Miscoding Potential of the N2-Ethyl-2‘-deoxyguanosine DNA Adduct by the Exonuclease-Free Klenow Fragment of Escherichia coli DNA Polymerase I

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Abstract
Acetaldehyde, a major metabolite of ethanol, reacts with dG residues in DNA, resulting in the formation of the N2-ethyl-2‘-deoxyguanosine (N2-Et-dG) adduct. This adduct has been detected in lymphocyte DNA of alcohol abusers. To explore the miscoding property of the N2-Et-dG DNA adduct, phosphoramidite chemical synthesis was used to prepare site-specifically modified oligodeoxynucleotides containing a single N2-Et-dG. These N2-Et-dG-modified oligodeoxynucleotides were used as templates for primer extension reactions catalyzed by the 3‘ → 5‘ exonuclease-free (exo-) Klenow fragment of Escherichia coli DNA polymerase I. The primer extension was retarded one base prior to the N2-Et-dG lesion and opposite the lesion; however, when the enzyme was incubated for a longer time or with increased amounts of this enzyme, full extension occurred. Quantitative analysis of the fully extended products showed the preferential incorporation of dGMP and dCMP opposite the N2-Et-dG lesion, accompanied by a small amounts of dAMP and dTMP incorporation and one- and two-base deletions. Steady-state kinetic studies were also performed to determine the frequency of nucleotide insertion opposite the N2-Et-dG lesion and chain extension from the 3‘ terminus from the dN·N2-Et-dG (N is C, A, G, or T) pairs. These results indicate that the N2-Et-dG DNA adduct may generate G → C transversions in living cells. Such a mutational spectrum has not been detected with other methylated dG adducts, including 8-methyl-2‘-deoxyguanosine, O6-methyl-2‘-deoxyguanosine, and N2-methyl-2‘-deoxyguanosine. In addition, N2-ethyl-2‘-deoxyguanosine triphosphate (N2-Et-dGTP) was efficiently incorporated opposite a template dC during DNA synthesis catalyzed by the exo- Klenow fragment. The utilization of N2-Et-dGTP was also determined by steady-state kinetic studies. N2-Et-dG DNA adducts are also formed by the incorporation of N2-Et-dGTP into DNA and may cause mutations, leading to the development of alcohol- and acetaldehyde-induced human cancers.