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. 2003 Jul 1;22(13):3461-71.
doi: 10.1093/emboj/cdg311.

Structure of a trapped endonuclease III-DNA covalent intermediate

J Christopher Fromme  1 , Gregory L Verdine
Affiliations

Structure of a trapped endonuclease III-DNA covalent intermediate

J Christopher Fromme et al. EMBO J. .

Abstract

Nearly all cells express proteins that confer resistance to the mutagenic effects of oxidative DNA damage. The primary defense against the toxicity of oxidative nucleobase lesions in DNA is the base-excision repair (BER) pathway. Endonuclease III (EndoIII) is a [4Fe-4S] cluster-containing DNA glycosylase with repair activity specific for oxidized pyrimidine lesions in duplex DNA. We have determined the crystal structure of a trapped intermediate that represents EndoIII frozen in the act of repairing DNA. The structure of the protein-DNA complex provides insight into the ability of EndoIII to recognize and repair a diverse array of oxidatively damaged bases. This structure also suggests a rationale for the frequent occurrence in certain human cancers of a specific mutation in the related DNA repair protein MYH.

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Figures

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Fig. 1. Reaction pathway catalysed by EndoIII. The Schiff base intermediate 2 can be intercepted in situ by borohydride to furnish the trapped complex 4, which is the subject of this study.
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Fig. 2. Overall structure of the trapped complex. (A) Ribbon diagram, with the two domains of EndoIII in blue and green; the [4Fe–4S] cluster is shown in CPK format (Fe, rust; sulfur, yellow). The DNA duplex is shown in gold, with the lesion nucleoside modelled in dark grey (see text) and the estranged nucleoside in red. (B) Same as in (A), but from a different perspective. (C) Ribbon diagram with distinct colours for α-helices, from the same perspective as in (A). (D) Electrostatic surface representation (GRASP) (Nicholls et al., 1991) of the protein–DNA interface, from the same perspective as in (B). Red represents negatively charged surface, blue represents positive charge; only formal charges are represented. (E) DNA duplex used for crystallization, with phosphate groups in purple, the estranged base in magenta and the lesion in grey. Enzyme interactions are denoted by arrows. The numbering system corresponds to that used in the text; phosphates are numbered according to the strand on which they reside (L, lesion strand; C, complementary strand). A dihydrouridine deoxynucleoside was present at the location of the lesion during enzymatic processing, and this nucleoside sugar has become covalently and irreversibly linked to the protein via borohydride reduction.
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Fig. 3. (A) Global stereoview of the protein–DNA interface modelled as a ball-and-stick representation. Colouring of DNA as in Figure 2A, and side chains of the protein are coloured teal, except for Lys121 (purple) and Asp139 (green). (B) Interactions of the complementary strand of DNA with the protein. Hydrogen bonds are shown as dashed lines, and distances are as indicated. Note that phosphate group pC0 caps the N-terminus of helix α-E. (C) Role of the HhH-GPD hairpin element in DNA binding. The central metal ion is modelled as Na+, but is probably a magnesium ion under physiological conditions (both are known to exhibit octahedral geometry). Hydrogen bonds are shown as dashed lines with distances, and metal coordination is shown as dashed lines without distances (measured metal–ligand distances range from 2.3 to 2.6 Å).
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Fig. 4. Estranged base recognition. (A) Recognition of an estranged guanine base. (B) Recognition of an estranged adenine base. Dashed lines denote hydrogen bonds, with the indicated heavy atom distances; blue dots denote a van der Waals interaction. The hydrogen bond between the carbonyl of Ile80 and the amide of Tyr83 can be seen in this panel (and forms part of a type II turn), but is absent in (A).
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Fig. 5. (A) Stereoview of the final 1.7 Å, σA-weighted (Read, 1986) 2Fo – Fc electron density map contoured at 1.25 σ, superimposed on the active site region of the final estranged-G model. (B) Active site region of the enzyme. The C-N bond of the reduced Schiff base is shown in pink. Contacts between the 3′-phosphate and the amide protons of Gln42 and Thr140, and between the 5′-phosphate and the amide proton of Asp45 are not shown explicitly, for reasons of illustrative clarity. The positions of the C2′ protons, though not visible in experimental electron density maps, are inferred from the geometry of the heavy atom backbone. (C) Structural superimposition of the non-covalent complex (THF-Iodine1) onto the covalent estranged-G complex. The non-covalent complex is coloured similarly to (B) and the covalent complex (estranged-G) is dark grey.
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Fig. 6. (A) Structure of the active site, with a thymine base modelled into the base-recognition pocket. (B) Same as in (A), from a different perspective.
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