Mechanisms of Insertion of dCTP and dTTP Opposite the DNA Lesion O6-Methyl-2'-deoxyguanosine by Human DNA Polymerase η

Abstract

O⁶-Methyl-2'-deoxyguanosine (O⁶-MeG) is a ubiquitous DNA lesion, formed not only by xenobiotic carcinogens but also by the endogenous methylating agent S-adenosylmethionine. It can introduce mutations during DNA replication, with different DNA polymerases displaying different ratios of correct or incorrect incorporation opposite this nucleoside. Of the "translesion" Y-family human DNA polymerases (hpols), hpol η is most efficient in incorporating equal numbers of correct and incorrect C and T bases. However, the mechanistic basis for this specific yet indiscriminate activity is not known. To explore this question, we report biochemical and structural analysis of the catalytic core of hpol η. Activity assays showed the truncated form displayed similar misincorporation properties as the full-length enzyme, incorporating C and T equally and extending from both. X-ray crystal structures of both dC and dT paired with O⁶-MeG were solved in both insertion and extension modes. The structures revealed a Watson-Crick-like pairing between O⁶-MeG and 2"-deoxythymidine-5"-[(α, β)-imido]triphosphate (approximating dT) at both the insertion and extension stages with formation of two H-bonds. Conversely, both the structures with O⁶- MeG opposite dCTP and dC display sheared configuration of base pairs but to different degrees, with formation of two bifurcated H-bonds and two single H-bonds in the structures trapped in the insertion and extension states, respectively. The structural data are consistent with the observed tendency of hpol η to insert both dC and dT opposite the O⁶-MeG lesion with similar efficiencies. Comparison of the hpol η active site configurations with either O⁶-MeG:dC or O⁶-MeG:dT bound compared with the corresponding situations in structures of complexes of Sulfolobus solfataricus Dpo4, a bypass pol that favors C relative to T by a factor of ∼4, helps rationalize the more error-prone synthesis opposite the lesion by hpol η.

Keywords: DNA damage; DNA polymerase; X-ray crystallography; enzyme kinetics; mass spectrometry (MS).

FIGURE 1.

LC-MS analysis of primer extension products. A–D, reconstructed chromatograms for the indicated ion. E–H, fragmentation patterns for each of the indicated primer extension products.

FIGURE 2.

Quality of the final models of ternary hpol η complexes with DNA template strands containing O⁶-MeG. Fourier (2F_o − F_c) sum electron density drawn at the 1σ threshold (blue meshwork; panels A, C, E, and G) and Fourier (F_o − F_c) omit electron density at the 3 σ level (magenta meshwork; panels B, D, F, and H) around the polymerase active site region. A and B, insertion stage with incoming dCTP opposite O⁶-MeG. C and D, insertion stage with incoming dTMP-NPP opposite O⁶-MeG. E and F, extension stage with dC opposite O⁶-MeG followed by dCMP-NPP opposite template dG. G and H, extension stage with dT opposite O⁶-MeG followed by dCMP-NPP opposite template dG. Selected active site residues are colored by atom with carbon atoms shown in purple (template O⁶-MeG and primer dC and dT in the extension complexes) or cyan (incoming nucleotide and template dG in the extension complexes). Base pairs at the −1 and −2 (panels A–D) or −2 (panels E–H) positions and template dT 5′-adjacent to the adducted residue are shown in yellow, with the DNA backbones highlighted by a ribbon, the protein is shown in light gray, and Mg²⁺ and Ca²⁺ ions are pink and brown spheres, respectively.

FIGURE 3.

Sheared configuration of incoming dCTP and O⁶-MeG in a hpol η insertion stage complex. A, the active site viewed into the DNA major groove. B, rotated by ∼180° around the vertical axis and viewed into the minor groove. Selected active site residues are colored by atom with carbon atoms shown in purple (O⁶-MeG), cyan (dCTP), or green (Arg-61 and Gln-38 from the finger domain as well as Asp/Glu coordinated to Ca²⁺ (brown spheres)). The remaining nucleotides are shown in yellow, except for dC9 (blue), which was added to the primer during crystallization. H-bonds involving the adducted residue are depicted as dashed lines.

FIGURE 4.

Watson-Crick-like configuration of incoming dTMP-NPP and O⁶-MeG in a hpol η insertion stage complex. A, the active site viewed into the DNA major groove. B, rotated by ∼90° around the horizontal axis and viewed perpendicular to the base planes of dTMP-NPP and O⁶-MeG. Selected active site residues are colored by atom with carbon atoms shown in purple (O⁶-MeG), cyan (dTMP-NPP), or green (Arg-61 and Gln-38 from the finger domain as well as Asp/Glu coordinated to Mg²⁺ (pink spheres)). The remaining nucleotides are shown in yellow, and H-bonds involving the adducted residue are depicted as dashed lines.

FIGURE 5.

Sheared configuration of primer dC and O⁶-MeG in a hpol η extension stage complex. A, the active site viewed into the DNA major groove. B, rotated by ∼90° around the horizontal axis and viewed perpendicular to the base planes of dCMP-NPP and dG. Selected active site residues are colored by atom with carbon atoms shown in purple (O⁶-MeG and dC at the −1 position), cyan (nascent dG:dCMP-NPP pair), or green (Arg-61 and Gln-38 from the finger domain as well as Asp/Glu coordinated to Mg²⁺ (pink spheres)). The remaining nucleotides are shown in yellow, and H-bonds involving the adducted residue are depicted as dashed lines.

FIGURE 6.

Sheared configuration of primer dT and O⁶-MeG in a hpol η extension stage complex. A, the active site viewed into the DNA major groove. B, rotated by ∼90° around the horizontal axis and viewed perpendicular to the base planes of dCMP-NPP and dG. Selected active site residues are colored by atom with carbon atoms shown in purple (O⁶-MeG and dT at the −1 position), cyan (nascent dG:dCMP-NPP pair), or green (Arg-61 and Gln-38 from the finger domain as well as Asp/Glu coordinated to Mg²⁺ (pink spheres)). The remaining nucleotides are shown in yellow, and H-bonds involving the adducted residue are depicted as dashed lines.

FIGURE 7.

Conformational flexibility of finger residue Arg-61 at the active site of ternary hpol η-DNA-dNTP complexes. View into the major groove of the DNA template (left):primer (right) duplex with dNTP visible to the right of Arg-61 side chains that are drawn in stick mode. Two divalent metal ions (Mg²⁺ or Ca²⁺) between the 3′-terminal nucleotide of the primer strand and the triphosphate moiety of the incoming nucleotide are located at the center of crossing dashed lines that mark their coordination geometry. A total of 23 crystal structures with the following PDB ID codes were included in the overlay: 5L1I, 5L1J, 5K1K, and 5L1L (O⁶-MeG, this work), 5DLF, 5DLG, 5DQG, 5DQH, and 5DQI (O⁴-methyl- and O⁴-ethyl-T) (22), 4RNM, 4RNN, and 4RNO (abasic site) (21), 4RU9 (MeFapy-dG) (23), 4O3N (native G:dCMP-NPP Mg²⁺ complex), 4O3O, 4O3P, 4O3Q, 4O3R, and 4O3S (8-oxoG) (17), 5DG7, 5DG8, and 5DG9 (1,N⁶-etheno-dA) (24), and 3MR3 (cys-syn-cyclobutane thymine dimer) (18). Arg-61 adopts various orientations that we have marked with numbers 1 (curled conformation and interacting with the α- and β-phosphate groups of the incoming 2′-deoxynucleotide triphosphate, the most common position), 2 (curled conformation and stacked in an offset manner on the nucleobase of the incoming dNTP), 3 (extended conformation and interacting with the major groove edge of the template base at the 0 position, e.g. 8-oxoG (magenta carbons)), 4 (curled conformation and interacting with the template base at the +1 position, e.g. the second T of cys-syn-cyclobutane thymine dimer (orange carbons)), and 5 (curled conformation and fully stacked on the nucleobase of the incoming dNTP, e.g. O⁶-MeG and dTMP-NPP, this work (lilac carbons)).