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

Abstract

<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