Replication of N²-Ethyldeoxyguanosine DNA Adducts in the Human Embryonic Kidney Cell Line 293

Abstract

N(2)-Ethyldeoxyguanosine (N(2)-ethyldGuo) is a DNA adduct formed by reaction of the exocyclic amine of dGuo with the ethanol metabolite acetaldehyde. Because ethanol is a human carcinogen, we assessed the biological consequences of replication of template N(2)-ethyldGuo, in comparison to the well-studied adduct O(6)-ethyldeoxyguanosine (O(6)-ethyldGuo). Single chemically synthesized N(2)-ethyldGuo or O(6)-ethyldGuo adducts were placed site specifically in the suppressor tRNA gene of the mutation reporting shuttle plasmid pLSX. N(2)-EthyldGuo and O(6)-ethyldGuo were both minimally mutagenic in double-stranded pLSX replicated in human 293 cells; however, the placement of deoxyuridines on the complementary strand at 5'- and 3'-positions flanking the adduct resulted in 5- and 22-fold enhancements of the N(2)-ethyldGuo- and O(6)-ethyldGuo-induced mutant fractions, respectively. The fold increase in the N(2)-ethyldGuo-induced mutant fraction in deoxyuridine-containing plasmids was similar after replication in 293T cells, a mismatch repair deficient variant of 293 cells, indicating that postreplication mismatch repair has little role in modulating N(2)-ethyldGuo-mediated mutagenesis. The mutation spectrum generated by N(2)-ethyldGuo consisted primarily of single base deletions and adduct site-targeted transversions, in contrast to the exclusive production of adduct site-targeted transitions by O(6)-ethyldGuo. The yield of progeny plasmids after replication in 293 cells was reduced by the presence of N(2)-ethyldGuo in parental plasmids with or without deoxyuridine to 39 or 19%, respectively. Taken together, these data indicate that N(2)-ethyldGuo in DNA exerts its principal biological activity by blocking translesion DNA synthesis in human cells, resulting in either failure of replication or frameshift deletion mutations.

Kinetics of DNA Adduct Formation in the Oral Cavity after Drinking Alcohol

“…Studies in vitro indicate that the closely related adduct N 2 -ethyl-dGuo blocks trans-lesion DNA synthesis catalyzed by a variety of DNA polymerases potentially resulting in failure of replication or frameshift deletion mutations. Studies on mammalian cells showed that the lesion can be bypassed by other specific polymerases, suggesting that mammalian cells can accurately replicate past N 2 -ethyl-dGuo which is thus considered non-mutagenic (27–29). However, this lesion could be mutagenic in particular contexts when polymerases are defective or reduced in their activity.…”

Bypass of N2-ethylguanine by human DNA polymerase κ☆

“…The blocking effect of N 2 -ethyl-Gua on DNA pols has been observed in both in vitro and cell-based assays [6–8]. In contrast to the blocking effects on replicative DNA pols the N 2 -ethyl-Gua adduct is bypassed by the Y-family DNA pols η, ι, and κ [6,9–12].…”

Lesion Bypass of N2-Ethylguanine by Human DNA Polymerase ι

“…The N 2 -ethyl-Gua adduct is a strong block to DNA replication by replicative DNA polymerases in vitro and in cells (6,7). Structures of bacteriophage DNA polymerase (pol) RB69, a homolog of human DNA pol ␣, indicate a possible mechanism of N 2 -ethyl-Gua blocked DNA replication.…”