Major Groove Orientation of the (2S)-N⁶-(2-Hydroxy-3-buten-1-yl)-2′-deoxyadenosine DNA Adduct Induced by 1,2-Epoxy-3-butene

Abstract

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 N6 exocyclic nitrogen of adenine to form N6-(hydroxy-3-buten-1-yl)-2′-dA ((2S)-N6-HB-dA) adducts (TretyakovaN.LinY.SangaiahR.UptonP. B.SwenbergJ. A.TretyakovaN.LinY.SangaiahR.UptonP. B.SwenbergJ. A.9054600Carcinogenesis199718137147). The structure of the (2S)-N6-HB-dA adduct has been determined in the 5′-d(C1G2G3A4C5Y6A7G8A9A10G11)-3′:5′-d(C12T13T14C15T16T17G18T19 C20C21G22)-3′ duplex [Y = (2S)-N6-HB-dA] containing codon 61 (underlined) of the human N-ras protooncogene, from NMR spectroscopy. The (2S)-N6-HB-dA adduct was positioned in the major groove, such that the butadiene moiety was oriented in the 3′ direction. At the Cα carbon, the methylene protons of the modified nucleobase Y6 faced the 5′ direction, which placed the Cβ carbon in the 3′ direction. The Cβ hydroxyl group faced toward the solvent, as did carbons Cγ and Cδ. The Cβ hydroxyl group did not form hydrogen bonds with either T16O4 or T17O4. The (2S)-N6-HB-dA nucleoside maintained the anti 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 (2S)-N6-HB-dA resulted in a 5 °C reduction in the Tm of the duplex, which is attributed to less favorable stacking interactions and adduct accommodation in the major groove.

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

“…Structural studies of (S) - N 6 -HB-dA I in 5′-d(C 1 G 2 G 3 A 4 C 5 Y 6 A 7 G 8 A 9 A 10 G 11 )-3′:5′-d(C 12 T 13 T 14 C 15 T 16 T 17 G 18 T 19 C 20 C 21 C 22 )-3′ using high field NMR ( Y 6 = (S) - N 6 -HB-dA I) 60 have revealed that the adduct is accommodated in the major grove of the DNA, maintaining standard Watson-Crick base pairing with T 17 . 60 Although (S) - N 6 -HB-dA I ( Y 6 ) did not stack well with its 5′ neighbor (C 5 ), it stacked normally with its 3′ neighbor (A 7 ), thereby leading to only minor perturbations in the overall DNA structure. Structurally analogous N 6 -THB-dA and N 6 -dA adducts are also readily accommodated in the DNA major grove, maintaining Watson-Crick base pairing.…”

Base Excision Repair of N6-Deoxyadenosine Adducts of 1,3-Butadiene

“…The overall B-DNA structure was maintained. 65,66 Strand incision assays conducted with site-specifically modified DNA duplexes and nuclear extracts from human fibrosarcoma (HT1080) cells (Figures 2 and 3) suggest that all three BD-dA adducts are efficiently recognized and processed by a BER pathway. In these experiments, 5′-radiolabeled DNA duplexes containing site-specific and stereospecific BD-dA adducts were incubated with nuclear protein extracts, and the cleavage products were detected as high-mobility bands on the gel.…”

“…Only small perturbations in base stacking and hydrogen bonding interactions were observed for ( S )- N 6 -HB-dA, while the presence of N 6 , N 6 -DHB-dA prevented its hydrogen bonding with thymidine in the opposite strand and disrupted stacking interactions of the adducted base with neighboring nucleotides. The overall B-DNA structure was maintained. , …”

Characterization of the Major Purine and Pyrimidine Adducts Formed after Incubations of 1-Chloro-3-buten-2-one with Single-/Double-Stranded DNA and Human Cells

“…The formation of CBO-dA adducts will disrupt Watson–Crick base paring, thus being likely mutagenic. Indeed, multiple studies have showed that the N 6 -dA adducts in DNA can disrupt Watson–Crick base pairing, − potentially inducing mutations . Recently, it has been demonstrated that N 6 -singly substituted dA lesions are not miscoding but that exocyclic N 6 , N 6 -dA adducts are strongly mispairing, probably due to their inability to form stable Watson–Crick pairs with dT .…”