Sequence‐Based Structural Stability Modulate Biological Processing of AFB 1 ‐Fapy‐dG Adduct by NEIL1 DNA glycosylase

Abstract

Dietary exposure to aflatoxin B1 (AFB1) is a significant risk factor for developing hepatocellular carcinomas (HCCs). Following ingestion and bioactivation by microsomal P450s, AFB1 reacts with the N7-position of guanine, leading to formation of highly genotoxic AFB1-Fapy-dG adducts. AFB1 5′-interface intercalation stabilizes DNA duplex significantly through strong base-stacking interactions with neighbor base-pairs. Also being sterically bulky DNA lesion, it was unexpectedly found to be excised by DNA glycosylase NEIL1, base-excision repair enzyme, from DNA in both synthetic oligodeoxynucleotides and liver DNA of exposed mice. We hypothesized that the DNA sequence context in which the AFB1-Fapy-dG adduct is formed might modulate duplex stability and consequently alter the efficiencies of NEIL1-initiated repair, ultimately contributing towards AFB1associated mutational spectrum. To test this, site-specific AFB1-Fapy-dG adducts were synthesized in three sequence contexts where 5′ neighbor base was varied. We observed differential DNA thermal stability specific to 5′-neighbor base-pair using UV absorbance and NMR-based melting studies. Furthermore, sequence-dependent stability differences also guided NEIL1-mediated base removal tendency where single turnover kinetic analyses showed an inverse correlation between the modified duplex stability and the NEIL1-catalyzed excision rates.