Conserved structural chemistry for incision activity in structurally non-homologous apurinic/apyrimidinic endonuclease APE1 and endonuclease IV DNA repair enzymes - PubMed (original) (raw)

Comparative Study

. 2013 Mar 22;288(12):8445-8455.

doi: 10.1074/jbc.M112.422774. Epub 2013 Jan 25.

David S Shin 2, Clifford D Mol 2, Tadahide Izumi 3, Andrew S Arvai 2, Anil K Mantha 4, Bartosz Szczesny 4, Ivaylo N Ivanov 5, David J Hosfield 2, Buddhadev Maiti 5, Mike E Pique 2, Kenneth A Frankel 1, Kenichi Hitomi 6, Richard P Cunningham 7, Sankar Mitra 4, John A Tainer 8

Affiliations

Comparative Study

Conserved structural chemistry for incision activity in structurally non-homologous apurinic/apyrimidinic endonuclease APE1 and endonuclease IV DNA repair enzymes

Susan E Tsutakawa et al. J Biol Chem. 2013.

Abstract

Non-coding apurinic/apyrimidinic (AP) sites in DNA form spontaneously and as DNA base excision repair intermediates are the most common toxic and mutagenic in vivo DNA lesion. For repair, AP sites must be processed by 5' AP endonucleases in initial stages of base repair. Human APE1 and bacterial Nfo represent the two conserved 5' AP endonuclease families in the biosphere; they both recognize AP sites and incise the phosphodiester backbone 5' to the lesion, yet they lack similar structures and metal ion requirements. Here, we determined and analyzed crystal structures of a 2.4 Å resolution APE1-DNA product complex with Mg(2+) and a 0.92 Å Nfo with three metal ions. Structural and biochemical comparisons of these two evolutionarily distinct enzymes characterize key APE1 catalytic residues that are potentially functionally similar to Nfo active site components, as further tested and supported by computational analyses. We observe a magnesium-water cluster in the APE1 active site, with only Glu-96 forming the direct protein coordination to the Mg(2+). Despite differences in structure and metal requirements of APE1 and Nfo, comparison of their active site structures surprisingly reveals strong geometric conservation of the catalytic reaction, with APE1 catalytic side chains positioned analogously to Nfo metal positions, suggesting surprising functional equivalence between Nfo metal ions and APE1 residues. The finding that APE1 residues are positioned to substitute for Nfo metal ions is supported by the impact of mutations on activity. Collectively, the results illuminate the activities of residues, metal ions, and active site features for abasic site endonucleases.

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Figures

FIGURE 1.

FIGURE 1.

Crystallographic structures of human APE1 and T. maritima Nfo showing active site details. A, the 2.4 Å structure of an APE1-product complex reveals a Mg2+-water cluster in the active site. Waters (red spheres) are shown with electron density from a PHENIX kicked map (1 σ, blue). B, simplified view of APE1 magnesium-water cluster shows tetrahedral geometry. C, the T. maritima Nfo active site structure, determined at 0.92 Å resolution. Electron density from a PHENIX kicked map (2.3 σ) is shown in blue. Mg2+ and Mn2+/Zn2+ atoms are shown as purple and dark blue spheres. A coordinated water is shown as a red sphere. D, the superimposition of the active sites from T. maritima Nfo and E. coli Nfo reveals a single difference in the active site. Ala-30 and two waters in E. coli Nfo are replaced with Gln and may account for greater thermostability. These residues are next to Glu-161 (E. coli), which is postulated to activate the attacking water.

FIGURE 2.

FIGURE 2.

Alignment of Nfo sequences shows that thermophilic Nfo has substituted Gln for Ala in the active site. The region around Ala-30 in E. coli (Eco) is shown, aligned with Bacteroides thetaiotaomicron (Bth), T. maritima (Tma), Aquifex aeolicus (Aae), mesophilic Bacillus subtilis (Bsu), and Thermus thermophilus (Tth), respectively.

FIGURE 3.

FIGURE 3.

Comparison of the fractal dimension in the DNA binding grooves of Nfo-product (PDB entry 1QUM), APE1-product (PDB entry 4IEM), and UDG (PDB entry 1EMH) highlights the shallow pockets of the AP endonucleases. Each protein's molecular surface (calculated using MS/MS with a 1.4-Å probe sphere) is colored by its local atomic fractal density (70). The density is calculated using Surfractal with a 1.0–10.0 Å radius range (71). This Hausdorff-Besicovitch dimension measures the change in packing density; 2.0 indicates a flat surface, and 3.0 indicates a fully packed volume. Intermediate values identify concave grooves and pockets. This figure was created in AVS (AudioVisualSystemsInc).

FIGURE 4.

FIGURE 4.

Structural superimposition of APE1-product DNA (PDB entry 4IEM) and Nfo-product DNA (PDB entry 1QUM) based on the tetrahydrofuran moiety reveals geometric similarity. A, superimposition based on the tetrahydrofuran moiety shows how similarly the tetrahydrofuran is deformed in the APE1 and Nfo structures and the close placement of the 3′-ribose oxygens. B, superimposition based on the scissile phosphate and the 3′-ribose oxygen shows how structurally similar the DNA products are, in contrast to the lack of conservation in tertiary structure of the proteins. C, close-up views of the active site showing the relative positioning of the scissile phosphate of the tetrahydrofuran and the 3′-ribose oxygen to the Mg in APE1 and the three zinc atoms in Nfo. The active site residues confirm of APE1 are shown relative to the Zn2+ atoms in Nfo.

FIGURE 5.

FIGURE 5.

Single turnover kinetics of WT and APE1 mutants shows the relative importance of Asp-210 and Asn-212. A, kinetics of product formation of three independent experiments for each enzyme at 10 °C. B, rate constants of AP site cleavage (nmol of product formation/min) at 10 °C. 52-nt THF-containing oligonucleotide duplex (10 n

m

) substrate and APE1 (100 n

m

) were used.

FIGURE 6.

FIGURE 6.

Catalytic mechanism for APE1 and Nfo. A, MD simulation of WT APE1-substrate DNA identified a persistent hydrogen bonding network, involving a water; residues His-309, Tyr-171, Asp-210, and Asn-212; and the abasic site phosphate. A snapshot from the MD trajectory shows the positions of the active site residues; hydrogen bonds (black dotted lines) and the nucleophilic attack direction (gray arrow) are shown; all distances are in Å. Tyr-171 is below but not labeled. B, model of Nfo-substrate, built from the substrate complex with E261Q (PDB entry 2NQJ) and with Glu-261 overlaid from the WT model (PDB entry 1qum).

References

    1. Lindahl T. (1993) Instability and decay of the primary structure of DNA. Nature 362, 709–715 - PubMed
    1. Lindahl T., Barnes D. E. (2000) Repair of endogenous DNA damage. Cold Spring Harb. Symp. Quant. Biol. 65, 127–133 - PubMed
    1. Ciccia A., Elledge S. J. (2010) The DNA damage response. Making it safe to play with knives. Mol. Cell 40, 179–204 - PMC - PubMed
    1. Huffman J. L., Sundheim O., Tainer J. A. (2005) DNA base damage recognition and removal. New twists and grooves. Mutat. Res. 577, 55–76 - PubMed
    1. Loeb L. A. (1985) Apurinic sites as mutagenic intermediates. Cell 40, 483–484 - PubMed

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