Structures of endonuclease V with DNA reveal initiation of deaminated adenine repair

Abstract

Endonuclease V (EndoV) initiates a major base-repair pathway for nitrosative deamination resulting from endogenous processes and increased by oxidative stress from mitochondrial dysfunction or inflammatory responses. We solved the crystal structures of Thermotoga maritima EndoV in complex with a hypoxanthine lesion substrate and with product DNA. The PYIP wedge motif acts as a minor groove-damage sensor for helical distortions and base mismatches and separates DNA strands at the lesion. EndoV incises DNA with an unusual offset nick 1 nucleotide 3' of the lesion, as the deaminated adenine is rotated approximately 90 degrees into a recognition pocket approximately 8 A from the catalytic site. Tight binding by the lesion-recognition pocket in addition to Mg(2+) and hydrogen-bonding interactions to the DNA ends stabilize the product complex, suggesting an orderly recruitment of downstream proteins in this base-repair pathway.

Figure 1

EndoV 3′ incision initiating deaminated adenine repair. (a) Deamination of the exocyclic amino group in adenine yields hypoxanthine. (b) EndoV dependent repair is initiated by cleavage at the second phosphodiester bond 3′ to the lesion (Hx, hypoxanthine) resulting from deamination of adenine.

Figure 2

EndoV overall fold, surface characteristics and protein-DNA complex structure. (a) Stereo pair showing protein fold and ternary structure of T. maritima EndoV. (b) Electrostatic potential of wild-type EndoV mapped onto the solvent accessible protein surface (blue positive regions, red negative regions). Electrostatic potential calculated using APBS. (c) Molecular surface showing conserved residues in the EndoV family [colored from dark burgundy (highly conserved) through neutral grey into dark cyan (highly variable)]. Degree of conservation calculated using ConSeq (http://conseq.tau.ac.il/). (d) Molecular surface with bound DNA (orange and yellow tubes and spheres) showing spatial relationships among key structural elements: the strand-separating PYIP wedge (cyan, left) protrudes out adjacent to residues Asp43, Glu89, Asp110, His214 involved in Mg²⁺ ion binding and phosphodiester incision (yellow, center), as well as hypoxanthine lesion and surrounding residues (Leu85, Gly111, Gln112, Gly113, Gly136, Leu142) forming the nucleobase pocket (red, center). (e) Molecular surface of wild-type Tma EndoV showing substantial bending of the bound duplex DNA (orange and yellow ball-and-stick representation). PYIP-wedge shown in cyan. Experimental electron density is shown for one of the DNA strands in the duplex (σ_A-weighted 2F_o-F_c map contoured at 1σ).

Figure 3

Protein-DNA contacts. (a) Protein-DNA contacts in the wild-type product complex. Hydrogen bond/ionic interactions (dashed lines, 3.75Å cut-off), main chain amide nitrogen atoms (N) and steric interactions (orange arcs, 4.25Å cut-off) involving side chains (yellow circles) are shown for one of the two EndoV molecules binding symmetrically to the DNA. (b) Close-up of the coordination around the phosphodiester insicion. (c) Close-up of the strand separating PYIP-wedge with selected distances to DNA bases stacking with residues defining the protein surface. The hypoxanthine (dark red) is partially buried behind the wedge.

Figure 4

Protein-DNA contacts in the base lesion pocket. (a) Diagram of interactions involved in hypoxanthine recognition, shown in both side-view and front-view. Hydrogen bonds and steric interactions shown with dashed lines; the van der Waals volumes of selected residues involved in hypoxanthine contacts represented by dotted surfaces. (b) Tautomeric forms of hypoxanthine and detailed hydrogen bonding network. (c) Tautomeric forms of xanthine and detailed hydrogen bonding network. (d) Model of uracil binding to EndoV.

Figure 5

Active site architecture of EndoV-DNA complex. (a) Stereo pair showing active site with the nicked product DNA free 3′ hydroxyl- and 5′ phosphate groups. A thin protein surface slab shows the steric separation of the recognition pocket and the catalytic centre (yellow Mg²⁺). (b) Stereo pair of the active site region with experimental electron density showing the DNA incision (density gap) and Mg²⁺ ion coordination (2Fo-Fc map contoured at 1.0σ).