Structural Factors That Determine Selectivity of a High Fidelity DNA Polymerase for Deoxy-, Dideoxy-, and Ribonucleotides^*

Ternary Complexes 1–8

A closed form ternary complex (PDB code 2HVI (37)) from which the active site residues on the O helix and preceding loop region (residues 681–727), incoming nucleotide substrate, metal ions, surface-bound sulfate (from the crystallization solution), and water molecules had been removed was used as the starting model for solving the structures of complexes 1, 2, 4, 5, and 7. Complexes 3, 6, and 8 are the Mg²⁺ or Mn²⁺ counterparts for complexes 4, 5, and 7. Preliminary refinement showed that the two complexes with different metal ions are almost identical. Therefore, fully built and refined structures of complexes 4, 5, and 7 with water molecules removed were used as the starting models for their metal-substituted counterparts.

Initial electron density maps for complexes 1, 2, 4, 5, and 7 were generated by Fourier synthesis after rigid body refinement using the modified starting model 2HVI in PHENIX (44). Missing residues, nucleotide substrates, metal ions, and surface small molecules for all complexes were manually built in Coot (45) into the electron density map generated by iterative rounds of model building and refinement with PHENIX (44). All residues in the complexes were evaluated by visual inspection to fit the map. Simulated annealing refinement was then applied to further improve the model. TLS groups were determined using PHENIX find_tls_groups (44), and TLS parameters were refined subsequently. Water molecules were added automatically in the refinement followed by visual inspection.

For the closed form ternary complexes 1–8, the two molecules in the asymmetric are not the same; molecule 1 (chains D, E, and F in the PDB) is more ordered than molecule 2 (chains A, B, and C) (chain naming follows previously published structures (37)). Each molecule in complexes 1, 2, 7, and 8 adopts a single unique conformation (closed, ajar, or open). In complexes 3 and 4, molecule 1 does not adopt a unique conformation (molecule 2 is open and empty), and it was modeled initially as a mixture of two distinct ajar O helix conformations. After refinement, additional densities around the O helix could be observed in difference maps, suggesting that there are additional conformations that have not been accounted for in the model. At least two distinct rCTP conformations were observed. In the final, deposited models for complexes 3 and 4, the conformationally diverse portions of the O helix, its preceding loop region, and rCTP were omitted. For complexes 5 and 6, molecule 1 adopted a single ajar conformation with bound rCTP, whereas molecule 2 adopted an ensemble of conformations similar to complexes 3 and 4; in the deposited structures for complexes 5 and 6, these also were omitted from the model.

Closed Form Binary Complex 9

The E658A mutant, DNA, dCTP, and Mg²⁺ complex was crystallized as described above for the closed form ternary complexes 1–8. However, the unit cell dimensions of this complex resemble those observed for an open, binary crystal form (31, 32), and it contains only one molecule in the asymmetric unit. The structure was determined by molecular replacement with Phaser (46), using as the search model the open, empty molecule of complex 7, which carries the same mutation and contains a DNA duplex with the same sequence. The output model was refined with simulated annealing in PHENIX (44) followed by iterative rounds of visual inspection, manual building, and refinement as described above. dCTP was not observed in the active site of this complex but was observed to bind at a remote surface site (37–39), confirming its presence in the crystal.

Open Form Binary Complex 10

an open form binary complex (PDB code 1L5U) with the base pair positioned at the postinsertion site surface-bound sulfate, and water molecules removed. The model for the rC nucleotide incorporated at the primer 3′ terminus was built into the electron density map. The structure was refined as described above. There is one molecule in the asymmetric unit with a single, open conformation.

Nucleotide Incorporation Selectivity

Nucleotide affinities and incorporation rates of dCTP, rCTP, and ddCTP were determined for wild-type BF and its E658A and F710Y mutants by pre-steady-state kinetics (Table 2). These values are consistent with those determined for other DNA polymerases in the A family (14, 19, 50, 51). Wild-type BF favors dCTP incorporation over rCTP or ddCTP by 4 and 3 orders of magnitude, respectively. The E658A mutant decreases the selectivity against rCTP by 2 orders of magnitude, primarily as a consequence of a 100-fold loss in the dCTP catalytic rate, consistent with the behavior of this mutation in other members of the DNA polymerase A family (Fig. 1). The F710Y mutation renders incorporation of ddCTP essentially as efficient as dCTP and slightly boosts the catalytic rate of the latter. This mutation also improves binding of rCTP but compromises its incorporation rate to a similar degree.

Nucleotide Positioning, O Helix Motion, and Water Structure

High resolution structures of dCTP, rCTP, and ddCTP complexes were obtained for the wild-type active site BF and the F710Y and E658A mutants using a primer lacking a 3′-OH of its terminus (Table 3). In all crystallized ternary complexes, a surface D598A mutation was included to destabilize a crystal contact that favors the open state in the wild-type crystal lattice (32). The resulting crystal form contains two molecules in the asymmetric unit. In all complexes, molecule 1 is more ordered than 2; accordingly, our comparative analysis is based on molecule 1. In all complexes, the nucleotide is positioned at the insertion site and forms hydrogen bonds with dG on the template strand. Mg²⁺ and Mn²⁺ were used in the rCTP complexes to determine the location of bound metal by anomalous scattering of the latter.

In the wild-type active site, the rCTP (Fig. 2) and ddCTP (Fig. 3) nucleotide triphosphates are displaced from the dCTP position. Consequently, the rCTP or ddCTP α-phosphates are not aligned properly with the primer strand terminus, consistent with the diminished k_pol rate of these nucleotides relative to dCTP. The structure of the rCTP complex with wild-type BF is complicated by the presence of a mixture of two rCTP conformations, both with misaligned α-phosphates; this situation is simplified in the rCTP complex of the F710Y variant (which retains a strong preference for dCTP over rCTP; Table 2) in which only one, misaligned conformation is observed (Fig. 2). In all misaligned rCTP or ddCTP complexes, the O helix fails to adopt the fully closed conformation (Figs. 2 (A and E) and 3A). Furthermore, these complexes contain a water layer that is absent in the fully closed wild-type dCTP and F710Y ddCTP complexes (Fig. 4). A water layer also is present in the fully open (e.g. PDB code 1L3S (32)) and ajar conformations (PDB codes 3HP6 (38) and 3PX4 (39)); its displacement is therefore part of the mechanism by which improper nucleotides are selected against.

ddCTP

The failure of ddCTP to displace the water layer present in the open state, with the accompanying lack of O helix closure and misalignment of the α-phosphate, is accompanied by the loss of two hydrogen bonds, formed between dCTP 3′-OH and its own β-phosphate oxygen and the backbone amide of Glu-658 (Fig. 3, C and D). These lost hydrogen bonds are compensated for in the F710Y mutant, which is located on the O helix surface facing the bound nucleotide. In the F710Y-ddCTP complex, the mutant phenolic hydroxyl substitutes for the 3′-OH to interact with the β-phosphate of bound ddCTP and backbone amide of Glu-658 (Fig. 3G). As a consequence, the ddCTP affinity improves sufficiently (∼40-fold; Table 2) to displace the water layer and close the O helix (Figs. 3 (E and F) and 4C). The F710Y-ddCTP complex is essentially indistinguishable from the wild-type dCTP complex, accounting for their nearly identical incorporation rates. We note that Tyr-710 is a natural variant in a number of family A DNA polymerases (e.g. T7 DNA polymerase) (52).

rCTP

In the wild-type enzyme in the presence of Mg²⁺ or Mn²⁺, rCTP adopts an ensemble of conformations, and the O helix exhibits an ensemble of states, none of which correspond to the fully closed form. This ensemble is simplified in the F710Y mutant in which both the bound rCTP and the O helix adopt a single, misaligned, non-closed conformation with either metal (Fig. 2A). In this complex, four effects are observed that misalign the α-phosphate and interfere with nucleotide incorporation. First, rather than forming a steric block, the Glu-658 carboxylate interacts with the rCTP 2′-OH (2.7 Å apart) (Fig. 2B). Consequently, the position of the entire rCTP is displaced from that of dCTP to such an extent that even the template dG base moves to retain pairing between the two bases, albeit with base pair parameters that deviate substantially from those of canonical Watson-Crick base pairs (Table 4). Furthermore, the 2′-OH forms a hydrogen bond with Arg-615, which normally forms a hydrogen bond with the sugar O4 atom (Fig. 2B). This residue also participates in recognizing cognate base pairs by reading out their minor groove hydrogen bonding pattern (31). Second, the rCTP displacement opens an aperture between the bound nucleotide and the wall of the active site opposite the O helix, which is filled with a water layer that adopts a structure different from what was observed in the ddCTP complex (Fig. 4D). Third, the triphosphate binds in an altered conformation that is stabilized by this water layer (Fig. 5, A and B). The absence of an anomalous scattering signal revealed that in this conformation, the Mg²⁺ (substituted with Mn²⁺ to obtain an anomalous signal) that normally assembles with the nucleotide is absent and replaced by two water molecules (Fig. 2C). The direct hydrogen bond between the 3′-OH and its own β-phosphate oxygen observed in the dCTP complex is replaced by the Tyr-710 phenolic hydroxyl (Fig. 2C). This cyclic, non-covalent interaction within the nucleotide triphosphate has also been observed in ternary complexes of B-, C-, X-, and Y-family DNA polymerases and reverse transcriptase family (Fig. 6). Finally, the direct interaction between 3′-OH and Glu-658 main-chain amide is replaced by a water-mediated hydrogen bond (Fig. 2C). The stabilizing interaction of the bound rCTP β-phosphate with Tyr-710 on the O helix and the Glu-658 backbone amide via a water molecule presumably together improve affinity sufficiently (Table 2) to select a single conformation of the ensemble present in the wild-type complex.

The loss of the interaction between the glutamate carboxylate and 2′-OH of the rCTP complex in the E658A single mutant does not suffice to form the closed state (Fig. 2E). The diminished selectivity between rCTP and dCTP (Table 2) is primarily a consequence of a 100-fold loss of activity for dCTP.

dCTP

To elucidate the structural mechanism for the loss of dCTP incorporation in the E658A mutant, we cocrystallized this mutant with dCTP and Mg²⁺. The resulting complex (Fig. 7) contains only one molecule in the asymmetric unit, compared with the two observed in all other ternary complexes. This complex does not bind a dCTP at its active site (dCTP is observed to bind at a remote surface site, confirming its presence in the crystal). The polymerase conformation is similar to the open state (31, 32), but the C-terminal segment of the O helix (residues 707–714) has bent toward the primer DNA 3′ terminus (Fig. 7A). A conserved water molecule that usually is present both in open structures (31, 32) (Fig. 7C) and in ternary complexes (37–39) is displaced by the backbone carbonyl of Val-713 (Fig. 7B). It also is present in all structures except this one, reported here. This water molecule normally interacts with the minor groove of the unincorporated dNTP-template base pair. Its hydrogen-bonding interactions are likely to be sufficiently strong that they perturb the intrinsic tautomeric equilibria of the free nucleotides (39). Loss of this water molecule can therefore be expected to have a significant impact on dNTP incorporation rates.

The loss of this important water molecule in the E658A mutant is accompanied by several rearrangements in the active site, which are compensated for by the 2′-OH in the rCTP complex. Normally, Gln-797, Asn-793, and Glu-658 form a hydrogen-bonded network that coordinates this water. This network is broken in E658A such that Gln-797 adopts a different side chain rotamer conformation, and the Val-713 carbonyl is displaced (Fig. 7D). These motions together destroy the water coordination sphere. However, in the rCTP complex, the 2′-OH is able to compensate for the loss of the carboxylate in the E658A, because in the properly formed closed state, this hydroxyl occupies the position vacated by the carboxylate, restoring the hydrogen-binding network (Fig. 4E). Despite the restoration of this critical water in the rCTP complex, the incorporation rate of this nucleotide remains low because it still adopts distorted sugar and triphosphate conformations (Figs. 2F and 5, C and D). The direct interaction between the 3′-OH and β-phosphate is replaced by water; there is no interaction between the 3′-OH and the E658A backbone amide; and the triphosphate binds Mg²⁺ (confirmed by Mn²⁺ anomalous signal) but not at full occupancy (Fig. 2G). The O helix remains open to a similar degree as observed for the F710Y-rCTP complex.

Base Pairing of the Incorporated rCTP

Covalently incorporated, cognate base pairs positioned at the postinsertion site form hydrogen bonds between their minor groove N3 (purines) or O2 atoms (pyrimidines) and Arg-615 and Gln-797 (31). We incorporated rCTP at the primer strand 3′ terminus by catalysis in solution in the presence of Mn²⁺ (53) prior to crystallization. This binary complex between wild-type BF and DNA was crystallized in the absence of additional nucleotide. The resulting crystals contain one molecule in the asymmetric unit in the open form (Table 3). The rC is positioned in the postinsertion site and retains the minor groove readout by Arg-615 and Gln-797 observed for deoxynucleotide (31) (Fig. 8), emphasizing the importance of selecting against rNTPs prior to their incorporation.