NMR Studies of the Exocyclic 1,N⁶-Ethenodeoxyadenosine Adduct (ϵdA) opposite Deoxyguanosine in a DNA Duplex. ϵdA(syn)·dG(anti) Pairing at the Lesion Site

Proton NMR studies are reported on the complementary d(C-A-T-G-G-G-T-A-C)-d(G-T-A- C-ϵA-C-A-T-G) nonanucleotide duplex (designated ϵdA·dG 9-mer duplex), which contains the exocyclic adduct 1,N⁶-ethenodeoxyadenosine positioned opposite deoxyguanosine in the center of the helix. The present study focuses on the alignment of dG5 and ϵdA14 at the lesion site in the ϵdA·dG 9-mer duplex at neutral pH. This alignment has been characterized by monitoring the NOEs originating from the NH1 proton of dG5 and the H2, H5, and H7/H8 protons of ϵdA14 in the central d(G4-G5-G6)·d(C13-ϵA14-C15) trinucleotide segment of the ϵdA·dG 9-mer duplex. These NOE patterns establish that ϵdA14 adopts a syn glycosidic torsion angle that positions the exocyclic ring toward the major groove edge while all the other bases including dG5 adopt anti glycosidic torsion angles. We detect a set of intra- and interstrand NOEs between protons (exchangeable and nonexchangeable) on adjacent residues in the d(G4-G5-G6)·d(C13- ϵA14-C15) trinucleotide segment which establish formation of right-handed helical conformations on both strands and stacking of the dG5(anti)·ϵdA14(syn) pair between stable dG4·dC15 and dG6·dC13 pairs. The energy-minimized conformation of the central d(G4-G5-G6)·d(C13-ϵA14-C15) segment establishes that the dG5(anti)·ϵdA14(syn) alignment is stabilized by two hydrogen bonds from the NH1 and NH₂-2 of dG5(anti) to N9 and N1 of ϵdA14(syn), respectively. The NH1 proton of dG5 resonates at 14.0 ppm, and its downfield shift is consistent with hydrogen bond formation with the ring nitrogen (N9) of ϵdA14 at the lesion site in the ϵdA·dG 9-mer duplex. The H6 and H5 base protons of dC13 and dC15 are broad in the ϵdA·dG 9-mer duplex, with some broadening also observed at the H2 and H5 base protons of ϵdA14 in NMR spectra recorded at ambient temperature. The observed broadening may originate in conformational averaging among ϵdA14 alignments on the intermediate time scale, which would affect the stacking in the d(C13- ϵA14-C15) segment centered about the lesion site. In summary, the dG5(anti)·ϵdA14(syn) alignment is readily accommodated into the DNA helix without disruption of flanking dG4·dC15 and dG6·dC13 base pairs and may account for the incorporation of dG opposite edA during in vitro replication by DNA polymerase I.