Major Groove Orientation of the (2<i>S</i>)‑<i>N</i>⁶‑(2-Hydroxy-3-buten-1-yl)-2′-deoxyadenosine DNA Adduct Induced by 1,2-Epoxy-3-butene

1,3-Butadiene (BD) is an environmental and occupational toxicant classified as a human carcinogen. It is oxidized by cytochrome P450 monooxygenases to 1,2-epoxy-3-butene (EB), which alkylates DNA. BD exposures lead to large numbers of mutations at A:T base pairs even though alkylation of guanines is more prevalent, suggesting that one or more adenine adducts of BD play a role in BD-mediated genotoxicity. However, the etiology of BD-mediated genotoxicity at adenine remains poorly understood. EB alkylates the <i>N</i>⁶ exocyclic nitrogen of adenine to form <i>N</i>⁶-(hydroxy-3-buten-1-yl)-2′-dA ((2<i>S</i>)-<i>N</i>⁶-HB-dA) adducts (Tretyakova, N., Lin, Y., Sangaiah, R., Upton, P. B., and Swenberg, J. A. (1997) Carcinogenesis 18, 137−147). The structure of the (2<i>S</i>)-<i>N</i>⁶-HB-dA adduct has been determined in the 5′-d­(C¹G²G³A⁴<u>C</u>^<u>5</u><u>Y</u>^<u>6</u><u>A</u>^<u>7</u>G⁸A⁹A¹⁰G¹¹)-3′:5′-d­(C¹²T¹³T¹⁴C¹⁵T¹⁶T¹⁷G¹⁸T¹⁹ C²⁰C²¹G²²)-3′ duplex [Y = (2<i>S</i>)-<i>N</i>⁶-HB-dA] containing codon 61 (underlined) of the human N-<i>ras</i> protooncogene, from NMR spectroscopy. The (2<i>S</i>)-<i>N</i>⁶-HB-dA adduct was positioned in the major groove, such that the butadiene moiety was oriented in the 3′ direction. At the C_α carbon, the methylene protons of the modified nucleobase Y⁶ faced the 5′ direction, which placed the C_β carbon in the 3′ direction. The C_β hydroxyl group faced toward the solvent, as did carbons C_γ and C_δ. The C_β hydroxyl group did not form hydrogen bonds with either T¹⁶ <i>O</i>⁴ or T¹⁷ <i>O</i>⁴. The (2<i>S</i>)-<i>N</i>⁶-HB-dA nucleoside maintained the <i>anti</i> conformation about the glycosyl bond, and the modified base retained Watson–Crick base pairing with the complementary base (T¹⁷). The adduct perturbed stacking interactions at base pairs C⁵:G¹⁸, Y⁶:T¹⁷, and A⁷:T¹⁶ such that the Y⁶ base did not stack with its 5′ neighbor C⁵, but it did with its 3′ neighbor A⁷. The complementary thymine T¹⁷ stacked well with both 5′ and 3′ neighbors T¹⁶ and G¹⁸. The presence of the (2<i>S</i>)-<i>N</i>⁶-HB-dA resulted in a 5 °C reduction in the <i>T</i>_m of the duplex, which is attributed to less favorable stacking interactions and adduct accommodation in the major groove

Polymerase Bypass of <i>N</i>⁶‑Deoxyadenosine Adducts Derived from Epoxide Metabolites of 1,3-Butadiene

<i>N</i>⁶-(2-Hydroxy-3-buten-1-yl)-2′-deoxyadenosine (<i>N</i>⁶-HB-dA I) and <i>N</i>⁶,<i>N</i>⁶-(2,3-dihydroxybutan-1,4-diyl)-2′-deoxyadenosine (<i>N</i>⁶,<i>N</i>⁶-DHB-dA) are exocyclic DNA adducts formed upon alkylation of the <i>N</i>⁶ position of adenine in DNA by epoxide metabolites of 1,3-butadiene (BD), a common industrial and environmental chemical classified as a human and animal carcinogen. Since the <i>N</i>⁶-H atom of adenine is required for Watson–Crick hydrogen bonding with thymine, <i>N</i>⁶-alkylation can prevent adenine from normal pairing with thymine, potentially compromising the accuracy of DNA replication. To evaluate the ability of BD-derived <i>N</i>⁶-alkyladenine lesions to induce mutations, synthetic oligodeoxynucleotides containing site-specific (<i>S</i>)-<i>N</i>⁶-HB-dA I and (<i>R</i>,<i>R</i>)-<i>N</i>⁶,<i>N</i>⁶-DHB-dA adducts were subjected to <i>in vitro</i> translesion synthesis in the presence of human DNA polymerases β, η, ι, and κ. While (<i>S</i>)-<i>N</i>⁶-HB-dA I was readily bypassed by all four enzymes, only polymerases η and κ were able to carry out DNA synthesis past (<i>R</i>,<i>R</i>)-<i>N</i>⁶,<i>N</i>⁶-DHB-dA. Steady-state kinetic analyses indicated that all four DNA polymerases preferentially incorporated the correct base (T) opposite (<i>S</i>)-<i>N</i>⁶-HB-dA I. In contrast, hPol β was completely blocked by (<i>R</i>,<i>R</i>)-<i>N</i>⁶,<i>N</i>⁶-DHB-dA, while hPol η and κ inserted A, G, C, or T opposite the adduct with similar frequency. HPLC-ESI-MS/MS analysis of primer extension products confirmed that while translesion synthesis past (<i>S</i>)-<i>N</i>⁶-HB-dA I was mostly error-free, replication of DNA containing (<i>R</i>,<i>R</i>)-<i>N</i>⁶,<i>N</i>⁶-DHB-dA induced significant numbers of A, C, and G insertions and small deletions. These results indicate that singly substituted (<i>S</i>)-<i>N</i>⁶-HB-dA I lesions are not miscoding, but that exocyclic (<i>R</i>,<i>R</i>)-<i>N</i>⁶,<i>N</i>⁶-DHB-dA adducts are strongly mispairing, probably due to their inability to form stable Watson–Crick pairs with dT