Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Crystal structure of RecBCD enzyme reveals a machine for processing DNA breaks

Abstract

RecBCD is a multi-functional enzyme complex that processes DNA ends resulting from a double-strand break. RecBCD is a bipolar helicase that splits the duplex into its component strands and digests them until encountering a recombinational hotspot (Chi site). The nuclease activity is then attenuated and RecBCD loads RecA onto the 3′ tail of the DNA. Here we present the crystal structure of RecBCD bound to a DNA substrate. In this initiation complex, the DNA duplex has been split across the RecC subunit to create a fork with the separated strands each heading towards different helicase motor subunits. The strands pass along tunnels within the complex, both emerging adjacent to the nuclease domain of RecB. Passage of the 3′ tail through one of these tunnels provides a mechanism for the recognition of a Chi sequence by RecC within the context of double-stranded DNA. Gating of this tunnel suggests how nuclease activity might be regulated.

Your institute does not have access to this article

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: The processing of double-strand breaks by RecBCD enzyme.
Figure 2: Structures of the individual RecBCD subunits.
Figure 3: Structure of the RecBCD–DNA complex.
Figure 4: Alternative exits from the 3′ tunnel.
Figure 5: Diagram outlining the changes in RecBCD that occur after encountering a Chi site.

References

  1. Kowalczykowski, S. C. Initiation of genetic recombination and recombination-dependent replication. Trends. Biochem. Sci. 25, 156–165 (2000)

    CAS  Article  Google Scholar 

  2. Ponticelli, A. S., Schultz, D. W., Taylor, A. F. & Smith, G. R. Chi-dependent DNA strand cleavage by the RecBC enzyme. Cell 41, 145–151 (1985)

    CAS  Article  Google Scholar 

  3. Taylor, A. F., Schultz, D. W., Ponticelli, A. S. & Smith, G. R. RecBC enzyme nicking at Chi sites during DNA unwinding: location and orientation-dependence of the cutting. Cell 41, 153–163 (1985)

    CAS  Article  Google Scholar 

  4. Bianco, P. R. & Kowalczykowski, S. C. The recombination hotspot Chi is recognized by the translocating RecBCD enzyme as the single strand of DNA containing the sequence 5′-GCTGGTGG-3′. Proc. Natl Acad. Sci. USA 94, 6706–6711 (1997)

    ADS  CAS  Article  Google Scholar 

  5. Spies, M. et al. A molecular throttle: the recombination hotspot Chi controls DNA translocation by the RecBCD helicase. Cell 114, 647–654 (2003)

    CAS  Article  Google Scholar 

  6. Dixon, D. A. & Kowalczykowski, S. C. The recombination hotspot Chi is a regulatory sequence that acts by attenuating the nuclease activity of the E. coli RecBCD enzyme. Cell 73, 87–96 (1993)

    CAS  Article  Google Scholar 

  7. Anderson, D. G. & Kowalczykowski, S. C. The recombination hot spot Chi is a regulatory element that switches the polarity of DNA degradation by the RecBCD enzyme. Genes Dev. 11, 571–581 (1997a)

    CAS  Article  Google Scholar 

  8. Taylor, A. F. & Smith, G. R. Strand specificity of nicking of DNA at Chi sites by RecBCD enzyme. J. Biol. Chem. 270, 24459–24467 (1995b)

    CAS  Article  Google Scholar 

  9. Anderson, D. G. & Kowalczykowski, S. C. The translocating RecBCD enzyme stimulates recombination by directing RecA protein onto ssDNA in a Chi-regulated manner. Cell 90, 77–86 (1997b)

    CAS  Article  Google Scholar 

  10. Taylor, A. F. & Smith, G. R. Monomeric RecBCD enzyme binds and unwinds DNA. J. Biol. Chem. 270, 24451–24458 (1995a)

    CAS  Article  Google Scholar 

  11. Boehmer, P. E. & Emmerson, P. T. Escherichia coli RecBCD enzyme: inducible overproduction and reconstitution of the ATP-dependent deoxyribonuclease from purified subunits. Gene 102, 1–6 (1991)

    CAS  Article  Google Scholar 

  12. Yu, M., Souaya, J. & Julin, D. A. The 30 kDa C-terminal domain of the RecB protein is critical for the nuclease activity, but not the helicase activity, of the RecBCD enzyme from Escherichia coli. Proc. Natl Acad. Sci. USA 95, 981–986 (1998)

    ADS  CAS  Article  Google Scholar 

  13. Handa, N., Ohashi, S., Kusano, K. & Kobayashi, I. Chi*, a chi-related 11-mer sequence partially active in an E. coli recC1004 strain. Genes Cells 2, 525–536 (1997)

    CAS  Article  Google Scholar 

  14. Dillingham, M. S., Spies, M. & Kowalczykowski, S. C. RecBCD enzyme is a bipolar DNA helicase. Nature 423, 893–897 (2003)

    ADS  CAS  Article  Google Scholar 

  15. Taylor, A. F. & Smith, G. R. RecBCD enzyme is a DNA helicase with fast and slow motors of opposite polarity. Nature 423, 889–893 (2003)

    ADS  CAS  Article  Google Scholar 

  16. Roman, L. J. & Kowalczykowski, S. C. Characterisation of the adenosinetriphosphatase activity of the Escherichia coli RecBCD enzyme: relationship of ATP hydrolysis to the unwinding of duplex DNA. Biochemistry 28, 2873–2881 (1989)

    CAS  Article  Google Scholar 

  17. Ganesan, S. & Smith, G. R. Strand-specific binding to duplex DNA ends by the subunits of the Escherichia coli RecBCD enzyme. J. Mol. Biol. 229, 67–78 (1993)

    CAS  Article  Google Scholar 

  18. Farah, J. A. & Smith, G. R. The RecBCD enzyme initiation complex for DNA unwinding: enzyme positioning and DNA opening. J. Mol. Biol. 272, 699–715 (1997)

    CAS  Article  Google Scholar 

  19. Boehmer, P. E. & Emmerson, P. T. The RecB subunit of the Escherichia coli RecBCD enzyme couples ATP hydrolysis to DNA unwinding. J. Biol. Chem. 267, 4981–4987 (1992)

    CAS  PubMed  Google Scholar 

  20. Gorbalenya, A. E. & Koonin, E. V. Helicases: amino acid sequence comparisons and structure-function relationships. Curr. Opin. Struct. Biol. 3, 419–429 (1993)

    CAS  Article  Google Scholar 

  21. Subramanya, H. S., Bird, L. E., Brannigan, J. A. & Wigley, D. B. Crystal structure of a DExx box helicase. Nature 384, 379–383 (1996)

    ADS  CAS  Article  Google Scholar 

  22. 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)

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  24. Dillingham, M. S., Wigley, D. B. & Webb, M. R. Unidirectional single-stranded DNA translocation by PcrA helicase: measurement of step size and translocation speed. Biochemistry 39, 205–212 (2000)

    CAS  Article  Google Scholar 

  25. Soultanas, P., Dillingham, M. S., Wiley, P., Webb, M. R. & Wigley, D. B. Uncoupling DNA translocation and helicase activity in PcrA: direct evidence for an active mechanism. EMBO J. 19, 3799–3810 (2000)

    CAS  Article  Google Scholar 

  26. Singleton, M. R. & Wigley, D. B. Modularity and specialisation in Superfamily 1 and 2 helicases. J. Bacteriol. 184, 1819–1826 (2002)

    CAS  Article  Google Scholar 

  27. Aravind, L., Makarova, K. S. & Koonin, E. V. Holliday junction resolvases and related nucleases: identification of new families, phyletic distribution and evolutionary trajectories. Nucleic Acids Res. 28, 3417–3432 (2000)

    CAS  Article  Google Scholar 

  28. Kovall, R. & Matthews, B. W. Toroidal structure of λ-exonuclease. Science 277, 1824–1827 (1997)

    CAS  Article  Google Scholar 

  29. Yu, M., Souaya, J. & Julin, D. A. Identification of the nuclease active site in the multifunctional RecBCD enzyme by creation of a chimeric enzyme. J. Mol. Biol. 283, 797–808 (1998)

    CAS  Article  Google Scholar 

  30. Rosamond, J., Telander, K. M. & Linn, S. Modulation of the action of the RecBC enzyme of Escherichia coli K-12 by Ca2+. J. Biol. Chem. 254, 8648–8652 (1979)

    Google Scholar 

  31. Holm, L. & Sander, C. Protein structure comparison by alignment of distance matrices. J. Mol. Biol. 233, 123–138 (1993)

    CAS  Article  Google Scholar 

  32. Amundsen, S. K., Taylor, A. F. & Smith, G. R. Domain of RecC required for assembly of the regulatory RecD subunit into the Escherichia coli RecBCD holoenzyme. Genetics 161, 483–492 (2002)

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Chen, H. W., Ruan, B., Yu, M., Wang, J. & Julin, D. A. The RecD subunit of the RecBCD enzyme from Escherichia coli is a single-stranded DNA dependent ATPase. J. Biol. Chem. 272, 10072–10079 (1997)

    CAS  Article  Google Scholar 

  34. Kuhn, B., Abdel-Monem, M., Krell, H. & Hoffmann-Berling, H. Evidence for two mechanisms for DNA unwinding catalyzed by DNA helicases. J. Biol. Chem. 254, 11343–11350 (1979)

    CAS  PubMed  Google Scholar 

  35. Dillingham, M. S., Soultanas, P., Wiley, P., Webb, M. R. & Wigley, D. B. Defining the roles of individual residues in the single-stranded DNA binding site of PcrA helicase. Proc. Natl Acad. Sci. USA 98, 8381–8387 (2001)

    ADS  CAS  Article  Google Scholar 

  36. Bianco, P. R. & Kowalczykowski, S. C. Translocation step size and mechanism of the RecBC DNA helicase. Nature 405, 368–372 (2000)

    ADS  CAS  Article  Google Scholar 

  37. Korangy, F. & Julin, D. A. Efficiency of ATP hydrolysis and DNA unwinding by the RecBC enzyme from Escherichia coli. Biochemistry 33, 9552–9560 (1994)

    CAS  Article  Google Scholar 

  38. Schultz, D. W., Taylor, A. F. & Smith, G. R. Escherichia coli RecBC pseudorevertants lacking Chi recombinational hotspot activity. J. Bacteriol. 155, 664–680 (1983)

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Arnold, D. A., Bianco, P. R. & Kowalczykowski, S. C. The reduced levels of Chi recognition exhibited by the RecBC1004D enzyme reflect its recombination defect in vivo. J. Biol. Chem. 273, 16476–16486 (1998)

    CAS  Article  Google Scholar 

  40. Arnold, D. A., Handa, N., Kobayashi, I. & Kowalczykowski, S. C. A novel, 11 nucleotide variant of Chi, Chi*: One of a class of sequences defining the Escherichia coli recombination hotspot Chi. J. Mol. Biol. 300, 469–479 (2000)

    CAS  Article  Google Scholar 

  41. Kulkarni, A. & Julin, D. A. Specific inhibition of the E. coli RecBCD enzyme by Chi sequences in single-stranded oligodeoxynucleotides. Nucleic Acids Res. 32, 3672–3682 (2004)

    CAS  Article  Google Scholar 

  42. Palas, K. M. & Kushner, S. R. Biochemical and physical properties of exonuclease V from Escherichia coli. J. Biol. Chem. 265, 3447–3454 (1990)

    CAS  PubMed  Google Scholar 

  43. Churchill, J. J. & Kowalczykowski, S. C. Identification of the RecA protein-loading domain of RecBCD enzyme. J. Mol. Biol. 297, 537–542 (2000)

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  45. Weeks, C. M. & Miller, R. Optimising Shake-and-bake for proteins. Acta Crystallogr. D 55, 492–500 (1999)

    CAS  Article  Google Scholar 

  46. de La Fortelle, E. & Bricogne, G. Maximum-likelihood heavy-atom parameter refinement for the multiple isomorphous replacement and multiwavelength anomalous diffraction methods. Methods Enzymol. 276, 472–494 (1997)

    CAS  Article  Google Scholar 

  47. Collaborative Computing Project No. 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994)

    Article  Google Scholar 

  48. Brunger, A. T. et al. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 904–925 (1998)

    Article  Google Scholar 

  49. Sanner, M. F., Spehner, J. C. & Olson, A. J. Reduced surface: an efficient way to compute molecular surfaces. Biopolymers 38, 305–320 (1996)

    CAS  Article  Google Scholar 

  50. Merritt, E. A. & Bacon, D. J. Raster3D: Photorealistic molecular graphics. Methods Enzymol. 277, 505–524 (1997)

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank V. Ramakrishnan and J. Lowe for advice on the use and supply of tantalum bromide clusters, S. Halford for discussions about nucleases, C. Flensburg for advice on using SHARP and a pre-release version of the program, V. Dearing for oligonucleotide synthesis and purification, and D. Hall and E. Gordon for time and assistance on ESRF beamlines. This work was supported by Cancer Research UK (D.B.W.), NIH (S.C.K.) and a Wellcome Trust Travelling Research Fellowship (M.S.D.).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Dale B. Wigley.

Ethics declarations

Competing interests

The authors declare that they have no competing financial interests.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Singleton, M., Dillingham, M., Gaudier, M. et al. Crystal structure of RecBCD enzyme reveals a machine for processing DNA breaks. Nature 432, 187–193 (2004). https://doi.org/10.1038/nature02988

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature02988

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing