A guardian residue hinders insertion of a Fapy•dGTP analog by modulating the open-closed DNA polymerase transition

Abstract

4,6-Diamino-5-formamidopyrimidine (Fapy•dG) is an abundant form of oxidative DNA damage that is mutagenic and contributes to the pathogenesis of human disease. When Fapy•dG is in its nucleotide triphosphate form, Fapy•dGTP, it is inefficiently cleansed from the nucleotide pool by the responsible enzyme in Escherichia coli MutT and its mammalian homolog MTH1. Therefore, under oxidative stress conditions, Fapy•dGTP could become a pro-mutagenic substrate for insertion into the genome by DNA polymerases. Here, we evaluated insertion kinetics and high-resolution ternary complex crystal structures of a configurationally stable Fapy•dGTP analog, β-C-Fapy•dGTP, with DNA polymerase β. The crystallographic snapshots and kinetic data indicate that binding of β-C-Fapy•dGTP impedes enzyme closure, thus hindering insertion. The structures reveal that an active site residue, Asp276, positions β-C-Fapy•dGTP so that it distorts the geometry of critical catalytic atoms. Removal of this guardian side chain permits enzyme closure and increases the efficiency of β-C-Fapy•dG insertion opposite dC. These results highlight the stringent requirements necessary to achieve a closed DNA polymerase active site poised for efficient nucleotide incorporation and illustrate how DNA polymerase β has evolved to hinder Fapy•dGTP insertion.

Figure 1.

Formation of formamidopyrimidines and 8-oxopurine lesions comes from a common intermediate, adapted from (26).

Figure 2.

Discrimination plot for insertion of β-C-Fapy•dGTP. The catalytic efficiencies (k_pol/K_d) for insertion of β-C-Fapy•dGTP opposite dC (green) or dA (red) for wild-type (WT) pol β are shown. The distance between the respective catalytic efficiencies is a measure of discrimination/fidelity. Each horizontal short bar represents the mean of duplicate independent determinations. Additionally, the value for each determination is plotted (small solid circle) to illustrate the reproducibility of these assays. The insertion efficiencies for dGTP opposite dC and dA were taken from (39).

Figure 3.

(A) Pre-catalytic ternary-open complex of wild-type pol β (gray) with β-C-Fapy•dGTP (green sticks) bound across from dC; Ca²⁺ ions are shown in orange. A polder map (green mesh) contoured at 3.0σ is shown for the incoming β-C-Fapy•dGTP. (B) β-C-Fapy•dGTP is shown as green sticks with the nucleotide binding pocket in surface representation looking into the minor groove behind the primer terminus (O3′). The Watson–Crick edge (W–C edge) is indicated. (C) Active site residues (gray sticks) that contact β-C-Fapy•dGTP (green) or Ca²⁺ (orange) are shown. Potential hydrogen bonds are shown as black dashed lines.

Figure 4.

(A) Pre-catalytic ternary-open complex of wild-type pol β (gray) with β-C-Fapy•dGTP (green sticks) bound across from dA; Ca²⁺ ions are shown in orange. A polder map (green mesh) contoured at 3.0σ is shown for the incoming β-C-Fapy•dGTP. (B) An alternate viewpoint demonstrating the two formamide conformations of β-C-Fapy•dGTP (green sticks) opposite adenine (gray sticks) with the polder map contoured to 3.0σ (green mesh). (C) An overlay between the pre-catalytic ternary-open structures of β-C-Fapy•dGTP across from dA (green) or dC (yellow). Key residues are shown as gray sticks and the N-helix in gray cartoon. (D) An overlay between the pre-catalytic ternary-open structures of β-C-Fapy•dGTP across from dA (green) or dC (yellow) highlighting altered amino acids in stick format with the N-helix subdomain shown as gray cartoon and potential hydrogen bonds as dashed lines.

Figure 5.

Discrimination plot for insertion of β-C-Fapy•dGTP. The catalytic efficiency of β-C-Fapy•dGTP insertion opposite dC or dA for wild-type pol β and the mutant where Asp276 was substituted with glycine (D276G) are shown. Each horizontal short bar represents the mean of duplicate independent determinations. Additionally, the value for each determination is plotted (small solid circle) to illustrate the reproducibility of these assays.

Figure 6.

(A) Pol β D276G mutant in a ternary-closed ground state (gray) with incoming β-C-Fapy•dGTP base (green sticks) pairing with dC in a canonical conformation. The polder map is shown as green mesh and contoured to 3.0σ. (B) Overlaid is PDB ID: 4UB4 demonstrating undamaged dGTP pre-catalytic insertion (cyan). The Watson–Crick edge (W–C edge) is indicated. Potential hydrogen bonds are shown as black dashed lines. The D276 and formamide/O8 clash is demonstrated via red dashed lines. (C) A rotated viewpoint of panel B highlighting the extent of the Asp276 and formamide/O8 clashes (red dashed lines).

Figure 7.

The pre-catalytic ternary-open pol β:β-C-Fapy•dGTP:dC (gray/green) structure is overlaid with the following: (A) A ternary-open complex (indigo) with an incoming nucleotide analog, dCMP(CF2)PP, opposite a templating 8-oxoGuo (PDBID: 4F5O). (B) A ternary-open complex (magenta) with an incoming nucleotide analog, dAMPCPP, opposite a templating dG (PDBID: 4F5P). (C) A ternary-closed complex (cyan) with an incoming analog, dUMPNPP, opposite a templating dA (PDBID: 2FMS).