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.

  • Article
  • Published:

The Mu transpososome structure sheds light on DDE recombinase evolution

Nature volume 491, pages 413–417 (2012)Cite this article

Subjects

Abstract

Studies of bacteriophage Mu transposition paved the way for understanding retroviral integration and V(D)J recombination as well as many other DNA transposition reactions. Here we report the structure of the Mu transpososome—Mu transposase (MuA) in complex with bacteriophage DNA ends and target DNA—determined from data that extend anisotropically to 5.2 Å, 5.2 Å and 3.7 Å resolution, in conjunction with previously determined structures of individual domains. The highly intertwined structure illustrates why chemical activity depends on formation of the synaptic complex, and reveals that individual domains have different roles when bound to different sites. The structure also provides explanations for the increased stability of the final product complex and for its preferential recognition by the ATP-dependent unfoldase ClpX. Although MuA and many other recombinases share a structurally conserved ‘DDE’ catalytic domain, comparisons among the limited set of available complex structures indicate that some conserved features, such as catalysis in trans and target DNA bending, arose through convergent evolution because they are important for function.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Transposition pathway and structure determination.
Figure 2: Transpososome structure.
Figure 3: Stereo close-up view of interactions near the Mu DNA–target junction.
Figure 4: Model for a transpososome assembled on full left (reddish) and right (blue) bacteriophage ends.
Figure 5: Comparison of DDE recombinase–DNA complexes.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Coordinates and structure factors were deposited at the Protein Data Bank under accession 4fcy.

References

  1. Yanagihara, K. & Mizuuchi, K. Mismatch-targeted transposition of Mu: a new strategy to map genetic polymorphism. Proc. Natl Acad. Sci. USA 99, 11317–11321 (2002)

    ADS  CAS  PubMed  Google Scholar 

  2. Haapa, S., Taira, S., Heikkinen, E. & Savilahti, H. An efficient and accurate integration of mini-Mu transposons in vitro: a general methodology for functional genetic analysis and molecular biology applications. Nucleic Acids Res. 27, 2777–2784 (1999)

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Mizuuchi, K. In vitro transposition of bacteriophage Mu: a biochemical approach to a novel replication reaction. Cell 35, 785–794 (1983)

    CAS  PubMed  Google Scholar 

  4. Montaño, S. P. & Rice, P. A. Moving DNA around: DNA transposition and retroviral integration. Curr. Opin. Struct. Biol. 21, 370–378 (2011)

    PubMed  PubMed Central  Google Scholar 

  5. Davies, D. R., Goryshin, I. Y., Reznikoff, W. S. & Rayment, I. Three-dimensional structure of the Tn5 synaptic complex transposition intermediate. Science 289, 77–85 (2000)

    ADS  CAS  PubMed  Google Scholar 

  6. Maertens, G. N., Hare, S. & Cherepanov, P. The mechanism of retroviral integration from X-ray structures of its key intermediates. Nature 468, 326–329 (2010)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  7. Richardson, J. M., Colloms, S. D., Finnegan, D. J. & Walkinshaw, M. D. Molecular architecture of the Mos1 paired-end complex: the structural basis of DNA transposition in a eukaryote. Cell 138, 1096–1108 (2009)

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Choi, W. & Harshey, R. M. DNA repair by the cryptic endonuclease activity of Mu transposase. Proc. Natl Acad. Sci. USA 107, 10014–10019 (2010)

    ADS  CAS  PubMed  Google Scholar 

  9. Chaconas, G., Kennedy, D. L. & Evans, D. Predominant integration end products of infecting bacteriophage Mu DNA are simple insertions with no preference for integration of either Mu DNA strand. Virology 128, 48–59 (1983)

    CAS  PubMed  Google Scholar 

  10. Lavoie, B. D., Chan, B. S., Allison, R. G. & Chaconas, G. Structural aspects of a higher order nucleoprotein complex: induction of an altered DNA structure at the Mu-host junction of the Mu type 1 transpososome. EMBO J. 10, 3051–3059 (1991)

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Surette, M. G., Buch, S. J. & Chaconas, G. Transpososomes: stable protein-DNA complexes involved in the in vitro transposition of bacteriophage Mu DNA. Cell 49, 253–262 (1987)

    CAS  PubMed  Google Scholar 

  12. Au, T. K., Pathania, S. & Harshey, R. M. True reversal of Mu integration. EMBO J. 23, 3408–3420 (2004)

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Mizuuchi, M., Rice, P. A., Wardle, S. J., Haniford, D. B. & Mizuuchi, K. Control of transposase activity within a transpososome by the configuration of the flanking DNA segment of the transposon. Proc. Natl Acad. Sci. USA 104, 14622–14627 (2007)

    ADS  CAS  PubMed  Google Scholar 

  14. Kruklitis, R., Welty, D. J. & Nakai, H. ClpX protein of Escherichia coli activates bacteriophage Mu transposase in the strand transfer complex for initiation of Mu DNA synthesis. EMBO J. 15, 935–944 (1996)

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Levchenko, I., Luo, L. & Baker, T. A. Disassembly of the Mu transposase tetramer by the ClpX chaperone. Genes Dev. 9, 2399–2408 (1995)

    CAS  PubMed  Google Scholar 

  16. Mhammedi-Alaoul, A., Pato, M., Gama, M. J. & Toussaint, A. A new component of bacteriophage Mu replicative transposition machinery: the Escherichia coli ClpX protein. Mol. Microbiol. 11, 1109–1116 (1994)

    Google Scholar 

  17. Abdelhakim, A. H., Oakes, E. C., Sauer, R. T. & Baker, T. A. Unique contacts direct high-priority recognition of the tetrameric Mu transposase-DNA complex by the AAA+ unfoldase ClpX. Mol. Cell 30, 39–50 (2008)

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Savilahti, H., Rice, P. A. & Mizuuchi, K. The phage Mu transpososome core: DNA requirements for assembly and function. EMBO J. 14, 4893–4903 (1995)

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Baker, T. A. & Mizuuchi, K. DNA-promoted assembly of the active tetramer of the Mu transposase. Genes Dev. 6, 2221–2232 (1992)

    CAS  PubMed  Google Scholar 

  20. Yuan, J. F., Beniac, D. R., Chaconas, G. & Ottensmeyer, F. P. 3D reconstruction of the Mu transposase and the Type 1 transpososome: a structural framework for Mu DNA transposition. Genes Dev. 19, 840–852 (2005)

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Savilahti, H. & Mizuuchi, K. Mu transpositional recombination: donor DNA cleavage and strand transfer in trans by the Mu transposase. Cell 85, 271–280 (1996)

    CAS  PubMed  Google Scholar 

  22. Aldaz, H., Schuster, E. & Baker, T. A. The interwoven architecture of the Mu transposase couples DNA synapsis to catalysis. Cell 85, 257–269 (1996)

    CAS  PubMed  Google Scholar 

  23. Krementsova, E., Giffin, M. J., Pincus, D. & Baker, T. A. Mutational analysis of the Mu transposase. Contributions of two distinct regions of domain II to recombination. J. Biol. Chem. 273, 31358–31365 (1998)

    CAS  PubMed  Google Scholar 

  24. Namgoong, S. Y., Sankaralingam, S. & Harshey, R. M. Altering the DNA-binding specificity of Mu transposase in vitro. Nucleic Acids Res. 26, 3521–3527 (1998)

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Zou, A. H., Leung, P. C. & Harshey, R. M. Transposase contacts with mu DNA ends. J. Biol. Chem. 266, 20476–20482 (1991)

    CAS  PubMed  Google Scholar 

  26. Tanaka, Y. et al. Crystal structure of the CENP-B protein-DNA complex: the DNA-binding domains of CENP-B induce kinks in the CENP-B box DNA. EMBO J. 20, 6612–6618 (2001)

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Watkins, S., van Pouderoyen, G. & Sixma, T. K. Structural analysis of the bipartite DNA-binding domain of Tc3 transposase bound to transposon DNA. Nucleic Acids Res. 32, 4306–4312 (2004)

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Craigie, R., Mizuuchi, M. & Mizuuchi, K. Site-specific recognition of the bacteriophage Mu ends by the Mu A protein. Cell 39, 387–394 (1984)

    CAS  PubMed  Google Scholar 

  29. Kuo, C. F., Zou, A. H., Jayaram, M., Getzoff, E. & Harshey, R. DNA-protein complexes during attachment-site synapsis in Mu DNA transposition. EMBO J. 10, 1585–1591 (1991)

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Mizuuchi, M., Baker, T. A. & Mizuuchi, K. DNase protection analysis of the stable synaptic complexes involved in Mu transposition. Proc. Natl Acad. Sci. USA 88, 9031–9035 (1991)

    ADS  CAS  PubMed  Google Scholar 

  31. Rice, P. & Mizuuchi, K. Structure of the bacteriophage Mu transposase core: a common structural motif for DNA transposition and retroviral integration. Cell 82, 209–220 (1995)

    CAS  PubMed  Google Scholar 

  32. Wu, Z. & Chaconas, G. A novel DNA binding and nuclease activity in domain III of Mu transposase: evidence for a catalytic region involved in donor cleavage. EMBO J. 14, 3835–3843 (1995)

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Abdelhakim, A. H., Sauer, R. T. & Baker, T. A. The AAA+ ClpX machine unfolds a keystone subunit to remodel the Mu transpososome. Proc. Natl Acad. Sci. USA 107, 2437–2442 (2010)

    ADS  CAS  PubMed  Google Scholar 

  34. Burton, B. M. & Baker, T. A. Mu transpososome architecture ensures that unfolding by ClpX or proteolysis by ClpXP remodels but does not destroy the complex. Chem. Biol. 10, 463–472 (2003)

    CAS  PubMed  Google Scholar 

  35. Naigamwalla, D. Z., Coros, C. J., Wu, Z. & Chaconas, G. Mutations in domain III α of the Mu transposase: evidence suggesting an active site component which interacts with the Mu-host junction. J. Mol. Biol. 282, 265–274 (1998)

    CAS  PubMed  Google Scholar 

  36. Yang, J. Y., Kim, K., Jayaram, M. & Harshey, R. M. A domain sharing model for active site assembly within the Mu A tetramer during transposition: the enhancer may specify domain contributions. EMBO J. 14, 2374–2384 (1995)

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Surette, M. G. & Chaconas, G. The Mu transpositional enhancer can function in trans: requirement of the enhancer for synapsis but not strand cleavage. Cell 68, 1101–1108 (1992)

    CAS  PubMed  Google Scholar 

  38. Mizuuchi, M. & Mizuuchi, K. Conformational isomerization in phage Mu transpososome assembly: effects of the transpositional enhancer and of MuB. EMBO J. 20, 6927–6935 (2001)

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Harshey, R. M. & Jayaram, M. The mu transpososome through a topological lens. Crit. Rev. Biochem. Mol. Biol. 41, 387–405 (2006)

    CAS  PubMed  Google Scholar 

  40. Craigie, R. & Mizuuchi, K. Role of DNA topology in Mu transposition: mechanism of sensing the relative orientation of two DNA segments. Cell 45, 793–800 (1986)

    CAS  PubMed  Google Scholar 

  41. Surette, M. G. & Chaconas, G. A protein factor which reduces the negative supercoiling requirement in the Mu DNA strand transfer reaction is Escherichia coli integration host factor. J. Biol. Chem. 264, 3028–3034 (1989)

    CAS  PubMed  Google Scholar 

  42. Allison, R. G. & Chaconas, G. Role of the A protein-binding sites in the in vitro transposition of Mu DNA. A complex circuit of interactions involving the Mu ends and the transpositional enhancer. J. Biol. Chem. 267, 19963–19970 (1992)

    CAS  PubMed  Google Scholar 

  43. Jiang, H., Yang, J. Y. & Harshey, R. M. Criss-crossed interactions between the enhancer and the att sites of phage Mu during DNA transposition. EMBO J. 18, 3845–3855 (1999)

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Craig, N. L. Mobile DNA II (ASM Press, 2002)

    Google Scholar 

  45. Pribil, P. A. & Haniford, D. B. Target DNA bending is an important specificity determinant in target site selection in Tn10 transposition. J. Mol. Biol. 330, 247–259 (2003)

    CAS  PubMed  Google Scholar 

  46. Swinger, K. K. & Rice, P. A. Structure-based analysis of HU-DNA binding. J. Mol. Biol. 365, 1005–1016 (2007)

    CAS  PubMed  Google Scholar 

  47. Levchenko, I., Yamauchi, M. & Baker, T. A. ClpX and MuB interact with overlapping regions of Mu transposase: implications for control of the transposition pathway. Genes Dev. 11, 1561–1572 (1997)

    CAS  PubMed  Google Scholar 

  48. Clubb, R. T., Schumacher, S., Mizuuchi, K., Gronenborn, A. M. & Clore, G. M. Solution structure of the Iγ subdomain of the Mu end DNA-binding domain of phage Mu transposase. J. Mol. Biol. 273, 19–25 (1997)

    CAS  PubMed  Google Scholar 

  49. Schumacher, S. et al. Solution structure of the Mu end DNA-binding Iβ subdomain of phage Mu transposase: modular DNA recognition by two tethered domains. EMBO J. 16, 7532–7541 (1997)

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Baker, T. A., Mizuuchi, M., Savilahti, H. & Mizuuchi, K. Division of labor among monomers within the Mu transposase tetramer. Cell 74, 723–733 (1993)

    CAS  PubMed  Google Scholar 

  51. Ducruix, A. & Giegg, R. in Preparation of Selenomethionyl Protein Crystals (eds Dublie, S. & Carter, C. W. ) (Oxford Univ. Press, 1992)

    Google Scholar 

  52. Otwinowski, Z. & Minor, W. in Methods in Enzymology Vol. 276 (eds Carter, C. W. & Sweet, R. M. ) 307–326 (Academic, 1997)

    Google Scholar 

  53. Sheldrick, G. A short history of SHELX. Acta Crystallogr. A 64, 112–122 (2008)

    ADS  CAS  PubMed  MATH  Google Scholar 

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

    PubMed  Google Scholar 

  55. CCP4. The CCP4 Suite: Programs for Protein Crystallography. Acta Crystallogr. D 50, 760–763 (1994)

  56. Zhang, K. Y., Cowtan, K. & Main, P. Combining constraints for electron-density modification. Methods Enzymol. 277, 53–64 (1997)

    CAS  PubMed  Google Scholar 

  57. Emsley, P., Lohkamp, B., Scott, W. & Cowtan, K. Features and Development of Coot. Acta Crystallogr. D 66, 486–501 (2010)

    CAS  Google Scholar 

  58. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010)

    CAS  PubMed  Google Scholar 

  59. Schröder, G. F., Levitt, M. & Brunger, A. T. Super-resolution biomolecular crystallography with low-resolution data. Nature 464, 1218–1222 (2010)

    ADS  PubMed  PubMed Central  Google Scholar 

  60. Strong, M. et al. Toward the structural genomics of complexes: crystal structure of a PE/PPE protein complex from Mycobacterium tuberculosis. Proc. Natl Acad. Sci. USA 103, 8060–8065 (2006)

    ADS  CAS  PubMed  Google Scholar 

  61. Zheng, G., Lu, X. J. & Olson, W. K. Web 3DNA–a web server for the analysis, reconstruction, and visualization of three-dimensional nucleic-acid structures. Nucleic Acids Res. 37, W240–W246 (2009)

    CAS  PubMed  PubMed Central  Google Scholar 

  62. Lavoie, B. D. & Chaconas, G. Site-specific HU binding in the Mu transpososome: conversion of a sequence-independent DNA-binding protein into a chemical nuclease. Genes Dev. 7, 2510–2519 (1993)

    CAS  PubMed  Google Scholar 

  63. Swinger, K. K. & Rice, P. A. IHF and HU: flexible architects of bent DNA. Curr. Opin. Struct. Biol. 14, 28–35 (2004)

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank K. Mizuuchi for initiating this project, K. K. Swinger and B. Vertessy for early crystallization efforts, and X. Yang and the staff of APS beamlines 14, 19 and 21 for assistance with data collection. This work was funded in part by NIH grant GM086826 (to P.A.R.).

Author information

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, 60637, Illinois, USA

    Sherwin P. Montaño, Ying Z. Pigli & Phoebe A. Rice

Authors
  1. Sherwin P. Montaño
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ying Z. Pigli
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Phoebe A. Rice
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

S.P.M. carried out most of the crystallographic work, Y.Z.P. grew the first diffracting transpososome crystals and assisted with all other aspects of the project, and P.A.R. designed the project and assisted in computational work and interpretation of the results.

Corresponding author

Correspondence to Phoebe A. Rice.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-4, Supplementary References and Supplementary Table 1. (PDF 1162 kb)

Ribbon drawing of the transpososome structure rotating 360°

The complex is rotating about the crystallographic twofold axis that relates the red and blue halves. Colours are as in the main text: bacteriophage Mu end DNAs are red and blue, target DNA black, and the scissile phosphate and active site residues are yellow. The darker-colored subunits catalyze DNA cleavage and strand transfer and the lighter-colored subunits aid in complex assembly and stability. (MPG 4785 kb)

Closeup view of the experimental electron density, after improvement with Parrot, and contoured at 1.3 and 2.3 Sigma, rotating 360°

The rotation axis and colors are as in the main text and Supplementary Video 1. (MPG 9535 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Montaño, S., Pigli, Y. & Rice, P. The Mu transpososome structure sheds light on DDE recombinase evolution. Nature 491, 413–417 (2012). https://doi.org/10.1038/nature11602

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature11602

This article is cited by

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

Advanced search

Quick links

Nature Briefing Microbiology

Sign up for the Nature Briefing: Microbiology newsletter — what matters in microbiology research, free to your inbox weekly.

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