Article etheno proton H6 H7 Table 3: Chemical Shift Comparison of Exocyclic Etheno Protons of the 1,N2-εdG Adduct in Different Oligodeoxynucleotide Duplexes 1,N2-εdG 3 dC duplex pH 8.6a 6.21 6.54 pH 5.2b 7.33 7.33 aChemical shift values taken from ref 50. bChemical shift values taken from ref 51. PdG also adopts the anti conformation about the glycosyl bond in the Dpo4 ternary complex and is accommodated in an intrahelical orientation (48). Recently, a similar structure for the malondial- dehyde-derived M1dG adduct has also been reported (49). The 1-BD duplex examined herein models the situation following the “type II” insertion of dATP in which the Dpo4 polymerase skips the damaged base in the 30-G(1,N2-εdG)TACT-50 template and instead utilizes the 50 neighbor thymine (underlined) to incorporate dATP (31), followed by further extension resulting in a bulged 1,N2-εdG lesion in the template, and a -1 deletion event, leading to the 50-CATGA-30 product (30). Structure of the 1-BD Duplex. 1,N2-εdG adopts the anti conformation about the glycosyl bond in the 1-BD duplex and is inserted into the duplex, similar to the series of binary template- primer 3 Dpo4 and ternary template-primer 3 Dpo4 3 (d)dATP complexes containing the 1,N2-εdG lesion in the same 30-G- (1,N2-εdG)TACT-50 template sequence (30). The intensity of the NOE cross-peak between 1,N2-εdG H2 and 1,N2-εdG H10 protons is similar to the intensities of the corresponding NOEs between either purine H8 or pyrimidine H6 protons and their attached deoxyribose H10 protons at the undamaged nucleotides in the duplex. The anti conformation at 1,N2-εdG is also consistent with the appearance of the sequential NOE between T5 H10 and X6 H2 protons. Similarly, the appearance of weak NOEs between the X6 H6 and H7 protons and the G7 N1H imino proton is consistent with the anti conformation of the glycosyl bond. The pattern of NOEs between the 1,N2-εdG H6 and H7 protons and the DNA protons is consistent with its accommoda- tion in an intrahelical orientation. For example, cross-strand NOEs are observed for the 1,N2-εdG H6 and H7 protons with both major groove C18 H6 and A19 H8 protons and the minor groove A19 H2 proton. The observation that the 1,N2-εdG H6 proton shows more cross-strand NOEs than does the H7 proton is consistent with the conclusion that the former proton is closer to the complementary strand than the latter proton is. The 5.84 and 5.55 ppm chemical shifts of the 1,N2-εdG H6 and H7 protons, respectively (Table S1 of the Supporting Information), also confirm their intrahelical orientation. Their resonances are shifted upfield by ∼2 ppm relative to their resonance frequencies in the monomer state. The structural refinement indicates that the 1,N2-εdG etheno moiety is located between the C18 and A19 nucleobases, which accounts for the upfield chemical shifts. The etheno protons are also shifted upfield compared to the situation in which 1,N2-εdG is placed opposite dC in the fully comple- mentary duplex. In the fully complementary duplex, 1,N2-εdG exists in equilibrium between the syn and anti conformers about the glycosyl bond (50, 51). In the anti conformation, 1,N2-εdG shifts toward the minor groove, thus weakening stacking inter- actions experienced by the etheno moiety (50), whereas in the syn conformation, 1,N2-εdG shifts into the major groove and the etheno moiety is not stacked into the helix (51) (Table 3). 1-BD duplex pH 7.0 5.55 5.84 Biochemistry, Vol. 49, No. 12, 2010 2623 1,N2-εdG Stabilizes the 1-BD Duplex. The deletion of dC from the complementary strand opposite 1,N2-εdG increases the duplex Tm compared to that of the complementary modified duplex (Table 1). The latter duplex exhibited a Tm of 39 C, whereas that of the 1,N2-εdG-modified 1-BD duplex increases to 45 C. At neutral pH, the complementary modified duplex exists as an equilibrium mixture of syn and anti conformers of the 1,N2- εdG lesion about the glycosyl bond (50, 51). Zaliznyak et al. (52) reported a similar conformational equilibrium for 1,N2-εdG in a 30-C(1,N2-εdG)C-50 sequence when placed opposite dC. In con- trast, the Tm for the unmodified 1-BD duplex is 37 C, which is 8 C lower than that of the modified 1-BD duplex (Table 1). These data are significant because one outcome of lesion bypass by the Dpo4 polymerase is the 50-CATGA-30 one-base deletion pro- duct (30). The formation of the one-base deletion product implies that the polymerase skips the damaged base in the 30-G(1,N2- εdG)TACT-50 template and instead utilizes the 50 neighbor thymine (underlined) to incorporate dATP (31), which may be attributed to the stability of the resulting one-base deletion product. Similarly, when the (þ)-cis-anti-[BP]dG adduct was placed opposite a one-base deletion in DNA (53), enhanced thermodynamic stability of the damaged duplex was also noted. In that instance, the enhanced thermodynamic stability of the duplex was also correlated with crystallographic studies using the Dpo4 polymerase, leading to the conclusion that the polymerase may exploit thermodynamic stabilization of the lesion for gene- ration of -1 frameshift mutations (54). Overall, enhanced stabilities of -1 base deletion duplexes containing aromatic lesions may be a more general phenomenon and perhaps not limited to 1,N2-εdG adducts. The increased stability of the 1-BD duplex compared to that of the corresponding unmodified 1-BD duplex is consistent with these NMR data. The observation of resonances from the thymine T5 N3H, T10 N3H, T16 N3H, T17 N3H, and T21 N3H imino protons and sequential NOE connectivity for imino protons from G2 N1H to T5 N3H, located in the 50 direction from 1,N2-εdG (X6);and from G7 N1H to G14 N3H, located in the 30 direction from 1,N2-εdG (X6);indicates formation of a stable duplex on both sides of the unpaired 1,N2-εdG lesion (Figure 1). In contrast, for the unmodified 1-BD duplex, only three sharp guanine imino resonances are observed (Figure 1B). Moreover, the peak assigned as G7, next to the modification site in the 30 direction, broadens in the unmodified 1-BD duplex (Figure 1B). When the temperature is increased to 47 C, the T16, T17, T20, G21, and G7 imino proton resonances for the 1-BD duplex persist (Figure 1A), unlike the case for the unmodified 1- BD duplex (Figure 1B) for which all of the peaks disappear. Our results provide a thermodynamic rationale for why the -1 frameshift event is facilitated when the 1,N2-εdG lesion in the 30-G- (1,N2-εdG)TACT-50 template is bypassed by the Dpo4 polymerase. Structural Comparisons with Two-Base Deletion Pro- ducts Opposite Six-Member 1,N2-dG Annelation Adducts. M1dG and PdG are six-member exocyclic adducts formed between the N1 and N2 atoms of dG. The M1dG 3 2BD (55) and PdG 3 2BD (56) duplexes were structurally characterized with respect to the observation of deletions in iterated CG repeat sequences (57), e.g., which occur in the Salmonella typhimurium hisD3052 gene (58, 59). The structure of the 1-BD duplex determined here shows similarities to the structures of the M1dG 3 2BD (55) and PdG 3 2BD (56) duplexes. In all the duplexes, the damaged nucleotides exist in the anti conformation about the glycosyl bond. Further, the exocyclic rings of the