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 AFB 1-N7-dG can rearrange to a formamidopyrimidine (AFB1-FAPY) derivative. Both AFB1-N7-dG and the β-anomer of the AFB 1-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′late neighbor dT from a primed AFB 1-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 AFB 1 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 AFB 1-N7-dG adduct. Ternary structures with AFB1-β-FAPY adducted template:primers show correct vs incorrect insertion of dATP vs dTTP opposite the 5′late 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. © 2011 American Chemical Society.