O6-Ethylguanine carcinogenic lesions in DNA: an NMR study of O6etG.cntdot.T pairing in dodecanucleotide duplexes

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Abstract
High-resolution two-dimensional NMR studies are reported on the self-complementary d-(Cl-G2-C3-O6etG4-A5-G6-C7-T8-T9-G10-C11-G12) duplex (designated O6etG.cntdot.T 12-mer) containing two symmetrically related O6etG.cntdot.T lesion sites located four base pairs in from either end of the duplex. Parallel studies were undertaken on a related sequence containing O6meG.cntdot.T lesion sites (designated O6meG.cntdot.T 12-mer) in order to evaluate the influence of the size of the alkyl substituent on the structure of the duplex and were undertaken on a related sequence containing G.cntdot.T mismatch sites (designated G.cntdot.T 12-mer duplex), which served as the control duplex. The exchangeable and nonexchangeable proton and the phosphorus nuclei have been assigned from an analysis of two-dimensional nuclear Overhauser enhancement (NOE) and correlated spectra of the O6etG.cntdot.T 12-mer, O6meG.cntdot.T 12-mer, and G.cntdot.T 12-mer duplexes in H2O and D2O solutions. The distances connectivities observed in the NOESY spectra of the O6alkG.cntdot.T 12-mer duplexes establish that the helix is right-handed and all of the bases adopt an anti conformation of the glycosidic torsion angle including the O6alkG4 and T9 bases at the lesion site. The imino proton of T9 at the O6alkG.cntdot.T lesion sites resonates at 8.85 ppm in the O6etG.cntdot.T 12-mer duplex and at 9.47 ppm in the O6meG.cntdot.T 12-mer duplex. The large upfield shift of the T9 imino proton resonance at the O6alkG4.cntdot.T9 lesion site relative to that of the same proton in the G4.cntdot.T9 wobble pair (11.99 ppm) and the A4.cntdot.T9 Watson-Crick pair (13.95 ppm) in related sequences establishes that the hydrogen bonding of the imino proton of T9 to O6alkG4 is either very weak or absent. The imino proton of T9 develops NOEs to the CH3 protons of the O6etG and O6meG alkyl groups across the base pair, as well as to the imino and H5 protons of the flanking C3.cntdot.G10 base pair and the imino and CH3 protons of the flanking A5.cntdot.T8 base pair in the O6alkG.cntdot.T 12-mer duplexes. These observations establish that the O6alkG4 and T9 residues are stacked into the duplex and that the O6CH3 and O6CH2CH3 groups of O6alkG4 adopt a syn orientation with respect to the N1 of the alkylated guanine. The syn orientation of the O6-alkyl group precludes wobble pairing at the O6alkG4.cntdot.T9 lesion site and favors a Watson-Crick alignment of O6alkG4 and T9 stabilized by one short hydrogen bond between the 4-amino group of O6alkG4 and the 2-carbonyl oxygen of T9 in the minor groove. Since the O6-alkyl group adopts a syn orientation, the separation between the O6 of O6alkG4 and the O4 of T9 in the major groove is increased, preventing the formation of a short hydrogen bond between the N1 ring nitrogen of O6alkG4 and the imino proton of T9. Only small proton and phosphorus chemical shift differences were detected when the O6etG.cntdot.T 12-mer and the O6meG.cntdot.T 12-mer duplexes are compared, which reflect small conformational differences between the two duplexes. Much larger chemical shift differences are detected when the O6alkG.cntdot.T 12-mer duplexes are compared with the G.cntdot.T 12-mer duplex.