Molecular and structural characterization of oxidized ribonucleotide insertion into DNA by human DNA polymerase β

Abstract

During oxidative stress, inflammation, or environmental exposure, ribo- and deoxyribonucleotides are oxidatively modified. 8-Oxo-7,8-dihydro-2'-guanosine (8-oxo-G) is a common oxidized nucleobase whose deoxyribonucleotide form, 8-oxo-dGTP, has been widely studied and demonstrated to be a mutagenic substrate for DNA polymerases. Guanine ribonucleotides are analogously oxidized to r8-oxo-GTP, which can constitute up to 5% of the rGTP pool. Because ribonucleotides are commonly misinserted into DNA, and 8-oxo-G causes replication errors, we were motivated to investigate how the oxidized ribonucleotide is utilized by DNA polymerases. To do this, here we employed human DNA polymerase β (pol β) and characterized r8-oxo-GTP insertion with DNA substrates containing either a templating cytosine (nonmutagenic) or adenine (mutagenic). Our results show that pol β has a diminished catalytic efficiency for r8-oxo-GTP compared with canonical deoxyribonucleotides but that r8-oxo-GTP is inserted mutagenically at a rate similar to those of other common DNA replication errors (i.e. ribonucleotide and mismatch insertions). Using FRET assays to monitor conformational changes of pol β with r8-oxo-GTP, we demonstrate impaired pol β closure that correlates with a reduced insertion efficiency. X-ray crystallographic analyses revealed that, similar to 8-oxo-dGTP, r8-oxo-GTP adopts an anti conformation opposite a templating cytosine and a syn conformation opposite adenine. However, unlike 8-oxo-dGTP, r8-oxo-GTP did not form a planar base pair with either templating base. These results suggest that r8-oxo-GTP is a potential mutagenic substrate for DNA polymerases and provide structural insights into how r8-oxo-GTP is processed by DNA polymerases.

Keywords: 8-oxoguanine (8-oxo-G); DNA damage; DNA polymerase; DNA repair; DNA replication; mutagenic nucleobase; nucleotidyl transferase reaction; oxidized ribonucleotide; r8-oxo-G lesion; structural biology.

Figure 1.

The chemical structure of various guanine nucleotides. Deviations from the canonical DNA polymerase substrate are highlighted in red.

Figure 2.

The ribonucleotide steric gate in DNA pol β. Helix M is shown as both surface and stick representation (gray). The ribose sugar O2′ (red sphere) was added to the pol β structure with an incoming dGTP (PDB code 4UB4). Steric clashes between the O2′ and helix M are indicated.

Figure 3.

Discrimination plot evaluating r8-oxo-GTP insertion. The catalytic efficiencies (k_pol/K_d) for insertion of r8-oxo-GTP opposite dC (yellow) or dA (green) for pol β are shown. The distance between the respective catalytic efficiencies is a measure of discrimination/fidelity. Each short horizontal bar represents the standard deviation of the mean of triplicate independent determinations. The insertion efficiencies for dGTP were reported in Refs. , and 8-oxo-dGTP in Ref. . The catalytic efficiency values are reported in Fig. S1C and are as follows: 2.4 × 10⁻¹ ± 3.0 × 10⁻² μm⁻¹·s⁻¹ (dGTP:dC), 1.0 × 10⁻⁴ ± 3.6 × 10⁻⁵ μm⁻¹·s⁻¹ (dGTP:dA), 3.9 × 10⁻³ ± 7.0 × 10⁻⁵ μm⁻¹·s⁻¹ (8-oxo-dGTP:dC), 4.1 × 10⁻² ± 4.1 × 10⁻³ μm⁻¹·s⁻¹ (8-oxo-dGTP:dA), 6.7 × 10⁻⁵ ± 5.7 × 10⁻⁶ μm⁻¹·s⁻¹ (rGTP:dC), 9.5 × 10⁻⁷ ± 1.8 × 10⁻⁷ μm⁻¹·s⁻¹ (r8-oxo-GTP:dC), 5.9 × 10⁻⁵ ± 7.9 × 10⁻⁵ μm⁻¹·s⁻¹ (r8-oxo-GTP:dA), 8.9 × 10⁻⁷ ± 8.3 × 10⁻⁸ μm⁻¹·s⁻¹ (Y271G:r8-oxo-GTP:dC), and 2.4 × 10⁻³ ± 2.9 × 10⁻⁴ μm⁻¹·s⁻¹ (Y271G:r8-oxo-GTP:dA).

Figure 4.

Calculated interprobe distances from FRET efficiencies during steady-state FRET experiments. Efficiencies from triplicate measurements are plotted for the following complexes opposite either a templating cytosine (dark blue) or templating adenine (red): binary; ternary with dGTP, rGTP, or r8-oxo-GTP; with error bars (black) calculated using standard deviation of the mean. Distance values are reported in Fig. S3 and are as follows: 42.6 ± 1.3 Å (binary:dC), 38.0 ± 0.3 Å (binary:dA), 30.7 ± 0.7 Å (dGTP:dC), 36.0 ± 0.5 Å (dGTP:dA), 38.1 ± 0.8 Å (rGTP:dC), 35.8 ± 0.4 Å (rGTP:dA), 38.1 ± 0.7 Å (8-oxo-dGTP:dC), 31.8 ± 0.5 Å (8-oxo-dGTP:dA), 37.7 ± 0.9 Å (r8-oxo-GTP:dC), and 35.2 ± 0.5 Å (r8-oxo-GTP:dA).

Figure 5.

X-ray crystallographic structure of Y271G pol β in complex with r8-oxo-GTP opposite dC. A, precatalytic closed ternary complex of Y271G pol β (yellow) with r8-oxo-GTP (blue sticks) bound across from dC. Mn²⁺ and Na⁺ ions are shown in purple, and potential hydrogen bonds are shown as black dashed lines. A polder map (green mesh) contoured at 3.0 σ is shown for the incoming r8-oxo-GTP. B, overlay of the incoming nucleotide and steric gating residues for the r8-oxo-GTP:dC complex (blue), the undamaged rGTP:dC complex (gray), and the dGTP:dC complex (white) (PDB code 4UB4). Potential clashes are highlighted by red dashed lines and potential hydrogen bonds as black dashed lines. C, overlay between the r8-oxo-GTP:dC complex (yellow/blue) and an undamaged rGTP:dC complex (gray). Key residues and DNA are shown as sticks and the N-helix as a cartoon. D, overlay between the r8-oxo-GTP:dC complex (yellow/blue) and a mismatched dAPCPP:dG complex (orange) (PDB code 3C2M). Key residues and DNA are shown as sticks and the N-helix as a cartoon.

Figure 6.

X-ray crystallographic structure of Y271G pol β in complex with r8-oxo-GTP opposite dA. A, precatalytic closed ternary complex of Y271G pol β (green) with r8-oxo-GTP (blue sticks) bound across from dA. Mn²⁺ ions are shown in purple, and potential hydrogen bonds are shown as black dashed lines. A polder map (green mesh) contoured at 3.0 σ is shown for the incoming r8-oxo-GTP. B, overlay between the r8-oxo-GTP:dA complex (green/blue) and the 8-oxo-dGTP:dA complex (purple) (PDB code 4UAW). Key residues and DNA are shown as sticks. Potential hydrogen bonds are shown as black dashed lines and the N-helix as a cartoon. C, overlay between the r8-oxo-GTP:dA complex (green/blue) and a rGTP:dC complex (gray). Key residues and DNA are shown as sticks and the N-helix as a cartoon. D, overlay of the incoming nucleotide and steric gating residues for the r8-oxo-GTP:dA complex (green), the undamaged rGTP:dC complex (gray), and the 8-oxo-dGTP:dA complex (blue) (PDB code 4UAW). Potential hydrogen bonds are shown as black dashed lines.

Figure 7.

Schematic of r8-oxo-GTP processing by human DNA polymerase β.