Polymerase Bypass of N6-Deoxyadenosine Adducts Derived from Epoxide Metabolites of 1,3-Butadiene

N-6-(2-Hydroxy-3-buten-1-yl)-2'-deoxyadenosine (N-6-HB-dA I) and N-6,N-6-(2,3-dihydroxybutan-1,4-diyl)-2'-deoxyadenosine (N-6,N-6-DHB-dA) are exocyclic DNA adducts formed upon alkylation of the N-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 N-6-H atom of adenine is required for Watson Crick hydrogen bonding with thymine, N-6-alkylation can prevent adenine from normal pairing with thymine, potentially compromising the accuracy of DNA replication. To evaluate the ability of BD-derived N-6-alkyladenine lesions to induce mutations, synthetic oligodeoxynucleotides containing site-specific (S)-N-6-HB-dA I and (R,R)-N-6,N-6-DHB-dA adducts were subjected to in vitro translesion synthesis in the presence of human DNA polymerases beta, eta, l, and kappa. While (S)-N-6-HB-dA I was readily bypassed by all four enzymes, only polymerases eta and kappa were able to carry out DNA synthesis past (R,R)-N-6,N-6-DHB-dA. Steady-state kinetic analyses indicated that all four DNA polymerases preferentially incorporated the correct base (T) opposite (S)-N-6-HB-dA I. In contrast, hPol beta was completely blocked by (R,R)-N-6,N-6-DHB-dA, while hPol eta and kappa 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 (S)-N-6-HB-dA I Was mostly error-free, replication of DNA containing (R,R)-N6,N6-DHB-dA induced significant numbers of A, C, and G insertions and small deletions. These results indicate that singly substituted (S)-N6-HB-dA I lesions are not miscoding, but that exocyclic (R,R)-N-6,N-6-DHB-dA adducts are strongly mispairing, probably due to their inability to form stable Watson-Crick pairs with dT.