-adenine (.epsilon.A) during DNA synthesis, we examined the base pairing potential of .epsilon.DATP during DNA synthesis catalyzed by Escherichia coli DNA polymerase I (Klenow fragment). An electrophoretic assay of chain elongation was used to assess the degree to which .epsilon.dATP could substitute for each of the normal dNTPs during elongation of a primer annealed to a bacteriophage template. Despite the fact that the etheno bridge completely blocks normal Watson-Crick pairing of .epsilon.A with T, we observed that .epsilon.dATP could substitue for dATP during primer elongation (although inefficiently). In addition, detectable substitution of .epsilon.dATP for dGTP and dCTP occurred, indicating that .epsilon.A exhibits ambiguous base pairing properties. The relative ease of .epsilon.dAMP incorporation (opposite template T, C and G) appeared to vary considerably at different positions along the template. The major form of .epsilon.A incorporation (replacement of A) was confirmed by measurements of .epsilon.ATP .fwdarw. .epsilon.dAMP turnover (a commonly used method for detecting misincorporation), and also by the demonstration that .epsilon.A was present in enzymatic hydrolysates prepared from DNA that was synthesized with .epsilon.dATP replacing dATP. A model for ambiguous base pairing of .epsilon.dATP is proposed, in which incorporation occurs via the protonated, syn form of .epsilon.dATP.

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