No abstract

Dietary exposure to aflatoxins is a significant risk factor in the development of hepatocellular carcinomas. Following bioactivation by microsomal P450s, the reaction of aflatoxin B 1 (AFB 1 ) with guanine (Gua) in DNA leads to the formation of stable, imidazole ring-opened 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4dihydropyrimid-5-yl-formamido)-9-hydroxyaflatoxin B 1 (AFB 1 -FapyGua) adducts. In contrast to most base modifications that result in destabilization of the DNA duplex, the AFB 1 -FapyGua adduct increases the thermal stability of DNA via 5′-interface intercalation and base-stacking interactions. Although it was anticipated that this stabilization might make these lesions difficult to repair relative to helix distorting modifications, prior studies have shown that both the nucleotide and base excision repair pathways participate in the removal of the AFB 1 -FapyGua adduct. Specifically for base excision repair, we previously showed that the DNA glycosylase NEIL1 excises AFB 1 -FapyGua and catalyzes strand scission in both synthetic oligodeoxynucleotides and liver DNA of exposed mice. Since it is anticipated that error-prone replication bypass of unrepaired AFB 1 -FapyGua adducts contributes to cellular transformation and carcinogenesis, the structural and thermodynamic parameters that modulate the efficiencies of these repair pathways are of considerable interest. We hypothesized that the DNA sequence context in which the AFB 1 -FapyGua adduct is formed might modulate duplex stability and consequently alter the efficiencies of NEIL1-initiated repair. To address this hypothesis, site-specific AFB 1 -FapyGua adducts were synthesized in three sequence contexts, with the 5′ neighbor nucleotide being varied. DNA structural stability analyses were conducted using UV absorbance-and NMR-based melting experiments. These data revealed differentials in thermal stabilities associated with the 5′neighbor base pair. Single turnover kinetic analyses using the NEIL1 glycosylase demonstrated corresponding sequence-dependent differences in the repair of this adduct, such that there was an inverse correlation between the stabilization of the duplex and the efficiency of NEIL1-mediated catalysis.

Dietary exposure to aflatoxin B1 (AFB1) is a recognized risk factor for developing hepatocellular carcinoma. The mutational signature of AFB1 is characterized by high‐frequency base substitutions, predominantly G>T transversions, in a limited subset of trinucleotide sequences. The 8,9‐dihydro‐8‐(2,6‐diamino‐4‐oxo‐3,4‐dihydropyrimid‐5‐yl‐formamido)‐9‐hydroxyaflatoxin B1 (AFB1‐FapyGua) has been implicated as the primary DNA lesion responsible for AFB1‐induced mutations. This study evaluated the mutagenic potential of AFB1‐FapyGua in four sequence contexts, including hot‐ and cold‐spot sequences as apparent in the mutational signature. Vectors containing site‐specific AFB1‐FapyGua lesions were replicated in primate cells and the products of replication were isolated and sequenced. Consistent with the role of AFB1‐FapyGua in AFB1‐induced mutagenesis, AFB1‐FapyGua was highly mutagenic in all four sequence contexts, causing G>T transversions and other base substitutions at frequencies of ~80%–90%. These data suggest that the unique mutational signature of AFB1 is not explained by sequence‐dependent fidelity of replication past AFB1‐FapyGua lesions.

O 6-Methyl-2′-deoxyguanosine (O 6-MeG) is one of the most common DNA lesions and arises as a consequence of both xenobiotic carcinogens and endogenous methylation by S-adenosylmethionine. O 6-MeG frequently causes G-to-A mutations during DNA replication due to the misincorporation of dTTP and continued DNA synthesis. Efforts to detect DNA adducts such as O 6-MeG, and to understand their impacts on DNA structure and function, have motivated the creation of nucleoside analogs with altered base moieties to afford a more favorable interaction with the adduct as compared to the unmodified nucleotide. Such analogs directed at O 6-MeG include benzimidazolinone and benzimidazole nucleotides, as well as their extended π surface analogs naphthimidazolinone and napthimidazole derivatives. These analogs form a more stable pair with O 6-MeG than with G, most likely due to a combination of H-bonding and stacking. While extending the π surface of the analogs enhances their performance as adduct-directed probes, the precise origins of the increased affinity between the synthetic analogs and O 6-MeG remain unclear. To better understand relevant conformational and pairing properties, we used X-ray crystallography and analyzed the structures of the DNA duplexes with naphthimidazolinone inserted opposite G or O 6-MeG. The structures reveal a complex interaction of the analog found either in an anti orientation and stacked inside the duplex, either above or below G or O 6-MeG, or in a syn orientation and paired opposite G with formation of a single H-bond. The experimental structural data are consistent with the stabilizing effect of the synthetic analog observed in UV melting experiments and calculations and moreover reveal that the origin of these observations appears to be superior stacking between O 6-MeG and the extended π system of the synthetic probe.

Recognition and repair of DNA lesions are critical for cell survival. Herein, we highlight recent advances in the sequencing, repair mechanisms, and biological consequences of DNA lesions presented at the 2022 Fall American Chemical Society meeting.