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

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 substitute 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 dATP----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.