Processing of N⁵-substituted formamidopyrimidine DNA adducts by DNA glycosylases NEIL1 and NEIL3

A variety of agents cause DNA base alkylation damage, including the known hepatocarcinogen aflatoxin B₁ (AFB₁) and chemotherapeutic drugs derived from nitrogen mustard (NM). The N7 site of guanine is the primary site of alkylation, with some N7-deoxyguanosine adducts undergoing imidazole ring-opening to stable mutagenic N⁵-alkyl formamidopyrimidine (Fapy-dG) adducts. These adducts exist as a mixture of canonical β- and unnatural α-anomeric forms. The β species are predominant in double-stranded (ds) DNA. Recently, we have demonstrated that the DNA glycosylase NEIL1 can initiate repair of AFB₁-Fapy-dG adducts both in vitro and in vivo, with Neil1^−/− mice showing an increased susceptibility to AFB₁-induced hepatocellular carcinoma. Here, we hypothesized that NEIL1 could excise NM-Fapy-dG and that NEIL3, a closely related DNA glycosylase, could excise both NM-Fapy-dG and AFB₁-Fapy-dG. Product formation from the reaction of human NEIL1 with ds oligodeoxynucleotides containing a unique NM-Fapy-dG followed a bi-component exponential function under single turnover conditions. Thus, two adduct conformations were differentially recognized by hNEIL1. The excision rate of the major form (∼13.0 min⁻¹), presumed to be the β-anomer, was significantly higher than that previously reported for 5-hydroxycytosine, 5-hydroxyuracil, thymine glycol (Tg), and AFB₁-Fapy-dG. Product generation from the minor form was much slower (∼0.4 min⁻¹), likely reflecting the rate of conversion of the α anomer into the β anomer. Mus musculus NEIL3 (MmuNEIL3Δ324) excised NM-Fapy-dG from single-stranded (ss) DNA (turnover rate of ∼0.4 min⁻¹), but not from ds DNA. Product formation from ss substrate was incomplete, presumably because of a substantial presence of the α anomer. MmuNEIL3Δ324 could not initiate repair of AFB₁-Fapy-dG in either ds or ss DNA. Overall, the data suggest that both NEIL1 and NEIL3 may protect cells against cytotoxic and mutagenic effects of NM-Fapy-dG, but NEIL1 may have a unique role in initiation of base excision repair of AFB₁-Fapy-dG.