PDBsum entry 2fbx

Transferase

PDB id

2fbx

Contents

			Protein chain

					195 a.a.

			Metals

					_MG ×2

			Waters ×203

References listed in PDB file

Key reference

Title

Wrn exonuclease structure and molecular mechanism imply an editing role in DNA end processing.

Authors

J.J.Perry, S.M.Yannone, L.G.Holden, C.Hitomi, A.Asaithamby, S.Han, P.K.Cooper, D.J.Chen, J.A.Tainer.

Ref.

Nat Struct Mol Biol, 2006, 13, 414-422. [DOI no: 10.1038/nsmb1088]

PubMed id

16622405

Abstract

WRN is unique among the five human RecQ DNA helicases in having a functional exonuclease domain (WRN-exo) and being defective in the premature aging and cancer-related disorder Werner syndrome. Here, we characterize WRN-exo crystal structures, biochemical activity and participation in DNA end joining. Metal-ion complex structures, active site mutations and activity assays reveal a nuclease mechanism mediated by two metal ions. The DNA end-binding Ku70/80 complex specifically stimulates WRN-exo activity, and structure-based mutational inactivation of WRN-exo alters DNA end joining in human cells. We furthermore establish structural and biochemical similarities of WRN-exo to DnaQ-family replicative proofreading exonucleases, describing WRN-specific adaptations consistent with double-stranded DNA specificity and functionally important conformational changes. These results indicate WRN-exo is a human DnaQ family member and support DnaQ-like proofreading activities stimulated by Ku70/80, with implications for WRN functions in age-related pathologies and maintenance of genomic integrity.



	Figure 2. Figure 2. WRN-exo metal-ion dependence and structural analyses. (a) Nuclease activity assays containing WRN-exo (50 pmol) and radiolabeled DNA substrate were incubated for 30 min with either no metal (control; lane 1) or the noted divalent cation(s). WRN-exo 3' arrow 5' dsDNA nuclease activity is supported by Mg^2+ or Mn^2+ ions, but not by Eu^2+ or in the absence of divalent cations. Addition of Eu^2+ inhibits nuclease activity in the presence of equimolar Mg^2+ or Mn^2+ ions. The DNA digestion pattern with equimolar Mg^2+ and Mn^2+ is indistinguishable from that of Mn^2+ alone. (b) Two Mn^2+ ions (purple) are chelated in the WRN active site in the absence of DNA; dashed magenta lines denote metal-ion bonds, with distances labeled. The inner metal ion, M[A], is directly coordinated by Asp82, Glu84 and Asp216, and the outer metal ion, M[B], directly ligates one side chain, Asp82, that bridges the two metal ions. Asp143 has an indirect interaction with the M[B] metal ion via two water molecules. (c) The WRN active site also accommodates two of the larger lanthanide Eu^3+ ions (blue) in the absence of substrate. Dashed blue lines denote metal-ion bonds. (d) Overlay of WRN Mn^2+ and Eu^3+ metal-ion complex structures, colored as in b and c. Incorporation of Eu^3+ metal ions at sites M[A] and M[B] does not cause appreciable changes in the WRN active site.		Figure 6. Figure 6. WRN-exo hexameric ring model and dGMP-binding site, and altered processing by the W145A mutant. (a) The WRN ring homology model, with differently colored WRN-exo subunits, was built by structural superimposition with the A. thaliana homolog (PDB entry 1VK0). The active site of the exonuclease (with gray spheres denoting metal ions) faces the center of the ring. The central cavity of the WRN ring is large enough (about 30 Å in diameter by 35 Å deep) to accommodate dsDNA and is similar to that observed in Ku70/80 (ref. 49). (b) DNA processing is altered in a WRN-exo W145A mutant. Control reactions with DNA alone or with 10 pmol of Ku70/80 are indicated. WRN-exo and W145A reactions contained 20 fmol of radiolabeled dsDNA substrate, approximately 200 pmol of each WRN nuclease variant and increasing amounts of Ku70/80 (0.06, 0.6 and 6 pmol), denoted by triangles. (c) F[o] - F[c] electron density map of WRN-exo dGMP soak (blue, 3 ; red, 5 ). dGMP stacks against Trp145, consistent with this region interacting with DNA substrate at the center of the ring. (d) Similar internal and external dimensions of the WRN-exo hexamer model (right) and Ku70/80 bound to DNA (left) suggest a possible interaction mode, which would place the protruding 2- 3 loop (left face) adjacent to the Ku dimer and/or allow Ku to provide a suitable DNA orientation.

PROCHECK

	Headers