Effective Utilization of N 2-Ethyl-2‘-deoxyguanosine Triphosphate during DNA Synthesis Catalyzed by Mammalian Replicative DNA Polymerases

Acetaldehyde is produced by metabolic oxidation of ethanol after drinking alcoholic beverages. This agent reacts with nucleosides and nucleotides, resulting in the formation of N 2-ethyl-guanine residues. N 2-ethyl-2‘-deoxyguanosine (N 2-ethyl-dG) adduct has been detected in the lymphocyte DNA of alcoholic patients [Fang, J. L., and Vaca, C. E. (1997) Carcinogenesis 18, 627−632]. Thus, the nucleotide pool is also expected to be modified by acetaldehyde. N 2-Ethyl-2‘-deoxyguanosine triphosphate (N 2-ethyl-dGTP) was chemically synthesized. The utilization of N 2-ethyl-dGTP during DNA synthesis was determined by steady-state kinetic studies. N 2-Ethyl-dGTP was efficiently incorporated opposite template dC in reactions catalyzed by mammalian DNA polymerase α and δ. When pol α was used, the insertion frequency of N 2-ethyl-dGTP was 400 times less than that of dGTP, but 320 times higher than that of 7,8-dihydro-8-oxo-2‘-deoxyguanosine triphosphate (8-oxo-dGTP), an oxidative damaged nucleotide. Using pol δ, the insertion frequency of N 2-ethyl-dGTP was only 37 times less than that of dGTP. The chain extension from dC:N 2-ethyl-dG pair occurred much more rapidly: the extension frequencies for pol α and pol δ were only 3.8 times and 6.3 times, respectively, lower than that of dC:dG pair. We also found that N 2-ethyl-dG can be detected in urine samples obtained from healthy volunteers who had abstained from drinking alcohol for 1 week before urine collection. This indicates that humans are exposed constantly to acetaldehyde even without drinking alcoholic beverages. Incorporation of N 2-ethyl-dG adducts into DNA may cause mutations and may be related to the development of alcohol- and acetaldehyde-induced human cancers.