Structural Investigations of N3-(2-Hydroxy-3-buten-1-yl)-2'-deoxyuridine DNA Adducts

Abstract

Butadiene monoepoxide (BDO) is the major metabolite of butadiene (BD), a widely used petrochemical and known human and animal carcinogen. BD metabolites may be involved in BD-induced carcinogenicity primarily through the production of DNA adducts. Under physiological conditions, BDO alkylates the N3 of deoxycytidine yielding stereoisomeric N3-(2-hydroxy-3-buten-1-yl)-2 f-deoxycytidine adducts. These adducts are relatively unstable leading to hydrolytic deamination, forming stereoisomeric N3-(2-hydroxy-3-buten-1-yl)-2 f-deoxyuridine (BD-N3-dU) adducts. Although, the BD-N3-dU adducts have not been isolated in vivo, suggesting a low-level occurrence in cellular DNA, they are highly mutagenic in mammalian cells. Mutagenesis studies, which revealed that the major mutations were C ¨T transitions and C ¨A transversions, also showed stereospecific differences in mutagenic frequencies. In the presence of the R adduct, there was a 2:1 preference for the insertion of dA vs. dT opposite the lesion. Whereas, in the presence of the S adduct, dA and dT were inserted at the same mutagenic frequency. It was hypothesized that structural differences in the orientation of the BD moiety may play a role in the differences observed in the mutagenesis results. Solution structures of R- and S-BD-N3-dU modified oligonucleotides, placing the modified nucleotides opposite dA and dT, were solved using NMR spectroscopy. These models represent the C ¨T and C ¨A mutations, respectively. NMR analysis revealed structural differences at the lesion site for the R-BD-N3-dU adduct when placed opposite dA vs. dT. For the S-BD-N3-dU adduct, however, the butadiene moieties assumed similar orientations when placed opposite dA and dT. These observations in duplex DNA correlate to the mutagenesis results and suggest that the orientation of the butadiene moiety may play a role in biological processing. Biochemical analyses of the BD-N3-dU adducts indicated the role of polymerase eta in the insertion of primarily dA and dG opposite the lesion site. NMR solution structures of the R- and S-BD-N3-dU modified nucleotides placed opposite dG, revealed that the butadiene moieties were oriented similarly when placed opposite dA. Again, suggesting a structural influence in DNA replication. X-ray crystallographic studies involving pol eta and incoming dNTPs will provide further insight into the mutagenicity of the R- and S-BD-N3-dU adducts.