DNA Sequence Modulates the Efficiency of NEIL1-Catalyzed Excision of the Aflatoxin B₁-Induced Formamidopyrimidine Guanine Adduct

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.

Synthesis and Characterization of 15N5-Labeled Aflatoxin B1–Formamidopyrimidines and Aflatoxin B1–N7-Guanine from a Partial Double-Stranded Oligodeoxynucleotide as Internal Standards for Mass Spectrometric Measurements

“… 4 , 37 , 38 In contrast, studies using human endonuclease VIII-like 1 (NEIL1) revealed that in addition to the well-known incision of the hydroxyl radical-induced 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) and 4,6-diamino-5-formamidopyrimidine (FapyAde), 39 , 40 both unedited NEIL1 (K242) and edited NEIL1 (K242R) recognize and excise AFB 1 –FapyGua from multiple sequence contexts in oligodeoxynucleotides and genomic DNAs. 4 , 41 − 44 Moreover, Neil1 –/– mice treated with AFB 1 accumulated significantly greater levels of AFB 1 –FapyGua in their liver DNA and developed HCCs at frequencies higher than wild-type or NER-deficient Xpa –/– mice. 41 Among human populations, there are known polymorphic variants of NEIL1 with altered DNA glycosylase, β-elimination, and δ-elimination activities.…”

“…As with other bulky adducts, AFB 1 –N7-Gua and AFB 1 –FapyGua are substrates for the nucleotide-excision repair (NER) pathway in Escherichia coli and mammalian cells. , Although several investigations have explored a role for base excision repair of aflatoxin adducts in E. coli, the data have been inconclusive. ,, In contrast, studies using human endonuclease VIII-like 1 (NEIL1) revealed that in addition to the well-known incision of the hydroxyl radical-induced 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) and 4,6-diamino-5-formamidopyrimidine (FapyAde), , both unedited NEIL1 (K242) and edited NEIL1 (K242R) recognize and excise AFB 1 –FapyGua from multiple sequence contexts in oligodeoxy­nucleotides and genomic DNAs. ,− Moreover, Neil1 –/– mice treated with AFB 1 accumulated significantly greater levels of AFB 1 –FapyGua in their liver DNA and developed HCCs at frequencies higher than wild-type or NER-deficient Xpa –/– mice . Among human populations, there are known polymorphic variants of NEIL1 with altered DNA glycosylase, β-elimination, and δ-elimination activities. − Data suggest that individuals carrying the variants of NEIL1 with reduced or no catalytic activity may be at an elevated risk of AFB 1 -induced HCCs.…”

The aflatoxin B1-induced imidazole ring-opened guanine adduct: high mutagenic potential that is minimally affected by sequence context

“…Thus, mechanism(s) that define sequence-dependent occurrence of AFB 1 -induced mutations likely operates prior to replication. Consistent with such possibility, we recently have demonstrated that sequence context modulates the catalytic efficiency of human glycosylase NEIL1 acting on AFB 1 -FapyGua, with rates of reactions being inversely correlated with thermal stability of the adduct-containing DNA (Tomar et al 2021). We speculate that AFB 1 -FapyGua in a more thermally-stable local environment would be more likely to escape repair not only by NEIL1, but also by nucleotide excision repair and thus, largely contribute to the final mutational signature of AFB 1 .…”

The aflatoxin B1‐induced imidazole ring‐opened guanine adduct: High mutagenic potential that is minimally affected by sequence context

“…These were annealed with the corresponding 6-mer oligodeoxynucleotides (5 0 -TAGCTA-3 0 , 5 0 -TAGCGA-3 0 , 5 0 -TAGCAA-3 0 , and 5 0 -TATCGA-3 0 ) to form partially double-stranded DNA structures and reacted with AFB 1 exo-8,9-epoxide. The conditions for chemical modifications, purification by reverse-phase HPLC, and characterization by mass spectrometry were previously described (Li et al, 2015;Tomar et al, 2021) (Figure S1).…”