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

Nature Structural & Molecular Biology volume 13, pages 414–422 (2006)Cite this article

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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References

  1. Goto, M. Hierarchical deterioration of body systems in Werner's syndrome: implications for normal ageing. Mech. Ageing Dev. 98, 239–254 (1997).

    Article  CAS  Google Scholar 

  2. Opresko, P.L., Cheng, W.H., von Kobbe, C., Harrigan, J.A. & Bohr, V.A. Werner syndrome and the function of the Werner protein; what they can teach us about the molecular aging process. Carcinogenesis 24, 791–802 (2003).

    Article  CAS  Google Scholar 

  3. Yu, C.E. et al. Positional cloning of the Werner's syndrome gene. Science 272, 258–262 (1996).

    Article  CAS  Google Scholar 

  4. von Kobbe, C. & Bohr, V.A. A nucleolar targeting sequence in the Werner syndrome protein resides within residues 949–1092. J. Cell Sci. 115, 3901–3907 (2002).

    Article  CAS  Google Scholar 

  5. Matsumoto, T., Shimamoto, A., Goto, M. & Furuichi, Y. Impaired nuclear localization of defective DNA helicases in Werner's syndrome. Nat. Genet. 16, 335–336 (1997).

    Article  CAS  Google Scholar 

  6. Moser, M.J. et al. WRN helicase expression in Werner syndrome cell lines. Nucleic Acids Res. 28, 648–654 (2000).

    Article  CAS  Google Scholar 

  7. Goto, M. et al. Immunological diagnosis of Werner syndrome by down-regulated and truncated gene products. Hum. Genet. 105, 301–307 (1999).

    Article  CAS  Google Scholar 

  8. Hickson, I.D. RecQ helicases: caretakers of the genome. Nat. Rev. Cancer 3, 169–178 (2003).

    Article  CAS  Google Scholar 

  9. Moser, M.J., Holley, W.R., Chatterjee, A. & Mian, I.S. The proofreading domain of Escherichia coli DNA polymerase I and other DNA and/or RNA exonuclease domains. Nucleic Acids Res. 25, 5110–5118 (1997).

    Article  CAS  Google Scholar 

  10. Mushegian, A.R., Bassett, D.E., Jr., Boguski, M.S., Bork, P. & Koonin, E.V. Positionally cloned human disease genes: patterns of evolutionary conservation and functional motifs. Proc. Natl. Acad. Sci. USA 94, 5831–5836 (1997).

    Article  CAS  Google Scholar 

  11. Huang, S. et al. The premature ageing syndrome protein, WRN, is a 3′ → 5′ exonuclease. Nat. Genet. 20, 114–116 (1998).

    Article  CAS  Google Scholar 

  12. von Kobbe, C., Thoma, N.H., Czyzewski, B.K., Pavletich, N.P. & Bohr, V.A. Werner syndrome protein contains three structure-specific DNA binding domains. J. Biol. Chem. 278, 52997–53006 (2003).

    Article  CAS  Google Scholar 

  13. Opresko, P.L., Laine, J.P., Brosh, R.M., Jr., Seidman, M.M. & Bohr, V.A. Coordinate action of the helicase and 3′ to 5′ exonuclease of Werner syndrome protein. J. Biol. Chem. 276, 44677–44687 (2001).

    Article  CAS  Google Scholar 

  14. Comai, L. & Li, B. The Werner syndrome protein at the crossroads of DNA repair and apoptosis. Mech. Ageing Dev. 125, 521–528 (2004).

    Article  CAS  Google Scholar 

  15. Cheng, W.H. et al. Linkage between Werner syndrome protein and the Mre11 complex via Nbs1. J. Biol. Chem. 279, 21169–21176 (2004).

    Article  CAS  Google Scholar 

  16. Yannone, S.M. et al. Werner syndrome protein is regulated and phosphorylated by DNA-dependent protein kinase. J. Biol. Chem. 276, 38242–38248 (2001).

    CAS  PubMed  Google Scholar 

  17. Baynton, K. et al. WRN interacts physically and functionally with the recombination mediator protein RAD52. J. Biol. Chem. 278, 36476–36486 (2003).

    Article  CAS  Google Scholar 

  18. Sakamoto, S. et al. Werner helicase relocates into nuclear foci in response to DNA damaging agents and co-localizes with RPA and Rad51. Genes Cells 6, 421–430 (2001).

    Article  CAS  Google Scholar 

  19. Cooper, M.P. et al. Ku complex interacts with and stimulates the Werner protein. Genes Dev. 14, 907–912 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Li, B. & Comai, L. Functional interaction between Ku and the werner syndrome protein in DNA end processing. J. Biol. Chem. 275, 39800 (2000).

    CAS  Google Scholar 

  21. Orren, D.K. et al. A functional interaction of Ku with Werner exonuclease facilitates digestion of damaged DNA. Nucleic Acids Res. 29, 1926–1934 (2001).

    Article  CAS  Google Scholar 

  22. Li, B. & Comai, L. Requirements for the nucleolytic processing of DNA ends by the Werner syndrome protein-Ku70/80 complex. J. Biol. Chem. 276, 9896–9902 (2001).

    Article  CAS  Google Scholar 

  23. Li, B. & Comai, L. Displacement of DNA-PKcs from DNA ends by the Werner syndrome protein. Nucleic Acids Res. 30, 3653–3661 (2002).

    Article  CAS  Google Scholar 

  24. Karmakar, P. et al. Werner protein is a target of DNA-dependent protein kinase in vivo and in vitro, and its catalytic activities are regulated by phosphorylation. J. Biol. Chem. 277, 18291–18302 (2002).

    Article  CAS  Google Scholar 

  25. Li, B., Navarro, S., Kasahara, N. & Comai, L. Identification and biochemical characterization of a Werner's syndrome protein complex with Ku70/80 and poly(ADP-ribose) polymerase-1. J. Biol. Chem. 279, 13659–13667 (2004).

    Article  CAS  Google Scholar 

  26. Beese, L.S. & Steitz, T.A. Structural basis for the 3′-5′ exonuclease activity of Escherichia coli DNA polymerase I: a two metal ion mechanism. EMBO J. 10, 25–33 (1991).

    Article  CAS  Google Scholar 

  27. Bruns, C.M., Hubatsch, I., Ridderstrom, M., Mannervik, B. & Tainer, J.A. Human glutathione transferase A4–4 crystal structures and mutagenesis reveal the basis of high catalytic efficiency with toxic lipid peroxidation products. J. Mol. Biol. 288, 427–439 (1999).

    Article  CAS  Google Scholar 

  28. Brautigam, C.A., Aschheim, K. & Steitz, T.A. Structural elucidation of the binding and inhibitory properties of lanthanide (III) ions at the 3′-5′ exonucleolytic active site of the Klenow fragment. Chem. Biol. 6, 901–908 (1999).

    Article  CAS  Google Scholar 

  29. Brautigam, C.A. & Steitz, T.A. Structural principles for the inhibition of the 3′-5′ exonuclease activity of Escherichia coli DNA polymerase I by phosphorothioates. J. Mol. Biol. 277, 363–377 (1998).

    Article  CAS  Google Scholar 

  30. Wang, J., Yu, P., Lin, T.C., Konigsberg, W.H. & Steitz, T.A. Crystal structures of an NH2-terminal fragment of T4 DNA polymerase and its complexes with single-stranded DNA and with divalent metal ions. Biochemistry 35, 8110–8119 (1996).

    Article  CAS  Google Scholar 

  31. Doublie, S., Tabor, S., Long, A.M., Richardson, C.C. & Ellenberger, T. Crystal structure of a bacteriophage T7 DNA replication complex at 2.2 A resolution. Nature 391, 251–258 (1998).

    Article  CAS  Google Scholar 

  32. Hopfner, K.P. et al. Crystal structure of a thermostable type B DNA polymerase from Thermococcus gorgonarius. Proc. Natl. Acad. Sci. USA 96, 3600–3605 (1999).

    Article  CAS  Google Scholar 

  33. Kamath-Loeb, A.S., Welcsh, P., Waite, M., Adman, E.T. & Loeb, L.A. The enzymatic activities of the Werner syndrome protein are disabled by the amino acid polymorphism R834C. J. Biol. Chem. 279, 55499–55505 (2004).

    Article  CAS  Google Scholar 

  34. Bernstein, D.A., Zittel, M.C. & Keck, J.L. High-resolution structure of the E. coli RecQ helicase catalytic core. EMBO J. 22, 4910–4921 (2003).

    Article  CAS  Google Scholar 

  35. Oshima, J., Huang, S., Pae, C., Campisi, J. & Schiestl, R.H. Lack of WRN results in extensive deletion at nonhomologous joining ends. Cancer Res. 62, 547–551 (2002).

    CAS  PubMed  Google Scholar 

  36. Crabbe, L., Verdun, R.E., Haggblom, C.I. & Karlseder, J. Defective telomere lagging strand synthesis in cells lacking WRN helicase activity. Science 306, 1951–1953 (2004).

    Article  CAS  Google Scholar 

  37. Laud, P.R. et al. Elevated telomere-telomere recombination in WRN-deficient, telomere dysfunctional cells promotes escape from senescence and engagement of the ALT pathway. Genes Dev. 19, 2560–2570 (2005).

    Article  CAS  Google Scholar 

  38. Melek, M., Gellert, M. & van Gent, D.C. Rejoining of DNA by the RAG1 and RAG2 proteins. Science 280, 301–303 (1998).

    Article  CAS  Google Scholar 

  39. Verkaik, N.S. et al. Different types of V(D)J recombination and end-joining defects in DNA double-strand break repair mutant mammalian cells. Eur. J. Immunol. 32, 701–709 (2002).

    Article  CAS  Google Scholar 

  40. Tauchi, H. et al. Nbs1 is essential for DNA repair by homologous recombination in higher vertebrate cells. Nature 420, 93–98 (2002).

    Article  CAS  Google Scholar 

  41. Chen, L. et al. WRN, the protein deficient in Werner syndrome, plays a critical structural role in optimizing DNA repair. Aging Cell 2, 191–199 (2003).

    Article  Google Scholar 

  42. Lan, L. et al. Accumulation of Werner protein at DNA double-strand breaks in human cells. J. Cell Sci. 118, 4153–4162 (2005).

    Article  CAS  Google Scholar 

  43. Monnat, R.J., Jr. & Saintigny, Y. Werner syndrome protein—unwinding function to explain disease. Sci. Aging Knowledge Environ. 2004, re3 (2004).

    Article  Google Scholar 

  44. Swanson, C., Saintigny, Y., Emond, M.J. & Monnat, R.J., Jr. The Werner syndrome protein has separable recombination and survival functions. DNA Repair (Amst.) 3, 475–482 (2004).

    Article  CAS  Google Scholar 

  45. Doublie, S., Sawaya, M.R. & Ellenberger, T. An open and closed case for all polymerases. Structure 7, R31–R35 (1999).

    Article  CAS  Google Scholar 

  46. Huang, S. et al. Characterization of the human and mouse WRN 3′ → 5′ exonuclease. Nucleic Acids Res. 28, 2396–2405 (2000).

    Article  CAS  Google Scholar 

  47. Karow, J.K., Newman, R.H., Freemont, P.S. & Hickson, I.D. Oligomeric ring structure of the Bloom's syndrome helicase. Curr. Biol. 9, 597–600 (1999).

    Article  CAS  Google Scholar 

  48. Xue, Y. et al. A minimal exonuclease domain of WRN forms a hexamer on DNA and possesses both 3′- 5′ exonuclease and 5′-protruding strand endonuclease activities. Biochemistry 41, 2901–2912 (2002).

    Article  CAS  Google Scholar 

  49. Walker, J.R., Corpina, R.A. & Goldberg, J. Structure of the Ku heterodimer bound to DNA and its implications for double-strand break repair. Nature 412, 607–614 (2001).

    Article  CAS  Google Scholar 

  50. Shin, D.S., Chahwan, C., Huffman, J.L. & Tainer, J.A. Structure and function of the double-strand break repair machinery. DNA Repair (Amst.) 3, 863–873 (2004).

    Article  CAS  Google Scholar 

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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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Authors and Affiliations

  1. Department of Molecular Biology and Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, 92037, California, USA

    J Jefferson P Perry, Lauren G Holden, Chiharu Hitomi & John A Tainer

  2. Life Sciences Division, Department of Molecular Biology, Lawrence Berkeley National Laboratory, Berkeley, 94720, California, USA

    J Jefferson P Perry, Steven M Yannone, Seungil Han, Priscilla K Cooper & John A Tainer

  3. Pfizer Global Research and Development, Exploratory Medicinal Sciences (EMS), MS4039 Eastern Point Road, Groton, 06340, Connecticut, USA

    Seungil Han

  4. Division of Molecular Radiation Biology, Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, 75390-9187, Texas, USA

    Aroumougame Asaithamby & David J Chen

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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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