2620 Biochemistry, Vol. 49, No. 12, 2010 Shanmugam et al. FIGURE 5: (A) Time-dependent 1H NMR spectra showing H-D exchange of the H7 etheno proton at 50 C. The NMR spectra were recorded at 25 C: (a) immediately after the sample was dissolved in buffer, (b) after 96 h, (c) after 216 h, and (d) after 576 h. (B) Change in the integrated intensity of H7 (5.84 ppm) and H6 (5.55 ppm) proton resonances with time. #The cytosine (C23) H5 proton reso- nance was taken as a reference. exchangeable protons of DNA (Figure 7), which were assigned to X6 H7 f T5 N3H, X6 H7 f G7 N1H, and X6 H6 f G7 N1H NOEs. Phosphorus Spectra. The proton-decoupled 31P spectrum of the 1-BD duplex is shown in Figure S2 of the Supporting Information. The spectrum shows a single phosphorus resonance shifted downfield as compared to those of the unmodified 1-BD duplex. The downfield-shifted 31P resonance was assigned to G 7 , from the proton-decoupled heteronuclear correlation spectrum. Chemical Shift Effects. Figure 8 shows a comparison of the chemical shifts of selected nonexchangeable protons between the 1-BD duplex and the unmodified 1-BD duplex. Chemical shift changes of >0.2 ppm were observed for the X6 H10, A19 H2, C18 H10, C18 H20, and C18 H200 protons. Compared to those of the unmodified 1-BD duplex, the X6 H2, G7 H8, and A19 H8 proton resonances shifted downfield 0.16, 0.12, and 0.18 ppm, respec- tively. The T5 CH3 signal shifted downfield 0.11 ppm, and the C18 H5 signal was shifted upfield 0.14 ppm. Relative to those of the unmodified 1-BD duplex, the A19 H2 signal shifted upfield by 0.41 ppm. In addition, several deoxyribose protons exhibited chemical shift changes from the nucleotides of both the modified and complementary strands; most of these were located adjacent to the 1,N2-εdG-modified site. For example, the X6 H10 and T5 H200 protons shifted upfield 0.36 and 0.17 ppm, respectively. G7 H10, G7 H20, and G7 H200 shifted downfield 0.14, 0.21, and 0.24 ppm, respectively, relative to those of the unmodified 1-BD duplex. A large chemical shift change was observed for C18 H10, which shifted 0.45 ppm downfield. The C18 H20 and H200 signals shifted upfield 0.23 and 0.31 ppm, respectively. Interestingly, chemical shift changes were observed for T17 H200, which was FIGURE 6: 1H NOESY spectrum (tile plot) showing the assignment of exocyclic etheno protons H7 and H6 and NOEs between the etheno and DNA protons. The NOESY spectrum was recorded at 25 C with a mixing time of 250 ms: (a) X6 H7 f X6 H6, (b) X6 H7 f C18 H20, (c) X6 H7 f C18 H200, (d) X6 H7 f A19 H50/H500, (e) X6 H7 f C18 H5, (f) X6 H7 f C18 H10, (g) X6 H7 f C18 H6, (h) X6 H7 f A19 H2, (i) X6 H6 f C18 H20, (j) X6 H6 f C18 H200, (k) X6 H6 f A19 H50/ H500, (l) X6 H6 f A19 H50/H500, (m) X6 H6 f A19 H40, (n) X6 H6 f C18 H10, (o) X6 H6 f A19 H10, (p) X6 H6 f A19 H2, (q) X6 H6 f C18 H6, and (r) X6 H6 f A19 H8. located at the second base from the adduction site in the 30 direction. In the case of exchangeable protons, the T5 N3H proton was shifted upfield 0.16 ppm, whereas the G7 N1H proton was shifted downfield 0.11 ppm. Experimental and Empirical Restraints. The structural refinement involved 567 restraints, which included distance, dihedral angle, sugar pucker, and Watson-Crick hydrogen bonding restraints. A total of 307 NOE-based distance restraints were used, consisting of 167 intranucleotide and 140 internucleo- tide distances. There were an average of 13 distance restraints per nucleotide. In addition, 45 empirical Watson-Crick hydrogen bonding restraints were used for all the base pairs except for the 1, N2-εdG lesion. The rationale for their inclusion was based on the NOESY experiments in which the observation of imino and amino proton resonances was consistent with a right-handed Watson-Crick paired duplex (Figure 3). Empirical sugar pucker restraints were not used for T5, X6, and G7 because the pattern of chemical shift perturbations (Figure 8) suggested a structural disturbance at the X6 nucleotide. No backbone torsion angle restraints were used at X6, C18, and A19. Elsewhere, backbone angles R, β, γ, δ, ε, and ξ were restrained to -60 ( 30 , 180 ( 30 , 60 ( 30 , 120 ( 30 , 180 ( 30 , and -90 ( 30 , respectively, to allow both A- and B-form DNA structures. A total of 24 distance