2622 Biochemistry, Vol. 49, No. 12, 2010 Table 2: Analysis of the rMD-Generated Structuresa of the 1,N2-εdG Adduct Opposite 1-BD in the 50-TXG-30 Sequence NMR restraints total no. of distance restraints no. of inter-residue distance restraints no. of intraresidue distance restraints empirical restraints no. of hydrogen bonding restraints no. of sugar pucker restraints no. of backbone torsion angle restraints initial structures IniA vs IniB rms shifts IniA vs ÆrMDAæb IniB vs ÆrMDBæb rms distributions ÆrMDAæ vs ÆrMDAæ ÆrMDBæ vs ÆrMDBæ ÆrMDAæ vs ÆrMDBæ ÆrMDAæ vs rMDc ÆrMDBæ vs rMDc atomic rmsd (A˚ ) 307 140 167 45 80 135 5.84 3.44 4.46 0.33 0.54 1.31 0.17 0.27 aRoot-mean-square deviations, excluding the end base pairs and includ- ing only heavy atoms from the rest of the residues, between various initial structures and averaged structures emergent from rMD. bÆrMDAæ and ÆrMDBæ represent the set of five structures that emerge from rMD calcula- tions starting with IniA and IniB, respectively. crMD represents the average minimized structure from all 10 rMD calculations. FIGURE 9: View of the central 50-TXG-30 segment of the 1-BD duplex as seen from the major groove. root factor, R1x, measured from the CORMA calculations, is shown in Figure S4 of the Supporting Information. The overall intra- and inter-residue sixth root residual R1x values for 1,N2- εdG duplex structures were 7.67 10-2 and 9.18 10-2, respectively. An average rMD structure was obtained using SUPPOSE, followed by 250 iterations of potential energy mini- mization using the conjugate gradient algorithm. A view normal to the helix axis and looking into the major groove of the central base pair segment (50-T5X6G7-30 3 50-C18A19-30) is shown in Figure 9. The 1,N2-εdG adduct adopted the anti conformation about the glycosyl bond. The 1,N2-εdG adduct was accommodated within the DNA helix. The 1,N2-εdG H6 etheno proton was close to the complementary strand compared to the 1, N2-εdG H7 etheno proton, consistent with the greater number of NOEs observed for the H6 proton versus the H7 etheno proton. Further, the etheno protons were close to the complementary strand C18 and A19 aromatic ring systems (Figure 10). Shanmugam et al. FIGURE 10: View from the top of the helix showing stacking inter- actions on the central segment of the duplex. (A) Interaction between the T5 3 A19 and X6 base pairs. (B) Interaction between the X6 and G7 3 C18 base pairs. DISCUSSION The error-prone replication bypass of the 1,N2-εdG lesion by the S. solfataricus P2 DNA polymerase IV (Dpo4) has been char- acterized (30). With the template sequence 30-G(1,N2-εdG)TACT- 50, preferential incorporation of dATP was observed. With a mixture of all four dNTPs, a mixture of full-length products 50-CAATGA-30 and 50-CATTGA-30 and deletion products 50-CATGA-30 and 50-CTGA-30 was observed (30). The formation of the -1 deletion product 50-CATGA-30 (30) is of particular interest with regard to this study. This is postulated to arise via the so-called “type II” Dpo4 insertion mechanism (31), in which the polymerase skips the damaged base in the 30-G(1,N2-εdG)TACT- 50 template and instead utilizes the 50 neighbor thymine (under- lined) to incorporate dATP. If further extension occurs without relaxation of the resulting bulged 1,N2-εdG lesion in the template, the result is a -1 deletion event, leading to the 50-CATGA-30 product (30). Crystallographic analyses of a 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 have been reported and provide insight into the mechanism of lesion bypass (30). In one case, 1,N2-εdG situated at position 5 of an 18-mer template stand paired with a 13-mer (-1) primer strand; crystallization with Dpo4 showed that the incoming (d)dATP paired with the 50 neighbor T of the template, as predicted by the “type II” mechan- ism (31) for the polymerase. Significantly, the 1,N2-εdG lesion adopts the anti conformation about the glycosyl bond and is inserted into the duplex. The incorporation of the 1,N2-propa- nodeoxyguanosine (PdG) adduct into the same 30-G(PdG)T- ACT-50 template sequence and the subsequent formation of ternary complexes with Dpo4 polymerase with dATP have also been reported (48). PdG differs from 1,N2-εdG in that it is a six-member as opposed to a five-member 1,N2-dG annela- tion product. In this case, the incoming dATP also pairs with the 50 neighbor T, as predicted by the “type II” mechanism (31).