Processing of N-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.