Replication of N2-ethyldeoxyguanosine DNA adducts in the human embryonic kidney cell line 293.
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.
Upton
Dana C
DC
Wake Forest University Health Sciences, Winston-Salem, North Carolina 27157, USA.
Wang
Xueying
X
Blans
Patrick
P
Perrino
Fred W
FW
Fishbein
James C
JC
Akman
Steven A
SA
D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D018736:DNA Adducts; D004261:DNA Replication; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D016368:Frameshift Mutation; D005822:Genetic Vectors; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D008969:Molecular Sequence Data; D009152:Mutagenicity Tests; D010957:Plasmids
D018736:DNA Adducts; C492934:N2-ethyl-2'-deoxyguanosine; D003849:Deoxyguanosine