The undamaged ND-50-bp and AAF-50-bp oligodeoxynucleotides were constructed as described (21). The Tg-modified 5′-d(GTGCGTgGTTTGT)-3′ (17) was characterized using a Voyager-DE MALDI-TOF mass spectrometer (PerSeptive Biosystems, Inc., Foster City, CA, USA). It was ligated with a 5′-³²P-labeled 20-mer, and a 19-mer on the 5′- and 3′-ends, respectively, to form the 5′-³²P-labeled Tg-51mer. The oligodeoxynucleotides 5′-d(GTGCGTGTTTGT)-3′ and 5′-d(ACAAACACGCAC)-3′ were purchased from the Midland Certified Reagent Co. (Midland, TX, USA) and purified by reverse phase HPLC.

Assays for the UvrA protein were performed as described (21). For the human NER proteins XPA and XPC/HR23B, the binding assay was performed as described for XPA binding (22), with minor modifications. The radioactivity of DNA bands on gels was quantified using a Fuji FLA-5000 phosphoimager. Dissociation constants (K_d) were determined from the binding curves as a concentration of the substrate at which half of DNA was bound to protein. Three experiments were performed for each binding curve.

The 5′-³²P-labeled substrates (2 nM) were incised by UvrABC nucleases (UvrA, 15 nM; UvrB, 250 nM; UvrC, 100 nM) in the UvrABC buffer with 1 mM ATP at 37°C and the products were resolved on a 12% polyacrylamide gel and analyzed as described (21). The initial rates were calculated by linear least-squares fits of data. The substrate incised in femtomoles was calculated based on the total molar amount of substrate employed in each reaction and the incision percentage of the substrate. Three experiments were performed to determine initial rates.

Oligodeoxynucleotides were annealed in 20 mM sodium phosphate, containing 100 mM NaCl, 10 μM NaN₃ and 50 μM Na₂EDTA (pH 7.0). Experiments were performed at 800 MHz. NOESY spectra for the nonexchangeable protons were recorded at 30°C with mixing times of 80, 150, 200 and 250 ms using States-TPPI phase cycling, 512 real data points in the d1 dimension with 32 scans per FID, 2K real data points in the d2 dimension, sweep width of 10 p.p.m., and a relaxation delay of 2.0 s. The water resonance was suppressed using presaturation. NOESY spectra of exchangeable protons were obtained at 5°C using watergate H₂O suppression (23). T₁ spin–lattice relaxation experiments were collected using the inversion recovery method (24,25). T₂ transverse relaxation experiments were collected using the CPMG method (24,25). ¹H spectra were referenced to 3-(trimethylsilyl)propionic-2,2,3,3-d₄ acid, sodium salt (3-TMSP). The program XWINNMR (Bruker Inc., Billerica, MA, USA) was used for data processing. A skewed sinebell-squared apodization function with a 90° phase shift was used for NOESY experiments; the same function with a 10° phase shift was used for COSY experiments.

NOE intensities were determined from volume integration using SPARKY (26). Intensities for the cis-(5R, 6S) epimer were corrected for the 7:3 cis-(5R,6S):trans-(5R,6R) molar ratio. These were combined with intensities generated from CORMA analysis of a B-form starting structure producing a hybrid intensity matrix (27,28), which was refined using MARDIGRAS (29–31) with the RANDMARDI function. Calculations at mixing times of 80, 150, 200 and 250 ms were run at correlation times of 2, 3, 4 and 5 ns. Distance restraints were divided into categories indicative of confidence levels. Empirical restraints were used to define Watson–Crick hydrogen bonding, but not for the Tg ⁶•A¹⁹ or G⁵•C²⁰ pairs.

³J ¹H coupling constants were obtained by amplitude constrained multiplet evaluation of COSY data (32). Electronegativity of substituent Karplus curves were generated and converted to phase angle space assuming a maximum pucker amplitude (Φ) of 44° (33,34). Scalar couplings were fit to the curves to determine pseudorotation ranges (P). The sugar pseudorotation and amplitude ranges were converted to restraints for the dihedral angles ν₀ to ν₄. Measurements of the mol fraction of sugar puckers in the N or S conformations were determined from the sum of J_1'2;' and J_1'2;'' scalar couplings (34). Nucleotides with <50% X_S were allowed to explore both N and S conformations during rMD calculations (ρ = 0°–210°). Nucleotides with X_S > 50% were restrained such that ρ = 125°–210°. Backbone torsion angles were restrained with data where available; otherwise they were restrained empirically based on canonical A-form and B-form values.

Partial charges for Tg were generated using GAUSSIAN (35). Geometry optimization and ESP calculations were performed using the Hartree–Fock method with the 6-31G* basis set (12). The output was formatted using ANTECHAMBER (36). Starting structures were generated using NAB (37) and energy minimized using the SANDER module of AMBER (36). Coordinate and topology files were generated with xLEaP (36) using ff99 parameters (38). The restraint function utilized square-well potentials (39). The generalized Born model was used for simulated annealing calculations, with a salt concentration of 0.2 mM (40,41). Temperature was maintained using the Berendsen algorithm (42). Complete relaxation matrix analysis (27,28) was performed to determine agreement with ¹H NOESY data. A refined structure from simulated annealing was neutralized with the addition of sodium ions and placed in a truncated octahedral TIP3P water box with periodic boundaries at a distance of 8 Å from the solute. After equilibration, a 10-ns isothermal rMD calculation was performed. The temperature was controlled using the Langevin thermostat (43,44) with a collision frequency of 1 ps⁻¹. Electrostatic interactions were treated with the PME method (45). A 15-Å cutoff for nonbonded interactions was used. Bond lengths involving hydrogen were fixed using SHAKE (46). A structural ensemble was extracted from the isothermal trajectory using PTRAJ (36). The heavy atoms were subjected to pairwise RMSD comparisons using SUPPOSE. Helicoidal analysis was performed using CURVES (47,48). Structures were rendered using Chimera (49). Solvent-excluded and solvent-accessible surface areas of individual bases as a function of probe radius were calculated using MSMS (50).

The 5′-d(GTGCGXGTTTGT)-3′, X = 5R-Tg, was subjected to mass spectrometric analysis, which yielded the anticipated molecular ion peak with mass 3732 (m/z). Capillary gel electrophoretic and HPLC analysis showed that it eluted as a single peak. The adducted oligodeoxynucleotide was pure and existed as a single chromatographically separable species.

A 51-bp substrate containing 5R-Tg (Supplementary Scheme S1) was utilized. A 50-bp substrate containing an AAF adduct (AAF-50 bp) that is recognized and incised by E. coli UvrABC (21), was used for comparison. A 50-bp substrate (ND-50 bp) was used as a negative control. Figure 1A and B show gel mobility shift assays for UvrA binding to Tg and AAF substrates, respectively. The slowly migrating bands represent the formation of DNA–UvrA₂ complexes. Binding isotherms (Figure 1C) showed that the affinity of UvrA to the 5R-Tg substrate was high, though the binding of UvrA to the AAF substrate was stronger (Table 1). The UvrA had a 2.4-fold lower affinity to the Tg-50-bp substrate (K_d = 24 ± 2 nM) than the AAF-51-bp substrate (K_d = 10 ± 1 nM). The K_d for UvrA binding to the AAF substrate agreed with the reported value (21). UvrA had lower affinity to the undamaged than to the damaged substrate, in agreement with previous observations (21). While the differing sequence contexts of the Tg- and AAF–DNA substrates (Scheme 2A) might affect their interactions with the UvrABC system (51–53), it was assumed that sequence specific effects were smaller than those caused by the difference in the types of DNA lesions. Figure 1.

Binding of UvrA to the duplex containing the site-specific 5R-Tg lesion paired with dA. In (A) and (B), UvrA at the indicated concentrations was incubated with 2-nM substrates at 37°C for 20 min in UvrABC binding buffer and then analyzed on a 3.5% polyacrylamide native gel by gel mobility shift assays. (A) TG-51 bp; (B) AAF-50 bp. (C) The binding curves generated based on the titration data in (A) and (B).

The binding of XPA and XPC/HR23B to the 5R-Tg damaged duplex was compared with the AAF-damaged duplex using EMSA (Figure 3). Neither XPA nor XPC/HR23B bound to the undamaged substrate. The shifted bands represent formation of DNA–XPA₂ complexes. At low concentrations XPA bound to AAF-50 bp with a greater affinity than to Tg-51 bp, which was evident from a comparison of the 25 nM XPA lanes for 5R-Tg and AAF. At concentrations >50 nM, XPA bound to both substrates with comparable affinities (Figure 3A). The binding of XPC/HR23B to 5R-Tg and AAF-damaged DNA is shown in Figure 3B. Figure 3C shows binding isotherms for XPA interacting with the 5R-Tg- and AAF–DNA substrates. The dissociation constants estimated for the 5R-Tg and AAF substrates were similar, 48 ± 4 nM and 44 ± 6 nM, respectively (Table 1). XPC/HR23B bound to Tg-51 bp with a greater affinity than to AAF-50 bp (Figure 3D). Unlike XPC/HR23B the E. coli NER protein UvrA bound better to AAF–DNA adduct than to Tg-DNA adduct (Figure 1). Figure 3.

Binding of human XPC/HR23B and XPA proteins to the site-specific 5R-Tg paired with dA substrate. In (A) and (B), XPA (A) or XPC/HR23B (B) proteins at the indicated concentrations were incubated with 4-nM substrates at 30°C for 30 min in XPA binding buffer and then analyzed on 3.5% polyacrylamide native gels by gel mobility shift assays (XPA, 4°C; XPC/HR23B, room temperature). (C) and (D) show the binding curves generated from the data in (A) and (B).

Data were collected upon preparation of the duplex containing the 5R-Tg lesion (Scheme 1), and again after 4 weeks. Spectral changes were not observed, suggesting that the cis-(5R,6S) and trans-(5R,6R) epimers had achieved equilibrium. The cis-(5R,6S) epimer was favored 7:3 over the trans-(5R,6R) epimer, as determined by integration of Tg CH₃ peaks (12). It was possible to obtain spectroscopic data for the cis-(5R,6S) epimer; the trans-(5R,6R) epimer was not present at sufficient levels to allow evaluation of its spectrum.

The T₁ relaxation times of the T², T⁸, T⁹, T¹⁰, & T¹² CH₃ and X⁶ CH₃ groups were compared to those of the unmodified duplex (Supplementary Figure S3). The X⁶ CH₃ T₁ relaxation time was 1.9 s faster than was the corresponding T⁶ CH₃ relaxation time of the unmodified duplex containing T⁶•A¹⁹. The X⁶ CH₃ relaxed an average of 1.4 s faster than other thymine CH₃ groups in the X⁶•A¹⁹ duplex.

The T₂ relaxation times of the T², T⁸, T⁹, T¹⁰, & T¹² CH₃ and X⁶ CH₃ groups were compared to those of the unmodified duplex (Supplementary Figure S4). The Tg CH₃ of the X⁶•A¹⁹ duplex relaxed ∼70 ms faster than the corresponding CH₃ of the unmodified sample.

Thirty starting structures were generated, of which half had the Tg CH₃ group in the axial conformation and half had the Tg CH₃ group in the equatorial conformation. These exhibited a maximum pairwise RMSD of 4.3 Å. The distance and torsion angle restraints used in the calculations are summarized in Supplementary Table S1. As Watson–Crick hydrogen bonding at the X⁶•A¹⁹ base pair was not observed, hydrogen bond restraints between X⁶ and A¹⁹ were not used. All but three of the 30 structures emerged from simulated annealing calculations with Tg CH₃ in the axial conformation. A refined structure with Tg CH₃ in the axial conformation was placed into a truncated octahedron TIP3P water box, and subjected to 10 ns of isothermal rMD calculations at 300 K (Supplementary Figure S5). At 4.38 ns the Tg CH₃–C5–C6–H6 torsion angle shifted from −40° to 50°. This corresponded to a change from the Tg CH₃ axial to the equatorial conformation. The glycosyl Tg O4′–H1′–N1–C2 torsion angle fluctuated between −90° and −140° during the first 4.38 ns; when the Tg CH₃ group shifted from axial to equatorial conformation, this angle fluctuated between −130° and −150°.

The distances between Tg HO5→G⁷ N7 (Supplementary Figure S5, panel C) and Tg HO6→G⁷ N7 (Supplementary Figure S5, panel D) were monitored during the isothermal calculations. Analysis of trajectories indicated intra-strand hydrogen bond stabilization of the X⁶•A¹⁹ base pair as predicted (13). Hydrogen bond occupancy was defined as hydrogen bond donors and acceptors being within 3.5 Å with an angle cutoff of 120°. During the first 4.38 ns, in which the Tg CH₃ group was in the axial conformation, the Tg HO6→G⁷ N7 hydrogen bond criteria were satisfied (Supplementary Figure S5, panel D). This induced altered propeller twist at the lesion site. There was 42% occupancy of the Tg OH6→G⁷ N7 hydrogen bond during the trajectory and the entirety of this occupancy occurred during the first 4.38 ns. In contrast, during the first 4.38 ns, in which the Tg CH₃ group was in the axial conformation, the distance between Tg HO5 and G⁷ N7 was 4.5 Å, which was not consistent with hydrogen bonding. As a consequence of Tg CH₃ reorienting to the equatorial conformation at 4.38 ns, Tg OH5 shifted to the axial conformation. This allowed for hydrogen bond formation with G⁷ N7. The Tg OH5→G⁷ N7 occupancy was 11% over 10 ns and was only observed after the conformational shift. During these isothermal calculations, the RMSD of the DNA backbone heavy atoms (Supplementary Figure S5, panel F) was 2 Å.

Structural ensembles representing both axial and equatorial conformations of Tg CH₃ were extracted for analyses (Supplementary Figure S6). The axial ensemble (PDB ID 2KH5) was extracted from the 120 ps before the conformation change at 4.38 ns; this ensemble had an RMSD of 0.56 Å for the core 6 bp. The equatorial ensemble (PDB ID 2KH6) was extracted 1 ns after the conformational change at 4.38 ns. This had an RMSD of 0.69 Å for the core 6 bp.

To evaluate the accuracies of the rMD ensembles with respect to the ¹H NOESY data, complete relaxation matrix analyses (27,28) were performed (Supplementary Figure S7). The ensemble with Tg CH₃ in the axial conformation had an average sixth root residual R₁^X value of 8.12 × 10⁻². The Tg lesion exhibited an R₁^X value of 7.19 × 10⁻² for inter-nucleotide cross-peaks with G⁵ and 7.61 × 10⁻² for intra-nucleotide cross-peaks. The ensemble with Tg CH₃ in the equatorial position had an average R₁^X value of 8.15 × 10⁻². The Tg lesion exhibited an R₁^X value of 1.77 × 10⁻¹ for inter-nucleotide cross peaks with G⁵ and 1.01 × 10⁻¹ for intra-nucleotide cross-peaks. This indicated that both ensembles of structures satisfied the ¹H NOESY data, although at the lesion site, the agreement between the structures with the Tg CH₃ axial conformation and the NOESY data was improved.

In both the Tg-axial and Tg-equatorial ensembles the cis-(5R,6S) lesion was positioned in the Watson–Crick orientation such that Tg NH₃ was proximate to A¹⁹ N1 and Tg O⁴ was proximate to A¹⁹ N⁶H (Figure 5). However, for the Tg-axial ensemble, the base pair opening at X⁶•A¹⁹ increased such that the Tg shifted toward the major groove. For the Tg-equatorial ensemble, Tg maintained Watson–Crick positioning. Irrespective of the conformation of Tg, the complementary dA remained in the anti conformation about the glycosyl bond and stacked into the duplex (Figure 6). The Tg base stacked below the 5′-neighbor base G⁵, while its complement A¹⁹ stacked below the 5′-neighbor base C²⁰. In the 3′-direction, base stacking between Tg and the G⁷•C¹⁸ base pair was disrupted (Figure 5). Helicoidal analyses of both the axial and equatorial conformations of Tg indicated disturbances in shear, opening, stagger and stretch at the lesion (Figure 6). For the Tg CH₃ axial conformation the disturbance of the glycosyl torsion angle (χ) alleviated steric interactions between Tg CH₃ and the 5′-neighbor purine G⁵. The solvent accessible surface of Tg in the duplex relative to that of a free Tg base was compared with bases A²³, T⁹, and T¹² (Supplementary Figure S8). Irrespective of whether the Tg CH₃ group was in the axial or equatorial conformation, Tg exhibited an increased solvent accessible surface relative to T⁹ and A²³ but was not as exposed to solvent as the terminal T¹² base. The average accessible surface area of the Tg base was 19% in the CH₃ axial structure and 18% in the CH₃ equatorial structure. Figure 5.

Base pair stacking interactions of the cis-(5R,6S) Tg lesion. Comparison stacking interactions in which Tg CH₃ is in the axial (PDB ID 2KH5) or equatorial (PDB ID 2KH6) conformations. The top panel shows the G⁵• C²⁰ base pair (black) stacked above the X⁶• A¹⁹ base pair (Tg is colored red and A¹⁹ is colored gray). The center panel shows the orientation of the X⁶ lesion (red) with respect the complementary nucleotide A¹⁹ (black). The bottom panel shows the X⁶• A¹⁹ base pair (Tg is colored red and A¹⁹ is colored black) stacked above the G⁷• C¹⁸ base pair (grey).