Mutation patterns of DNA adducts, such as mutational spectra and signatures, are useful tools for diagnostic and prognostic purposes. Mutational spectra of carcinogens derive from three sources: adduct formation, replication bypass, and repair. Here, we consider the repair aspect of 1,N⁶-ethenoadenine (εA) by the 2-oxoglutarate/Fe(II)-dependent AlkB family enzymes. Specifically, we investigated εA repair across 16 possible sequence contexts (5′/3′ flanking base to εA varied as G/A/T/C). The results revealed that repair efficiency is altered according to sequence, enzyme, and strand context (ss- versus ds-DNA). The methods can be used to study other aspects of mutational spectra or other pathways of repair.

The AlkB-family enzymes have been shown to oxidize alkylative DNA adducts to different extents dependent upon the pH conditions to which they are subject in vitro. Reduced activity of enzymes such as ABH2 and ABH3 in humans, could contribute to accumulation of the cytotoxic DNA adducts, 1-methyladenine or 3-methylcytosine. This study will investigate the in vitro pH

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.

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 A (purine) and T (pyrimidine) as representative 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.