Decoupling of Bypass Efficiency and Mutagenicity of the 2-Acetylaminofluorene C8-Guanine Adduct by DNA Sequence Context

DNA sequence context plays a critical role in modulating the mutational effects of DNA damage. Here, we investigated how base identity influences the replication bypass and mutagenicity of a site-specific dG-AAF (2-acetylaminofluorene) bulky adduct in the well-defined AG*N and TG*N sequence contexts. By selecting adenine and thymine as 5′-flanking bases and systematically varying the 3′-base, we established a controlled system to examine sequence-dependent lesion replication. We found that the 5′-flanking base strongly affects the bypass profile, with AG*N sequences exhibiting uniformly low bypass (≤9.7%) and TG*N sequences showing markedly elevated bypass (23.6–50.4%) with strong sequence dependence. These differences may arise from the structure of the dG-AAF, whose conformation heterogeneities are sensitive to the flanking sequence context. In contrast, mutagenicity remains consistently low across all sequences examined, with a low frequency of point mutations and no detectable frameshift events. These results reveal a clear decoupling between lesion bypass efficiency and replication fidelity, where sequence context strongly controls lesion tolerance but has limited impact on mutagenicity. In total, our findings demonstrate that DNA sequence affects lesion processing, providing insights into how local sequence context shapes genome stability and mutational processes.