Article CA). Proton-decoupled 31P spectra were recorded at 25 C. The 31P- 1 H COSY heterocorrelation spectrum was measured with presaturation of protons and selective excitation. The data matrix consisted of 96 data points in the t1 dimension and 1024 data points in the t2 dimension. The spectra were zero- filled during processing to create a matrix of 128 512 real points. NMR Restraints. Cross-peak intensities were determined by volume integrations of the NOESY spectra. These were com- bined as necessary with intensities generated from complete relaxation matrix analysis of a B-DNA structure to generate a hybrid intensity matrix (35, 36). The distances were obtained from the NOE volumes using MARDIGRAS version 5.2 (37). The RANDMARDI algorithm (38) conducted 50 iterations for each set of data, randomizing peak volumes within limits specified by the input noise level. The molecular motion was assumed to be isotropic. The volume error was one-half the volume of the weakest cross-peak. Calculations were performed using a B-DNA starting structure, NOE intensities derived from experiments at three mixing times, and three isotropic correlation times (2, 3, and 4 ns), yielding nine sets of distances. Analysis of these data yielded the experimental distance restraints and their standard deviations used in subsequent restrained molecular dynamics calculations. For partially overlapped cross-peaks, the upper bounds on the distances were increased. Distance restraints were classified according to the error assessed in their measurements. Five classes were defined on the basis of com- pletely resolved, slightly overlapped, partially overlapped, intermediately overlapped, and heavily overlapped cross-peaks, which were at least 0.5 ppm from the water resonance or from the diagonal line of the spectrum. Additional empirical restraints preserving Watson-Crick hydrogen bonding and preventing excessive propeller twisting of base pairs were in- cluded since the NMR data indicated that the duplex conserved Watson-Crick base pairing except at the unpaired 1,N2-εdG lesion. Structural Refinement. The calculations were conducted with AMBER (39). Classical B-DNA and A-DNA (40) were used as reference structures to create starting structures. The 1,N2-εdG adduct was constructed using INSIGHT II. The restrained electrostatic potential (RESP) charges for 1,N2-εdG were calcu- lated using GAUSSIAN (41) (Figure S1 of the Supporting Information). The input data for the calculations were obtained from ANTECHAMBER (42). The complementary strand of the 1-BD duplex was created by deletion of dC opposite 1,N2-εdG and creation of a bond between O30 of the 30-flanking dC and P of the 50-flanking dA. The coordinates, connectivity, and para- meters were obtained with xLEaP. Initially constructed A- and B-DNA starting structures were energy-minimized by the con- jugate gradient method without experimental restraints for 250 iterations using SANDER (43). This process yielded the structures used for subsequent relaxation matrix analysis and restrained molecular dynamics calculations. For rMD calcula- tions, the restraint energy function included terms describing distance restraints as square-well potentials. The generalized Born solvent model was used with a salt concentration of 0.1 M, and the SHAKE algorithm was used (44, 45). The nonbonded pair list was updated if any atom moved more than 0.5 A˚ , and the cutoff radius for nonbonded interactions was 18 A˚ . The effective energy function included terms describing distance restraints, in the form of square-well potentials. A total of 20000 steps were used. Calculations were initiated by coupling to a virtual heating Biochemistry, Vol. 49, No. 12, 2010 2617 Table 1: Melting Temperatures of the 1-BD Duplexa and Other Related Oligodeoxynucleotide Duplexesa aThe duplex contained ∼0.3 OD unit in 10 mM phosphate buffer, 100 mM NaCl, and 5 μM Na2EDTA (pH 7). bMelting temperatures as previously reported (50). cX = 1,N2-εdG. bath, with a target temperature of 600 K. The force constants were 20 kcal mol-1 A˚ -2 for empirical hydrogen bonding and 32 kcal mol-1 A˚ -2 for class 1, class 2, class 3, class 4, and class 5 NOE restraints. The force constants of the restraints were scaled from 3.2 to 32 kcal mol-1 A˚ -2 during the first 5000 steps and were maintained at 32 kcal mol-1 A˚ -2 during the rest of calculations. The target temperature was reached and maintained for 5000 steps. The molecules were cooled to 100 K over 13000 steps with a time constant of 2.0 ps. During the final 2000 steps, the temperature was reduced to 0 K. An average structure was subjected to 500 cycles of conjugate gradient energy minimiza- tion, yielding the final structure. The NOE intensities obtained from the structures emergent from rMD simulations were compared with the experimental intensities using Complete Relaxation Matrix Calculations (CORMA version 5.3) (36). Input volumes (intensities) were normalized for the intensities of protons with fixed internuclear distances (cytosine H5-H6 protons). An isotropic correlation time (τc) of 3 ns was used. The rotation of thymine CH3 groups was modeled using a three-jump site model. The sixth root residual (R1x) factor was calculated for the averaged structure. RESULTS Thermal Melting Studies of the 1-BD Duplex. Table 1 shows the thermal denaturation for the 1-BD duplex and a series of related duplexes. The melting temperature (Tm) of the 1-BD duplex was 45 C, and the corresponding Tm for the unmodified 1-BD duplex under the same conditions was 37 C. Thus, the presence of the 1,N2-εdG lesion increased the Tm by ∼8 C. Deletion of dC from the complementary strand increased the Tm compared to that of the fully complementary modified duplex (Table 1). The Tm of the duplex containing the 1,N2-εdG adduct paired opposite dC of a fully complementary strand was 39 C under the same conditions. Thus, the Tm of the 1,N2-εdG- containing duplex was 6 C higher when paired opposite a one- base deletion than a fully complementary strand. The increased stability of the 1-BD duplex compared to that of the corresponding unmodified 1-BD duplex was confirmed in NMR experiments. Figure 1A shows the imino proton region of a series of 1H NMR spectra, recorded as a function of tempera- ture. At 7 C, four sharp resonances for guanine imino protons (11.5-13.2 ppm) and five sharp resonances for thymine imino protons (13.2-13.9 ppm) were observed. When the temperature