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

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.

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Figure 1: WRN-exo fold, structure and active site structural chemistry.
Figure 2: WRN-exo metal-ion dependence and structural analyses.
Figure 3: WRN-exo DNA-binding cleft.
Figure 4: Nuclease activities of WRN, WRN-exo and WRN-exo point mutants, and nuclease modulation by Ku70/80.
Figure 5: Nuclease and helicase activity act together, according to an in vivo plasmid end–joining assay.
Figure 6: WRN-exo hexameric ring model and dGMP-binding site, and altered processing by the W145A mutant.

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Acknowledgements

We thank J. Campisi and S. Huang (Berkeley Lab) for the full-length WRN clone and WRN cell lines used in the microhomology repair assay, D. King for MS/MS analysis of proteolytic digests and D. McRee and Syrrx Inc. for use of the Syrrx Inc. robotic crystallization screens to discover initial WRN-exo crystallization conditions. We thank S. Williams and J. Tubbs for critical reading of the manuscript. This work was supported by US National Institutes of Health grants CA104660 (J.A.T., S.M.Y.), CA63503 (P.K.C.) and CA92584 (J.A.T., P.K.C., D.J.C.).

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Correspondence to Steven M Yannone or John A Tainer.

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Supplementary Fig. 1

WRN-exo active site structure and experimental electron density (PDF 332 kb)

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Perry, J., Yannone, S., Holden, L. et al. WRN exonuclease structure and molecular mechanism imply an editing role in DNA end processing. Nat Struct Mol Biol 13, 414–422 (2006). https://doi.org/10.1038/nsmb1088

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