Translesion Synthesis of the N-2-2 '-Deoxyguanosine Adduct of the Dietary Mutagen IQ in Human Cells: Error -Free Replication by DNA Polymerase kappa and Mutagenic Bypass by DNA Polymerases eta, zeta, and Rev1

摘要

Translesion synthesis (TLS) of the N-2-2'-deoxyguanosine (dG-N-2-IQ) adduct of the carcinogen 2-amino-3-methylimidazo [4,5-f]quinoline (IQ) was investigated in human embryonic kidney 293T cells by replicating plasmid constructs in which the adduct was individually placed at each guanine (G(1), G(2), or G(3)) of the NanI sequence (5'-CG(1)G(2)CG(3)CC-3'). TLS efficiency was 38%, 29%, and 25% for the dG-N-2-IQ located at G(1), G(2), and G(3), respectively, which suggests that dG-N-2-IQ is bypassed more efficiently by one or more DNA polymerases at G(1) than at either G(2), or G(3). TLS efficiency was decreased 8-35% in cells with knockdown of pol eta, pol kappa, pol iota, pol zeta or Rev1. Up to 75% reduction in TLS occurred when pol eta, pol zeta, and Rev1 were simultaneously knocked down, suggesting that these three polymerases play important roles in dG-N-2-IQ bypass. Mutation frequencies (MFs) of dG-N-2-IQ at G(1), G(2), and G(3) were 23%, 17%, and 11%, respectively, exhibiting a completely reverse trend of the previously reported MF of the C8-dG adduct of IQ(dG-C8-IQ), which is most mutagenic at G(3) ((2015) Nucleic Acids Res. 43, 8340-8351). The major type of mutation induced by dG-N-2-IQ was targeted G -> T, as was reported for dG-C8-IQ In each site, knockdown of pol kappa resulted in an increase in MF, whereas MF was reduced when pol eta, pol iota, pol zeta or Rev1 was knocked down. The reduction in MF was most pronounced when pol eta, pol zeta and Rev1 were simultaneously knocked down and especially when the adduct was located at G(3), where MF was reduced by 90%. We conclude that pol kappa predominantly performs error-free TLS of the dG-N-2-IQ adduct, whereas pols eta, pol zeta and Rev1 cooperatively carry out the error-prone TLS. However, in vitro experiments using yeast pol zeta and kappa showed that the former was inefficient in full-length primer extension on dG-N-2-IQ templates, whereas the latter was efficient in both error-free and error prone extensions. We believe that the observed differences between the in vitro experiments using purified DNA polymerases, and the cellular results may arise from several factors including the crucial roles played by the accessory proteins in TLS.