Dna Sequence Modulates The Efficiency Of Neil1-Catalyzed Excision Of The Aflatoxin B-1-Induced Formamidopyrimidine Guanine Adduct

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-3,4-dihydropyrimid-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.