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Recognition and processing of the origin of transfer DNA by conjugative relaxase TrwC

Nature Structural & Molecular Biology volume 10, pages 1002–1010 (2003)Cite this article

A Corrigendum to this article was published on 01 January 2004

Abstract

Relaxases are DNA strand transferases that catalyze the initial and final stages of DNA processing during conjugative cell-to-cell DNA transfer. Upon binding to the origin of transfer (oriT) DNA, relaxase TrwC melts the double helix. The three-dimensional structure of the relaxase domain of TrwC in complex with its cognate DNA at oriT shows a fold built on a two-layer α/β sandwich, with a deep narrow cleft that houses the active site. The DNA includes one arm of an extruded cruciform, an essential feature for specific recognition. This arm is firmly embraced by the protein through a β-ribbon positioned in the DNA major groove and a loop occupying the minor groove. It is followed by a single-stranded DNA segment that enters the active site, after a sharp U-turn forming a hydrophobic cage that traps the N-terminal methionine. Structural analysis combined with site-directed mutagenesis defines the architecture of the active site.

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Figure 1: Sequence alignment and scheme of DNA processing by TrwC.
Figure 2: TrwC binding to oriT DNA.
Figure 3: Structure of TrwC-N293 in complex with DNA.
Figure 4: Protein-DNA interactions.
Figure 5: Details of TrwC-DNA interactions.
Figure 6: Electron density map and active site.

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References

  1. Zechner, E.L. et al. Conjugative DNA transfer processes. In The Horizontal Gene Pool: Bacterial Plasmids and Gene Spread (ed. Thomas, M.) 87–173 (Harwood Academic, London, 2000).

    Google Scholar 

  2. Errington, J., Bath, J. & Wu, L.J. DNA transport in bacteria. Nat. Rev. Mol. Cell Biol. 2, 538–545 (2001).

    Article  CAS  Google Scholar 

  3. Llosa, M., Gomis-Ruth, F.X., Coll, M. & de la Cruz Fd, F. Bacterial conjugation: a two-step mechanism for DNA transport. Mol. Microbiol. 45, 1–8 (2002).

    Article  CAS  Google Scholar 

  4. Gomis-Rüth, F.X. et al. The bacterial conjugation protein TrwB resembles ring helicases and F1-ATPase. Nature 409, 637–641 (2001).

    Article  Google Scholar 

  5. Clewell, D.B. & Helinski, D.R. Supercoiled circular DNA–protein complex in Escherichia coli: purification and induced conversion to an open circular DNA form. Proc. Natl. Acad. Sci. USA 62, 1159–1166 (1969).

    Article  CAS  Google Scholar 

  6. Lanka, E. & Wilkins, B.M. DNA processing reactions in bacterial conjugation. Annu. Rev. Biochem. 64, 141–169 (1995).

    Article  CAS  Google Scholar 

  7. Llosa, M., Grandoso, G., Hernando, M.A. & de la Cruz, F. Functional domains in protein TrwC of plasmid R388: dissected DNA strand transferase and DNA helicase activities reconstitute protein function. J. Mol. Biol. 264, 56–67 (1996).

    Article  CAS  Google Scholar 

  8. Grandoso, G. et al. Two active-site tyrosyl residues of protein TrwC act sequentially at the origin of transfer during plasmid R388 conjugation. J. Mol. Biol. 295, 1163–1172 (2000).

    Article  CAS  Google Scholar 

  9. Gomis-Rüth, F.X. et al. The structure of plasmid-encoded transcriptional repressor CopG unliganded and bound to its operator. EMBO J. 17, 7404–7415 (1998).

    Article  Google Scholar 

  10. Moncalián, G., Valle, M., Valpuesta, J.M. & de la Cruz, F. IHF protein inhibits cleavage but not assembly of plasmid R388 relaxosomes. Mol. Microbiol. 31, 1643–1652 (1999).

    Article  Google Scholar 

  11. Champoux, J.J. DNA topoisomerases: structure, function, and mechanism. Annu. Rev. Biochem. 70, 369–413 (2001).

    Article  CAS  Google Scholar 

  12. Wang, J.C. DNA topoisomerases. Annu. Rev. Biochem. 65, 635–692 (1996).

    Article  CAS  Google Scholar 

  13. Novick, R.P. Contrasting lifestyles of rolling-circle phages and plasmids. Trends Biochem. Sci. 23, 434–438 (1998).

    Article  CAS  Google Scholar 

  14. Ide, H., Kow, Y.W. & Wallace, S.S. Thymine glycols and urea residues in M13 DNA constitute replicative blocks in vitro. Nucleic Acids Res. 13, 8035–8052 (1985).

    Article  CAS  Google Scholar 

  15. Campos-Olivas, R., Louis, J.M., Clerot, D., Gronenborn, B. & Gronenborn, A.M. The structure of a replication initiator unites diverse aspects of nucleic acid metabolism. Proc. Natl. Acad. Sci. USA 99, 10310–10315 (2002).

    Article  CAS  Google Scholar 

  16. Hickman, A.B., Ronning, D.R., Kotin, R.M. & Dyda, F. Structural unity among viral origin binding proteins: crystal structure of the nuclease domain of adeno-associated virus Rep. Mol. Cell 10, 327–337 (2002).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  18. Shindyalov, I.N. & Bourne, P.E. Protein structure alignment by incremental combinatorial extension (CE) of the optimal path. Protein Eng. 11, 739–747 (1998).

    Article  CAS  Google Scholar 

  19. Koonin, E.V. & Ilyina, T.V. Computer-assisted dissection of rolling circle DNA replication. Biosystems 30, 241–268 (1993).

    Article  CAS  Google Scholar 

  20. Uliel, S., Fliess, A. & Unger, R. Naturally occurring circular permutations in proteins. Protein Eng. 14, 533–542 (2001).

    Article  CAS  Google Scholar 

  21. Jung, J. & Lee, B. Circularly permuted proteins in the protein structure database. Protein Sci. 10, 1881–1886 (2001).

    Article  CAS  Google Scholar 

  22. Changela, A., DiGate, R.J. & Mondragon, A. Crystal structure of a complex of a type IA DNA topoisomerase with a single-stranded DNA molecule. Nature 411, 1077–1081 (2001).

    Article  CAS  Google Scholar 

  23. Chen, S.-J. & Wang, J.C. Identification of active site residues in Escherichia coli DNA topoisomerase I. J. Biol. Chem. 273, 6050–6056 (1998).

    Article  CAS  Google Scholar 

  24. Krogh, B.O. & Shuman, S. Proton relay mechanism of general acid catalysis by DNA topoisomerase IB. J. Biol. Chem. 277, 5711–7714 (2002).

    Article  CAS  Google Scholar 

  25. Sherratt, D.J. & Wigley, D.B. Conserved themes but novel activities in recombinases and topoisomerases. Cell 93, 149–152 (1998).

    Article  CAS  Google Scholar 

  26. Liu, Q. & Wang, J.C. Similarity in the catalysis of DNA breakage and rejoining by type IA and IIA DNA topoisomerases. Proc. Natl. Acad. Sci. USA 96, 881–886 (1999).

    Article  CAS  Google Scholar 

  27. Raumann, B.E., Rould, M.A., Pabo, C.O. & Sauer, R.T. DNA recognition by β-sheets in the Arc repressor–operator crystal structure. Nature 367, 754–757 (1994).

    Article  CAS  Google Scholar 

  28. Somers, W.S. & Phillips, S.E. Crystal structure of the met repressor–operator complex at 2.8 Å resolution reveals DNA recognition by β-strands. Nature 359, 387–393 (1992).

    Article  CAS  Google Scholar 

  29. Müller, C.W. & Herrmann, B.G. Crystallographic structure of the T domain–DNA complex of the Brachyury transcription factor. Nature 389, 884–888 (1997).

    Article  Google Scholar 

  30. Coll, M., Seidman, J.G. & Muller, C.W. Structure of the DNA–bound T-box domain of human TBX3, a transcription factor responsible for ulnar-mammary syndrome. Structure 10, 343–356 (2002).

    Article  CAS  Google Scholar 

  31. Newman, M., Strzelecka, T., Dorner, L.F., Schildkraut, I. & Aggarwal, A.K. Structure of Bam HI endonuclease bound to DNA: partial folding and unfolding on DNA binding. Science 269, 656–663 (1995).

    Article  CAS  Google Scholar 

  32. Llosa, M., Bolland, S. & de la Cruz, F. Structural and functional analysis of the origin of conjugal transfer of the broad-host-range IncW plasmid R388 and comparison with the related IncN plasmid R46. Mol. Gen. Genet. 226, 473–483 (1991).

    Article  CAS  Google Scholar 

  33. Llosa, M., Bolland, S. & de la Cruz, F. Genetic organization of the conjugal DNA processing region of the IncW plasmid R388. J. Mol. Biol. 235, 448–464 (1994).

    Article  CAS  Google Scholar 

  34. Sarkar, G. & Sommer, S.S. The “megaprimer” method of site-directed mutagenesis. Biotechniques 8, 404–407 (1990).

    CAS  PubMed  Google Scholar 

  35. Miroux, B. & Walker, J.E. Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J. Mol. Biol. 260, 289–298 (1996).

    Article  CAS  Google Scholar 

  36. Budisa, N. et al. High-level biosynthetic substitution of methionine in proteins by its analogs 2-aminohexanoic acid, selenomethionine and ethionine in Escherichia coli. Eur. J. Biochem. 230, 788–796 (1995).

    Article  CAS  Google Scholar 

  37. Matthews, B.W. Solvent content of protein crystals. J. Mol. Biol. 33, 491–497 (1968).

    Article  CAS  Google Scholar 

  38. Leslie, A.G.W. Macromolecular data processing. In Crystallographic Computing V (eds. Moras, D., Podjarny, A.D. & Thierry, J.C.) 27–38 (Oxford Univ. Press, Oxford, 1991).

    Google Scholar 

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

  40. Weeks, C.M., DeTitta, G.T., Hauptman, H.A., Thuman, P. & Miller, R. Structure solution by minimal-function phase refinement and Fourier filtering. II. Implementation and applications. Acta Crystallogr. A 50, 210–220 (1994).

    Article  Google Scholar 

  41. Cowtan, K.D. & Main, P. Phase combination and cross validation in iterated density-modification calculations. Acta Crystallogr. D 52, 43–48 (1996).

    Article  CAS  Google Scholar 

  42. Terwilliger, T.C. Automated structure solution, density modification and model building. Acta Crystallogr. D 58, 1937–1940 (2002).

    Article  Google Scholar 

  43. Roussel, A. & Cambilleau, C. Turbo-Frodo. In Silicon Graphics Geometry Partners Directory 77–79 (Silicon Graphics, Mountain View, California, USA, 1989).

    Google Scholar 

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

    Article  CAS  Google Scholar 

  45. Navaza, J. AMoRe: an automated package for molecular replacement. Acta Crystallogr. A 50, 157–163 (1994).

    Article  Google Scholar 

  46. Gouet, P., Courcelle, E., Stuart, D.I. & Metoz, F. ESPript: analysis of multiple sequence alignments in PostScript. Bioinformatics 15, 305–308 (1999).

    Article  CAS  Google Scholar 

  47. Evans, S.V. SETOR: hardware-lighted three-dimensional solid model representations of macromolecules. J. Mol. Graph. 11, 134–138 (1993).

    Article  CAS  Google Scholar 

  48. Nicholls, A., Bharadwaj, R. & Honig, B. GRASP: graphical representation and analysis of surface proteins. Biophys. J. 64, A166 (1993).

    Google Scholar 

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Acknowledgements

This study was supported by the Spain Ministerio de Educación y Ciencia, the Generalitat de Catalunya and the European Union (EU). Synchrotron data collection was supported by the European Synchrotron Radiation Facility (ESRF) and the EU. We thank A.G. Blanco for generating Figure 1b and helping with data collection.

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Authors and Affiliations

  1. Institut de Biologia Molecular de Barcelona, CSIC, Jordi Girona, 18-26, Barcelona, 08034, Spain

    Alicia Guasch, Rosa Pérez-Luque, F Xavier Gomis-Rüth & Miquel Coll

  2. Departamento de Biología Molecular (Laboratorio asociado al CIB, CSIC), Universidad de Cantabria, Herrera Oria, s/n, Santander, 39011, Spain

    María Lucas, Gabriel Moncalián, Matilde Cabezas & Fernando de la Cruz

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  1. Alicia Guasch
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Correspondence to Fernando de la Cruz or Miquel Coll.

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Guasch, A., Lucas, M., Moncalián, G. et al. Recognition and processing of the origin of transfer DNA by conjugative relaxase TrwC. Nat Struct Mol Biol 10, 1002–1010 (2003). https://doi.org/10.1038/nsb1017

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