2-Amino-3-methylimidazo[4,5-f]quinoline (IQ), a highly mutagenic and carcinogenic heterocyclic amine found in cooked meats, forms a major DNA adduct at the C8-position of 2'-deoxyguanosine (dG-C8-IQ). IQ induces frameshift mutations in bacteria. We have constructed three plasmid vectors containing the dG-C8-IQ adduct into the G1-, G2-, or G3-positions of the NarI recognition sequence (5'-G1G2CG3CC-3'), which is a hotspot for arylamine modification. The control and adducted plasmids were replicated in human embryonic kidney 293T cells and the progeny were analyzed for mutations. Although the dG-C8-IQ adduct was found to be mutagenic at each guanine of the NarI site,the location of the adduct strongly influenced the mutational frequency (MF), which followed the order G3>>G2>G1. Unlike in bacteria, dG-C8-IQ did not induce frameshift mutations in human cells. The major types of mutations observed were G?T transversions followed by G?A transitions. Bulky DNA adducts like dG-C8-IQ usually block or stall DNA replication by replicative DNA polymerases and translesion (TLS) DNA polymerases (e.g., Y-family DNA polymerases) are involved in bypassing them. To investigate the identity of the TLS polymerases responsible for mutagenesis of dG-C8-IQ, we have employed siRNA knock-down approach, which showed that the fidelity of bypass is highly dependent on specific TLS polymerases. Mutation frequency (MF) was reduced by 40-70% when pol ? was knocked down. The reduction in MF was most pronounced when the adduct was located at G2. MF was also reduced in pol ?, pol ?, or REV1 knock-down cells, although the magnitude of reduction was less pronounced compared to pol ? knock-down cells. In contrast, a 10-24% increase in MF was observed when pol ? was knocked down. These results suggest a major role of pol ? in the mutagenic TLS of dG-C8-IQ, although pol ?, pol ?, and REV1 also play important roles. Conversely, pol ? is involved in the error-free bypass of dG-C8-IQ. [This research was supported by the NIEHS grants ES009127 to AKB and ES016561 to CJR.]

Translesion synthesis of the C8-deoxyguanosine adduct of the dietary mutagen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) in the NarI sequence in human cells

248th National Meeting (2014)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.