Bypass of Aflatoxin B1 Adducts by the Sulfolobus solfataricus DNA Polymerase IV

Abstract

Aflatoxin B₁ (AFB₁) is oxidized to an epoxide in vivo, which forms an N7-dG DNA adduct (AFB₁–N7-dG). The AFB₁–N7-dG can rearrange to a formamidopyrimidine (AFB₁–FAPY) derivative. Both AFB₁–N7-dG and the β-anomer of the AFB₁–FAPY adduct yield G→T transversions in Escherichia coli, but the latter is more mutagenic. We show that the Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) bypasses AFB₁–N7-dG in an error-free manner but conducts error-prone replication past the AFB₁–FAPY adduct, including misinsertion of dATP, consistent with the G→T mutations observed in E. coli. Three ternary (Dpo4–DNA–dNTP) structures with AFB₁–N7-dG adducted template:primers have been solved. These demonstrate insertion of dCTP opposite the AFB₁–N7-dG adduct, and correct vs incorrect insertion of dATP vs dTTP opposite the 5′-template neighbor dT from a primed AFB₁–N7-dG:dC pair. The insertion of dTTP reveals hydrogen bonding between the template N3 imino proton and the O² oxygen of dTTP, and between the template T O⁴ oxygen and the N3 imino proton of dTTP, perhaps explaining why this polymerase does not efficiently catalyze phosphodiester bond formation from this mispair. The AFB₁–N7-dG maintains the 5′-intercalation of the AFB₁ moiety observed in DNA. The bond between N7-dG and C8 of the AFB₁ moiety remains in plane with the alkylated guanine, creating a 16° inclination of the AFB₁ moiety with respect to the guanine. A binary (Dpo4–DNA) structure with an AFB₁–FAPY adducted template:primer also maintains 5′-intercalation of the AFB₁ moiety. The β-deoxyribose anomer is observed. Rotation about the FAPY C5–N⁵ bond orients the bond between N⁵ and C8 of the AFB₁ moiety out of plane in the 5′-direction, with respect to the FAPY base. The formamide group extends in the 3′-direction. This improves stacking of the AFB₁ moiety above the 5′-face of the FAPY base, as compared to the AFB₁–N7-dG adduct. Ternary structures with AFB₁–β-FAPY adducted template:primers show correct vs incorrect insertion of dATP vs dTTP opposite the 5′-template neighbor dT from a primed AFB₁–β-FAPY:dC pair. For dATP, the oxygen atom of the FAPY formamide group participates in a water-mediated hydrogen bond with Arg332. The insertion of dTTP yields a structure similar to that observed for the AFB₁–N7-dG adduct. The differential accommodation of these AFB₁ adducts within the active site may, in part, modulate lesion bypass.