Structural and functional determinants of the archaeal 8-oxoguanine-DNA glycosylase AGOG for DNA damage recognition and processing

Abstract

8-Oxoguanine (GO) is a major purine oxidation product in DNA. Because of its highly mutagenic properties, GO absolutely must be eliminated from DNA. To do this, aerobic and anaerobic organisms from the three kingdoms of life have evolved repair mechanisms to prevent its deleterious effect on genetic integrity. The major way to remove GO is the base excision repair pathway, usually initiated by a GO-DNA glycosylase. First identified in bacteria (Fpg) and eukaryotes (OGG1), GO-DNA glycosylases were more recently identified in archaea (OGG2 and AGOG). AGOG is the less documented enzyme and its mode of damage recognition and removing remains to be clarified at the molecular and atomic levels. This study presents a complete structural characterisation of apo AGOGs from Pyrococcus abyssi (Pab) and Thermococcus gammatolerans (Tga) and the first structure of Pab-AGOG bound to lesion-containing single- or double-stranded DNA. By combining X-ray structure analysis, site directed mutagenesis and biochemistry experiments, we identified key amino acid residues of AGOGs responsible for the specific recognition of the lesion and the base opposite the lesion and for catalysis. Moreover, a unique binding mode of GO, involving double base flipping, never observed for any other DNA glycosylases, is revealed. In addition to unravelling the properties of AGOGs, our study, through comparative biochemical and structural analysis, offers new insights into the evolutionary plasticity of DNA glycosylases across all three kingdoms of life.

Figure 1.

Comparative enzyme activities of archaeal, bacterial and eukaryotic GO-DNA glycosylases. As indicated, 5’-[³²P]-labeled GO- (or AP site)-containing single (ss) or double-stranded (GO:C or AP:C) 24-mer DNA duplex was incubated for 15 min at 37°C alone (lanes 1, 6 and 11), with 2 nM (lanes 2, 4, 7, 9, 12 and 14) or with 200 nM (lanes 3, 5, 8, 10, 13 and 15) of indicated enzyme. Reaction mixtures were analyzed by urea–PAGE as described in Materials and Methods. Representative gel autoradiographs are shown. S and P are for DNA substrate and cleavage end-product, respectively. MTO, STO, Fpg, OGG1, Pab, Tga, Pae, h and Ll are defined in abbreviation list.

Figure 2.

Primary and secondary structures of AGOGs. (A) Sequence alignment of AGOG proteins with known 3D structures. Secondary structures are colored according to Lingaraju et al. (N/C domain in blue, HhH motif in yellow and HhH domain in red) (20). Red dots indicate the positions of the catalytic residues K142 and D174 and green triangles indicate the position of the 8-oxodG-interacting residues. Cysteine residues are highlighted in magenta. (B) Sequence identity between Pab-AGOG and Tga-AGOG, Pfu-AGOG or Pae-AGOG.

Figure 3.

apo AGOG 3D structures. (A) Ribbon overall 3D structure of Pab-AGOG. The N/C domain is shown in blue, the HhH domain is shown in red and the HhH motif is highlighted in yellow. (B) Superposition of Pab-AGOG with Tga-AGOG (pink), Pfu-AGOG (cyan) or Pae-AGOG (green). (C) Rmsd values between Cα atoms of Pab-AGOG and Tga-AGOG, Pfu-AGOG or Pae-AGOG calculated using Matchmaker of ChimeraX.

Figure 4.

Overview of the Pab-AGOG binding site. (A) The 8oxo-dG is shown in green. Pab-AGOG interacting residues and the catalytic lysine (K142) are represented as blue sticks (but Cα as balls). Electron density mFo-DFc composite omit map is contoured at 5σ. Hydrogen bonds are shown with dashed lines. (B) Superposition of apo (orange and yellow) and 8-oxodG bound (blue and green) Pab-AGOG.

Figure 5.

Trapping of the imino Pab-AGOG-DNA covalent intermediate. (A) Schematic representation of the GO-DNA glycosylase/AP lyase catalytic mechanism. K142 and D174 are the catalytic residues of Pab-AGOG. (B) Comparative trapping assays with Pab-AGOG, hOGG1 and LlFpg. As indicated, 20 nM of 5’[³²P]-labeled GO- or AP site-containing single (ss-GO or ss-AP) or double-stranded (GO:C or AP:C) 24-mer DNA fragment was incubated for 15 min at 37°C alone (lanes 1 and 6), with 50 nM of indicated enzyme only (lanes 2, 4, 7 and 9) and 0.1 M NaBH₄ (lanes 3, 5, 8, 10). Reactions were then analyzed by urea–PAGE as described in Materials and Methods. Representative autoradiographs are presented. The stable reduced Schiff base (rSB) is indicated by an orange arrow and, S and P are for DNA substrate and cleaved end-product, respectively.

Figure 6.

3D structure of Pab-AGOG trapped to single-stranded DNA. (A) Overwiew of the complex. mFo – DFc simulated annealing omit map contoured at 3σ (DNA and K142 residue omitted) is shown as black mesh. The phosphate group with two conformations is denoted by an arrow. (B) Details of the direct interactions between Pab-AGOG and single-stranded DNA. H-bonds are represented as black dash lines. (C) Superposition of the single-stranded DNA trapped on Pab-AGOG (violet) with apo Pab-AGOG (dark cyan) or Pab-AGOG + 8oxodG (yellow/green).

Figure 7.

Comparative analysis of the effect of the base opposite the damage on the activities of several GO-DNA glycosylases under MTO conditions. As indicated, 20 nM of radiolabeled 24-mer DNA duplex containing GO or AP site opposite C, T, A or G was incubated at 37°C alone (empty dashed circles, GO:C and AP:C, lanes 1 and 6, and 11, respectively) or with 2 nM enzyme (full grey circles; lanes 2–5, 7–10 and 12–15 for DNA glycosylase, DNA glycosylase/AP lyase and AP lyase activity, respectively). Incubation times were: 2 min for DNA glycosylase of LlFpg, 5 min for DNA glycosylase of Pab- and Tga-AGOG and for DNA glycosylase/AP lyase and AP lyase of LlFpg, 20 min for DNA glycosylase/AP lyase and AP lyase of Pab-AGOG, 60 min for all activities of hOGG1 and for DNA glycosylase/AP lyase and AP lyase of Tga-AGOG. Reaction mixtures were then analyzed by urea–PAGE as described inMaterials & Methods. Representative autoradiographs are shown.

Figure 8.

3D structure of covalently trapped Pab-AGOG-AP-dsDNA complexes. (A) Overview of the Pab-AGOG/AP dsDNA-C complex. (B) Details of the direct interactions between Pab-AGOG and AP dsDNA-C. (C) Opposite base recognition.

Figure 9.

3D structure of K142Q-Pab-AGOG complexed with GO-containing double-stranded DNA. (A) Overview of the Pab-AGOG/dsDNA-GO:C complex. (B) Zoom of the double base flipping (GO and T3) in the K142Q-Pab-AGOG complex. Around the DNA, the 2Fo – Fc electron density map contoured at 1σ is shown as black mesh. (C) Close-up view of the binding pocket of the Pab-AGOG/dsDNA-GO:C complex (protein in rosy and DNA in green) with the Pab-AGOG + 8-oxodG complex overlaid (in dark cyan). (D) Comparison of AP- (cyan) and GO-DNA (rosy) conformation at the lesion site. (E) Overview of Pab-AGOG, h-OGG1 and Mja-OGG2 complexed with GO-containing DNA in the same orientation. HhH motif in yellow, GO in green and estranged cytosine in red. The arrow indicates the conserved loop motif in the three OGG subgroup family.

Figure 10.

Substrate specificity of Pab-AGOG for several oxidized purines. 20 nM of radiolabeled 24-mer single (ss) and double (ds) stranded DNA containing GO, AO or F (opposite C and/or T) was incubated 15 min at 37°C with 200 nM of Pab-AGOG, hOGG1 and LlFpg as described in Materials and Methods for DNA glycosylase. Mean value of base excision were obtained from at least three independent experiments.