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 a lcoholic beverages. This agent reacts with nucleosides and nucleotides, res ulting in the formation of N-2-ethyl-guanine residues. N-2-ethyl-2'-deoxygu anosine (N-2-ethyl-dG) adduct has been detected in the lymphocyte DNA of al coholic patients [Fang, J. L., and Vaca, C. E. (1997) Carcinogenesis 18, 62 7-632]. Thus, the nucleotide pool is also expected to be modified by acetal dehyde. N-2-Ethyl-2'-deoxyguanosine triphosphate (N-2-ethyl-dGTP) was chemi cally synthesized. The utilization of N-2-ethyl-dCTP during DNA synthesis w as determined by steady-state kinetic studies. N-2-Ethyl-dGTP was efficient ly incorporated opposite template dC in reactions catalyzed by mammalian DN A polymerase alpha and delta. When pol alpha was used, the insertion freque ncy of N-2-ethyl-dGTP was 400 times less than that of dGTP, but 320 times h igher than that of 7,8-dihydro-8-oxo-2'-deoxyguanosine triphosphate (8-oxo- dGTP), an oxidative damaged nucleotide. Using pol delta, the insertion freq uency 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 extens ion frequencies for pol alpha and pol delta were only 3.8 times and 6.3 tim es, respectively, lower than that of dC:dG pair. We also found that N-2-eth yl-dG can be detected in urine samples obtained from healthy volunteers who had abstained from drinking alcohol for 1 week before urine collection. Th is indicates that humans are exposed constantly to acetaldehyde even withou t drinking alcoholic beverages. Incorporation of N-2-ethyl-dG adducts into DNA may cause mutations and may be related to the development of alcohol- a nd acetaldehyde-induced human cancers.