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Structural basis for DNA duplex separation by a superfamily-2 helicase

Nature Structural & Molecular Biology volume 14, pages 647–652 (2007)Cite this article

Abstract

To reveal the mechanism of processive strand separation by superfamily-2 (SF2) 3′→5′ helicases, we determined apo and DNA-bound crystal structures of archaeal Hel308, a helicase that unwinds lagging strands and is related to human DNA polymerase θ. Our structure captures the duplex-unwinding reaction, shows that initial strand separation does not require ATP and identifies a prominent β-hairpin loop as the unwinding element. Similar loops in hepatitis C virus NS3 helicase and RNA-decay factors support the idea that this duplex-unwinding mechanism is applicable to a broad subset of SF2 helicases. Comparison with ATP-bound SF2 enzymes suggests that ATP promotes processive unwinding of 1 base pair by ratchet-like transport of the 3′ product strand. Our results provide a first structural framework for strand separation by processive SF2 3′→5′ helicases and reveal important mechanistic differences from SF1 helicases.

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Figure 1: Structure of the Hel308–DNA complex.
Figure 2: Details of selected DNA-recognition sites.
Figure 3: Model of processive unwinding by archaeal Hel308.
Figure 4: Test for large-scale conformational changes by limited proteolyis.
Figure 5: Comparison of Hel308 to other SF2 and SF1 helicases.

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References

  1. Linder, P. & Kowalczykowski, S. Helicases and NTP-driven nucleic acid machines: structure, function and roles in human disease. Nucleic Acids Res. 34, 4081 (2006).

    Article  CAS  Google Scholar 

  2. Houseley, J., LaCava, J. & Tollervey, D. RNA-quality control by the exosome. Nat. Rev. Mol. Cell Biol. 7, 529–539 (2006).

    Article  CAS  Google Scholar 

  3. Frick, D.N. Step-by-step progress toward understanding the hepatitis C virus RNA helicase. Hepatology 43, 1392–1395 (2006).

    Article  Google Scholar 

  4. Gorbalenya, A.E., Koonin, E.V., Donchenko, A.P. & Blinov, V.M. Two related superfamilies of putative helicases involved in replication, recombination, repair and expression of DNA and RNA genomes. Nucleic Acids Res. 17, 4713–4730 (1989).

    Article  CAS  Google Scholar 

  5. Velankar, S.S., Soultanas, P., Dillingham, M.S., Subramanya, H.S. & Wigley, D.B. Crystal structures of complexes of PcrA DNA helicase with a DNA substrate indicate an inchworm mechanism. Cell 97, 75–84 (1999).

    Article  CAS  Google Scholar 

  6. Korolev, S., Hsieh, J., Gauss, G.H., Lohman, T.M. & Waksman, G. Major domain swiveling revealed by the crystal structures of complexes of E. coli Rep helicase bound to single-stranded DNA and ADP. Cell 90, 635–647 (1997).

    Article  CAS  Google Scholar 

  7. Lee, J.Y., Yang, W. & Uvr, D. Helicase unwinds DNA one base pair at a time by a two-part power stroke. Cell 127, 1349–1360 (2006).

    Article  CAS  Google Scholar 

  8. Fischer, C.J., Maluf, N.K. & Lohman, T.M. Mechanism of ATP-dependent translocation of E. coli UvrD monomers along single-stranded DNA. J. Mol. Biol. 344, 1287–1309 (2004).

    Article  CAS  Google Scholar 

  9. Bennett, R.J. & Keck, J.L. Structure and function of RecQ DNA helicases. Crit. Rev. Biochem. Mol. Biol. 39, 79–97 (2004).

    Article  CAS  Google Scholar 

  10. Seki, M., Marini, F. & Wood, R.D. POLQ (Pol theta), a DNA polymerase and DNA-dependent ATPase in human cells. Nucleic Acids Res. 31, 6117–6126 (2003).

    Article  CAS  Google Scholar 

  11. Oshige, M., Aoyagi, N., Harris, P.V., Burtis, K.C. & Sakaguchi, K. A new DNA polymerase species from Drosophila melanogaster: a probable mus308 gene product. Mutat. Res. 433, 183–192 (1999).

    Article  CAS  Google Scholar 

  12. Dumont, S. et al. RNA translocation and unwinding mechanism of HCV NS3 helicase and its coordination by ATP. Nature 439, 105–108 (2006).

    Article  CAS  Google Scholar 

  13. Levin, M.K., Gurjar, M. & Patel, S.S. A Brownian motor mechanism of translocation and strand separation by hepatitis C virus helicase. Nat. Struct. Mol. Biol. 12, 429–435 (2005).

    Article  CAS  Google Scholar 

  14. Serebrov, V. & Pyle, A.M. Periodic cycles of RNA unwinding and pausing by hepatitis C virus NS3 helicase. Nature 430, 476–480 (2004).

    Article  CAS  Google Scholar 

  15. Sengoku, T., Nureki, O., Nakamura, A., Kobayashi, S. & Yokoyama, S. Structural basis for RNA unwinding by the DEAD-box protein Drosophila Vasa. Cell 125, 287–300 (2006).

    Article  CAS  Google Scholar 

  16. Singleton, M.R., Scaife, S. & Wigley, D.B. Structural analysis of DNA replication fork reversal by RecG. Cell 107, 79–89 (2001).

    Article  CAS  Google Scholar 

  17. Durr, H., Korner, C., Muller, M., Hickmann, V. & Hopfner, K.P. X-ray structures of the Sulfolobus solfataricus SWI2/SNF2 ATPase core and its complex with DNA. Cell 121, 363–373 (2005).

    Article  Google Scholar 

  18. Marini, F. & Wood, R.D. A human DNA helicase homologous to the DNA cross-link sensitivity protein Mus308. J. Biol. Chem. 277, 8716–8723 (2002).

    Article  CAS  Google Scholar 

  19. McCaffrey, R., St Johnston, D. & Gonzalez-Reyes, A. Drosophila mus301/spindle-C encodes a helicase with an essential role in double-strand DNA break repair and meiotic progression. Genetics 174, 1273–1285 (2006).

    Article  CAS  Google Scholar 

  20. Yoshimura, M. et al. Vertebrate POLQ and POLbeta cooperate in base excision repair of oxidative DNA damage. Mol. Cell 24, 115–125 (2006).

    Article  CAS  Google Scholar 

  21. Koonin, E.V., Wolf, Y.I. & Aravind, L. Prediction of the archaeal exosome and its connections with the proteasome and the translation and transcription machineries by a comparative-genomic approach. Genome Res. 11, 240–252 (2001).

    Article  CAS  Google Scholar 

  22. Guy, C.P. & Bolt, E.L. Archaeal Hel308 helicase targets replication forks in vivo and in vitro and unwinds lagging strands. Nucleic Acids Res. 33, 3678–3690 (2005).

    Article  CAS  Google Scholar 

  23. Fujikane, R., Komori, K., Shinagawa, H. & Ishino, Y. Identification of a novel helicase activity unwinding branched DNAs from the hyperthermophilic archaeon, Pyrococcus furiosus. J. Biol. Chem. 280, 12351–12358 (2005).

    Article  CAS  Google Scholar 

  24. Singleton, M.R., Dillingham, M.S., Gaudier, M., Kowalczykowski, S.C. & Wigley, D.B. Crystal structure of RecBCD enzyme reveals a machine for processing DNA breaks. Nature 432, 187–193 (2004).

    Article  CAS  Google Scholar 

  25. Bono, F., Ebert, J., Lorentzen, E. & Conti, E. The crystal structure of the exon junction complex reveals how it maintains a stable grip on mRNA. Cell 126, 713–725 (2006).

    Article  CAS  Google Scholar 

  26. Andersen, C.B. et al. Structure of the exon junction core complex with a trapped DEAD-box ATPase bound to RNA. Science 313, 1968–1972 (2006).

    Article  CAS  Google Scholar 

  27. Kim, J.L. et al. Hepatitis C virus NS3 RNA helicase domain with a bound oligonucleotide: the crystal structure provides insights into the mode of unwinding. Structure 6, 89–100 (1998).

    Article  CAS  Google Scholar 

  28. Levin, M.K., Gurjar, M.M. & Patel, S.S. ATP binding modulates the nucleic acid affinity of hepatitis C virus helicase. J. Biol. Chem. 278, 23311–23316 (2003).

    Article  CAS  Google Scholar 

  29. Lam, A.M., Keeney, D. & Frick, D.N. Two novel conserved motifs in the hepatitis C virus NS3 protein critical for helicase action. J. Biol. Chem. 278, 44514–44524 (2003).

    Article  CAS  Google Scholar 

  30. Jankowsky, E., Gross, C.H., Shuman, S. & Pyle, A.M. Active disruption of an RNA-protein interaction by a DExH/D RNA helicase. Science 291, 121–125 (2001).

    Article  CAS  Google Scholar 

  31. Kabsch, W. Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J. Appl. Cryst. 26, 795–800 (1993).

    Article  CAS  Google Scholar 

  32. Schneider, T.R. & Sheldrick, G.M. Substructure solution with SHELXD. Acta Crystallogr. D Biol. Crystallogr. 58, 1772–1779 (2002).

    Article  Google Scholar 

  33. Turk, D. Weiterentwicklung eines programms fuer molekuelgraphik und elektrondichte-manipulation und seine anwendung auf verschiedene protein-strukturaufklaerungen. Thesis, Technical Univ. Munich, (1992).

    Google Scholar 

  34. Brunger, A.T. et al. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54, 905–921 (1998).

    Article  CAS  Google Scholar 

  35. Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760–763 (1994).

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Acknowledgements

We thank S. Cui for comments on the manuscript, F. Uhlmann for collaborative efforts and scientific discussions, and the staff of the Swiss Light Source (beamline PXI) and the European Synchrotron Radiation Facility for generous beamtime allocation and help with data collection. K.-P.H. acknowledges grant support from the German research council (Deutsche Forschungsgemeinschaft HO2489/3-1 and SFB455), Human Frontiers of Science and the European Commission (integrated project 'DNA Repair').

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Authors and Affiliations

  1. Gene Center and Department of Chemistry and Biochemistry, Center for Integrated Protein Science, Ludwig-Maximilians-University Munich, Feodor-Lynen-Str. 25, Munich, 81377, Germany

    Katharina Büttner, Sebastian Nehring & Karl-Peter Hopfner

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  1. Katharina Büttner
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  2. Sebastian Nehring
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  3. Karl-Peter Hopfner
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Contributions

K.B. crystallized DNA-bound Hel308, determined structures and designed research; S.N. cloned the gene for A. fulgidus Hel308 and crystallized apo-Hel308; K.-P.H. designed research, participated in model building and evaluation, and wrote manuscript.

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Correspondence to Karl-Peter Hopfner.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Sequence alignment of A. fulgidus Hel308 with selected SF2/Ski2 family helicases. (PDF 629 kb)

Supplementary Fig. 2

Composit simulated annealed omit electron density shows how strands of a DNA duplex are separated by archaeal Hel208. (PDF 749 kb)

Supplementary Fig. 3

Comparison of apo- and DNA-bound archaeal Hel308. (PDF 688 kb)

Supplementary Fig. 4

Test for large-scale DNA- and ATP-induced conformational changes in archaeal Hel308 by limited proteolysis digestion using trypsin and chymotrypsin. (PDF 339 kb)

Supplementary Methods (PDF 68 kb)

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Büttner, K., Nehring, S. & Hopfner, KP. Structural basis for DNA duplex separation by a superfamily-2 helicase. Nat Struct Mol Biol 14, 647–652 (2007). https://doi.org/10.1038/nsmb1246

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