Translesion Synthesisof the N2-2′-DeoxyguanosineAdduct of the Dietary MutagenIQ in Human Cells: Error-Free Replication by DNA Polymerase κand Mutagenic Bypass by DNA Polymerases η ζ and Rev1

摘要

Translesion synthesis (TLS) of the N²-2′-deoxyguanosine (dG-N²-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 (G1, G2, or G3) of the NarI sequence (5′-CG1G2CG3CC-3′). TLS efficiency was 38%, 29%, and 25% for the dG-N²-IQ located at G1, G2, and G3, respectively, which suggests that dG-N²-IQ is bypassed more efficiently by one or more DNA polymerases at G1 than at either G2 or G3. TLS efficiency was decreased 8–35% in cells with knockdown of pol η, pol κ, pol ι, pol ζ, or Rev1. Up to 75% reduction in TLS occurred when pol η, pol ζ, and Rev1 were simultaneously knocked down, suggesting that these three polymerases play important roles in dG-N²-IQ bypass. Mutation frequencies (MFs) of dG-N²-IQ at G1, G2, and G3 were 23%, 17%, and 11%, respectively,exhibiting a completely reverse trend of the previously reported MFof the C8-dG adduct of IQ (dG-C8-IQ), which is most mutagenic at G3 ((2015) Nucleic Acids Res. 43, 8340−8351 [] [] []). The major type of mutationinduced by dG-N²-IQ was targeted G →T, as was reported for dG-C8-IQ. In each site, knockdown of pol κresulted in an increase in MF, whereas MF was reduced when pol η,pol ι, pol ζ, or Rev1 was knocked down. The reductionin MF was most pronounced when pol η, pol ζ, and Rev1were simultaneously knocked down and especially when the adduct waslocated at G₃, where MF was reduced by 90%. We concludethat pol κ predominantly performs error-free TLS of the dG-N²-IQ adduct, whereas pols η, pol ζ,and Rev1 cooperatively carry out the error-prone TLS. However, in vitro experiments using yeast pol ζ and κshowed that the former was inefficient in full-length primer extensionon dG-N²-IQ templates, whereas the latterwas efficient in both error-free and error-prone extensions. We believethat the observed differences between the in vitro experiments using purified DNA polymerases, and the cellular resultsmay arise from several factors including the crucial roles playedby the accessory proteins in TLS.