Unexpected behavior of DNA polymerase Mu opposite template 8-oxo-7,8-dihydro-2'-guanosine

Abstract

DNA double-strand breaks (DSBs) resulting from reactive oxygen species generated by exposure to UV and ionizing radiation are characterized by clusters of lesions near break sites. Such complex DSBs are repaired slowly, and their persistence can have severe consequences for human health. We have therefore probed DNA break repair containing a template 8-oxo-7,8-dihydro-2'-guanosine (8OG) by Family X Polymerase μ (Pol μ) in steady-state kinetics and cell-based assays. Pol μ tolerates 8OG-containing template DNA substrates, and the filled products can be subsequently ligated by DNA Ligase IV during Nonhomologous end-joining. Furthermore, Pol μ exhibits a strong preference for mutagenic bypass of 8OG by insertion of adenine. Crystal structures reveal that the template 8OG is accommodated in the Pol μ active site with none of the DNA substrate distortions observed for Family X siblings Pols β or λ. Kinetic characterization of template 8OG bypass indicates that Pol μ inserts adenosine nucleotides with weak sugar selectivity and, given the high cellular concentration of ATP, likely performs its role in repair of complex 8OG-containing DSBs using ribonucleotides.

Figure 1.

Comparison of catalytic efficiencies for nucleotide insertion by Pol μ opposite template dG versus 8OG. (A) Values were determined for catalytic efficiency (k_cat/K_m, s⁻¹ × μM⁻¹) of steady-state nucleotide (dCTP in blue; dATP in red; CTP in purple; ATP in green) incorporation by full-length wildtype Pol μ on a 1-nt gapped DNA substrate containing either dG or 8OG as the templating base. Reaction conditions are given in Supplementary Table S1, and full kinetic parameters are given in Supplementary Table S5. Values are calculated from multiple independent experimental replicates, as indicated in Supplementary Table S1. (B) Theoretical values for nucleotide (dCTP in blue; dATP in red; CTP in purple; ATP in green) incorporation efficiency on a 1-nt gapped DNA substrate containing either dG or 8OG as the templating base, normalized (calculated catalytic efficiency multiplied by cellular concentration) for previously determined cellular concentrations of nucleoside triphosphates (42).

Figure 2.

In vivo NHEJ repair of 8OG-containing DNA substrates by Pol μ. (A) Schematic representation of the input DNA substrates (left) containing a template 8OG (blue), an abasic site (tetrahydrofuran; THF) and repaired products (right) of NHEJ in MEF cells. The position of the nucleotide newly inserted by the DNA polymerase is indicated by a red question mark. (B) DNA products are recovered from MEFs after NHEJ and amplified by PCR. Gap-filling synthesis via incorporation of adenine nucleotides, followed by ligation, generates an NruI restriction site in amplified NHEJ products (Top panel, +A product), which is indicated by the appearance of a lower molecular weight band after cleavage. Gap-filling synthesis using cytidine, followed by ligation, creates a sequence context that is sensitive to BssHII (Bottom panel, +C product). (C) Quantitation of relative proportions of BssHII-sensitive (+C insertion opposite template 8OG, white bars) and NruI-sensitive (+A insertion opposite template 8OG, black bars) bands in NHEJ products in wildtype (WT), Polm^−/−, and Polm^−/− cells rescued by addition of Pol μ. Products resistant to both NruI and BssHII likely have heterogeneous deletions of overhang sequence.

Figure 3.

Pol μ is structurally predisposed for mutagenic adenine incorporation opposite 8OG. (A) Wildype hPol μΔ2 was crystallized in either binary (no incoming nucleotide) or pre-catalytic ternary (with incoming nonhydrolyzable nucleotide, as indicated) complex with a 1-nt gapped DNA substrate containing either a canonical or 8-oxo-7,8-dihydroguanine (red). Post-catalytic product complexes could be obtained by soaking binary or pre-catalytic ternary complex crystals with a hydrolyzable nucleotide, as indicated (Supplementary Table S3). (B) Global superposition of the hPol μΔ2 binary complex with undamaged (protein in dark gray, DNA in light gray) or 8OG-containing (protein in dark green, DNA in light green, 8OG in red) 1-nt gapped DNA substrates. The subdomains of the catalytic domain are labeled, and the location of the Loop2 deletion is indicated by a black asterisk. (C) The template 8OG nucleotide (light green) is observed in the syn conformation in the hPol μΔ2 binary complex, as indicated by the 2F_o-F_c electron density map (gray mesh, contoured at 1σ). The side chain of Gln441 (dark green) makes a putative hydrogen bond (dashed line) with the O8 atom, in an altered conformation from that usually found in previously published hPol μΔ2 binary or ternary complex crystal structures (gray, PDB ID code 4LZG (27)). Other stabilizing interactions include putative hydrogen bonds between the N2 atom of the 8OG base and the non-bridging OP1 atom of its 5′-phosphate, or with a neighboring water molecule (red sphere). (D) The template 8OG in binary complex with Pol β (PDB ID: code 3RJE (24)) is observed in a mixture of syn (yellow) and anti (white) conformations, with the position of the latter resulting in a rearrangement of the phosphate backbone (measured distance between phosphorus atoms, magenta). 2F_o-F_celectron density for the 8OG (residue T6) and preceding template base (residue T5) is shown as a gray mesh (contoured at 1σ).

Figure 4.

Analysis of deoxycytidine incorporation opposite template 8OG. (A) Superimposed ribbon diagrams of pre-catalytic ternary complexes of hPol μΔ2 with DNA substrate containing a template 8OG (protein in orange, DNA in light orange, dCMPNPP in magenta, Mg2+ ions in green) versus with a DNA substrate containing a canonical template guanine (protein in dark gray, DNA and dCMPNPP in light gray). The Gln441 side chain is drawn in stick from each complex (8OG in orange, guanine in gray), along with the conformation observed in the template 8OG binary complex (green). (B) Detailed comparison of the dCMPNPP:8OG base pairing in the template 8OG (orange, dCMPNPP in magenta, Mg²⁺ ions in green) and template guanine (gray) complexes. The location of the O8 atom is indicated by a green circle, and its putative interactions are shown as black dashed lines, along with their measured interatomic distances. 2F_o-F_c electron density for the 8OG base pair is shown as a gray mesh (contoured at 1σ). (C) Ribbon diagram of the post-catalytic product complex of hPol μΔ2 (brown) with a DNA substrate containing a template 8OG (khaki). Two magnesium ions (green) and the pyrophosphate leaving group remain in the active site after catalysis.

Figure 5.

Analysis of deoxyadenosine incorporation opposite template 8OG. (A) Superposition of the ribbon diagrams of the hPol μΔ2 dAMPNPP:8OG (protein in purple, DNA in lavender, dAMPNPP in cyan, Mg²⁺ ions in green) and dCMPNPP:8OG (protein in orange, DNA and dCMPNPP in khaki) pre-catalytic ternary complexes. The positions of the Gln441 side chain from each complex are compared (dAMNPNPP:8OG in purple, dCMPNPP:8OG in orange, and 8OG binary complex in green). The interatomic distance between the primer terminal 3′-OH and the α-phosphate of the incoming dAMPNPP is marked by a black dashed line. (B) Superposition of the hPol μΔ2 nascent base pair binding site in the dAMPNPP:8OG (protein in purple, DNA in lavender, dAMPNPP in cyan, Mg²⁺ ions in green) and dCMPNPP:8OG (protein in orange, DNA in khaki, and dCMPNPP in magenta) pre-catalytic ternary complexes. Hydrogen bonding interactions are marked by black dashed lines and labeled with their measured interatomic distances. (C) Stacking and hydrogen bonding interactions of the Arg442 side chain and the template 8OG nucleotide in the dAMPNPP:8OG (protein in purple, DNA in lavender, dAMPNPP in cyan, Mg²⁺ ions in green) and dCMPNPP:8OG (protein in orange, DNA in khaki and dCMPNPP in magenta) pre-catalytic ternary complexes. (D) The hPol μΔ2 post-catalytic dAMP:8OG product complex (protein in dark purple, DNA in lavender, pyrophosphate in orange, A site divalent metal in pink (contaminant metal based on anomalous scattering is modeled as Mn²⁺), and B site Mg²⁺ in green).

Figure 6.

Characterization of ribonucleotide binding opposite template 8OG. Ribbon diagrams of the hPol μΔ2 AMPNPP:8OG (A, protein in red, DNA in pink, AMPNPP in green, Mg²⁺ ions as green spheres) and CMPCPP:8OG (B, protein in blue, DNA in light blue, CMPCPP in yellow, Mg²⁺ ions as green spheres—superimposed with a correctly paired reference CMPCPP:dG structure in transparent gray) pre-catalytic ternary complexes. Magenta inset: comparison of interactions in the nascent base pair binding site with an incoming ribonucleotide (AMPNPP in green or CMPCPP in yellow opposite 8OG in pink or light blue, respectively) versus a deoxyribonucleotide (dAMPNPP in cyan, dCMPNPP in magenta opposite 8OG in either purple or light orange, respectively). 2Fo-Fc electron density maps for the ribonucleotide-containing ternary complexes are shown as a gray mesh (contoured at 1σ). Cyan inset: comparison of interactions between the 2′-OH and the Gly433 backbone carbonyl in the ribonucleotide-containing complexes (AMPNPP in green or CMPCPP in yellow opposite 8OG, with protein in red or blue, respectively; and CMPCPP opposite template dG in light gray) versus a deoxyribonucleotide (dAMPNPP in cyan opposite 8OG, with protein in purple; and dCMPNPP opposite template G in dark gray). Putative hydrogen bonds and their interatomic distances are shown as dashed lines. Yellow inset: Primer terminal heterogeneity in the AMPNPP:8OG ternary complex, with the predominant catalytically relevant conformation shown in pink (occupancy = 0.6), and the noncatalytic flipped conformation shown in white (occupancy = 0.4).

Figure 7.

In crystallo incorporation of ATP opposite template 8OG is more efficient than that of CTP. Ribbon diagrams of the hPol μΔ2 post-catalytic product complexes for incorporation hydrolyzable ATP (A, protein in dark red, DNA in pink) or CTP (B, protein in dark blue, DNA in light blue, unreacted CMPCPP in yellow, pyrophosphate in orange) opposite template 8OG. Na⁺ ions in the metal B sites are shown as purple spheres. The hydrolyzable ribonucleotide stocks used for these soaks contain an unknown metal contaminant with anomalous scattering, which now occupies the metal A site in both structures. Given the observed coordination geometry and ionic distances, this metal has been modeled as Mn²⁺ (pink spheres).

Figure 8.

Gradient of DNA backbone distortions in Family X polymerases as a consequence of guanosine oxidation. Stick representation of distortions (or lack thereof) in the template strand in pre-catalytic ternary complex structures of Pol β (top row), Pol λ (middle row), and Pol μ (bottom row), with either incoming nonhydrolyzable cytidine analogs (A–C, magenta, PDB ID codes 3RJI (24), 5IIJ (25), and 6P1P) or nonhydrolyzable deoxyadenosine (D–F, cyan, PDB ID codes 3RJF (24), 5III (25), and 6P1N; inset: Pol β ternary complex in green with dAMP:8OG in open conformation PDB ID code 1MQ2 (26)) opposite template 8OG in comparison to a correctly paired reference structure (gray, PDB ID codes 1BPY (51), 2PFO (52), and 6P1V). (H–I) Superpositions of the same template DNA region in pre-catalytic ternary complex structures of incoming nonhydrolyzable dC (magenta) versus dA (cyan) opposite a template 8OG. The naming convention for base pairing is that of incoming nucleotide:template nucleotide, as is used elsewhere in this manuscript.