Article | Published:

The FtsK γ domain directs oriented DNA translocation by interacting with KOPS

Nature Structural & Molecular Biology volume 13, pages 965972 (2006) | Download Citation

Subjects

Abstract

The bacterial septum-located DNA translocase FtsK coordinates circular chromosome segregation with cell division. Rapid translocation of DNA by FtsK is directed by 8-base-pair DNA motifs (KOPS), so that newly replicated termini are brought together at the developing septum, thereby facilitating completion of chromosome segregation. Translocase functions reside in three domains, α, β and γ. FtsKαβ are necessary and sufficient for ATP hydrolysis–dependent DNA translocation, which is modulated by FtsKγ through its interaction with KOPS. By solving the FtsKγ structure by NMR, we show that γ is a winged-helix domain. NMR chemical shift mapping localizes the DNA-binding site on the γ domain. Mutated proteins with substitutions in the FtsKγ DNA-recognition helix are impaired in DNA binding and KOPS recognition, yet remain competent in DNA translocation and XerCD-dif site-specific recombination, which facilitates the late stages of chromosome segregation.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

Primary accessions

References

  1. 1.

    Order and disorder in bacterial genomes. Curr. Opin. Microbiol. 7, 519–527 (2004).

  2. 2.

    & Chromosome dimer resolution. in The Bacterial Chromosome (ed. Higgins, N.P.) 513–524 (ASM Press, Washington, DC, 2005).

  3. 3.

    , , & The two Escherichia coli chromosome arms locate to separate cell halves. Genes Dev. 20, 1727–1731 (2006).

  4. 4.

    et al. The complete genome sequence of Escherichia coli K-12. Science 277, 1453–1474 (1997).

  5. 5.

    , , , & Roles for replichores and macrodomains in segregation of the Escherichia coli chromosome. EMBO Rep. 6, 557–562 (2005).

  6. 6.

    , , & Decatenation of DNA circles by FtsK-dependent Xer site-specific recombination. EMBO J. 22, 6399–6407 (2003).

  7. 7.

    , & Genetic recombination and the cell cycle: what we have learned from chromosome dimers. Mol. Microbiol. 54, 1151–1160 (2004).

  8. 8.

    , & DNA transport in bacteria. Nat. Rev. Mol. Cell Biol. 2, 538–545 (2001).

  9. 9.

    , , & Fast, DNA-sequence independent translocation by FtsK in a single-molecule experiment. EMBO J. 23, 2430–2439 (2004).

  10. 10.

    et al. Sequence-directed DNA translocation by purified FtsK. Science 307, 586–590 (2005).

  11. 11.

    et al. KOPS: DNA motifs that control E. coli chromosome segregation by orienting the FtsK translocase. EMBO J. 24, 3770–3780 (2005).

  12. 12.

    et al. Identification of oligonucleotide sequences that direct the movement of the Escherichia coli FtsK translocase. Proc. Natl. Acad. Sci. USA 102, 17618–17623 (2005).

  13. 13.

    , & Dancing around the divisome: asymmetric chromosome segregation in Escherichia coli. Genes Dev. 19, 2367–2377 (2005).

  14. 14.

    et al. Dissection of a functional interaction between the DNA translocase, FtsK, and the XerD recombinase. Mol. Microbiol. 59, 1754–1766 (2006).

  15. 15.

    , , , & Double-stranded DNA translocation: structure and mechanism of hexameric FtsK. Mol. Cell 23, 457–469 (2006).

  16. 16.

    , , & Identification of the FtsK-recognition domain. Nat. Struct. Mol. Biol., advance online publication 15 October 2006 (doi:10.1038/nsmb1157).

  17. 17.

    & Winged helix proteins. Curr. Opin. Struct. Biol. 10, 110–116 (2000).

  18. 18.

    et al. The many faces of the helix-turn-helix domain: transcription regulation and beyond. FEMS Microbiol. Rev. 29, 231–262 (2005).

  19. 19.

    & Searching protein structure databases has come of age. Proteins 19, 165–173 (1994).

  20. 20.

    , , , & Crystal structure of the Za domain of the human editing enzyme ADAR1 bound to left-handed Z-DNA. Science 284, 1841–1845 (1999).

  21. 21.

    et al. Structure of the MecI repressor from Staphylococcus aureus in complex with the cognate DNA operator, mec. Acta Crystallograph. Sect. F. Struct. Biol. Cryst. Commun. 62, 320–324 (2006).

  22. 22.

    et al. FtsK is a DNA motor protein that activates chromosome dimer resolution by switching the catalytic state of the XerC and XerD recombinases. Cell 108, 195–205 (2002).

  23. 23.

    et al. Conformational changes induced by nucleotide binding in Cdc6/ORC from Aeropyrum pernix. J. Mol. Biol. 343, 547–557 (2004).

  24. 24.

    , , , & Structure-function analysis of the three domains of RuvB DNA motor protein. J. Biol. Chem. 280, 30504–30510 (2005).

  25. 25.

    , , & Extension of G-quadruplex DNA by ciliate telomerase. EMBO J. 25, 1148–1159 (2006).

  26. 26.

    , , & Tight regulation modulation and high level expression by vectors containing the arabinose pBAD promoter. J. Bacteriol. 177, 4121–4130 (1995).

  27. 27.

    , , , , & Crystal structure of the site-specific recombinase, XerD. EMBO J. 16, 5178–5187 (1997).

  28. 28.

    , & Switching catalytic activity in the XerCD site-specific recombination machine. J. Mol. Biol. 312, 45–57 (2001).

  29. 29.

    NMR of protein and nucleic acids (John Wiley & Sons, New York, 1986).

  30. 30.

    , , , & Stereospecific nuclear magnetic resonance assignments of the methyl groups of valine and leucine in the DNA-binding domain of the 434 repressor by biosynthetically directed fractional 13C labeling. Biochemistry 28, 7510–7516 (1989).

  31. 31.

    , , , & Solution structure and dynamics of Ras p21·GDP determined by heteronuclear three- and four-dimensional NMR spectroscopy. Biochemistry 33, 3515–3531 (1994).

  32. 32.

    , & Protein backbone angle restraints from searching a database for chemical shift and sequence homology. J. Biomol. NMR 13, 289–302 (1999).

  33. 33.

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

  34. 34.

    , , , & FtsK-dependent and -independent pathways of Xer site-specific recombination. EMBO J. 18, 5724–5734 (1999).

  35. 35.

    & Measuring motion on DNA by the type I restriction endonuclease EcoR124 using triplex displacement. EMBO J. 19, 2094–2102 (2000).

Download references

Acknowledgements

Research was supported by the Medical Research Council (Cambridge) and the Wellcome Trust (Oxford). We acknowledge our collaborators in the N.R. Cozzarelli and C. Bustamante laboratories (University of California, Berkeley) and would like to dedicate this paper to N.R. Cozzarelli, who died while this work was in progress. We thank our Oxford colleagues, P. Antrobus for his help with mass spectroscopy and I. Grainge for valuable discussions. V.S. was supported by an Oxford University Clarendon Postgraduate Award.

Author information

Author notes

    • Viknesh Sivanathan
    •  & Mark D Allen

    These authors contributed equally to this work.

Affiliations

  1. Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK.

    • Viknesh Sivanathan
    • , Charissa de Bekker
    • , Rachel Baker
    • , Lidia K Arciszewska
    •  & David J Sherratt
  2. Centre for Protein Engineering, Medical Research Council, Hills Road, Cambridge CB2 2QH, UK.

    • Mark D Allen
    • , Stefan M Freund
    •  & Mark Bycroft
  3. Laboratory of Molecular Biology, Medical Research Council, Hills Road, Cambridge CB2 2QH, UK.

    • Jan Löwe

Authors

  1. Search for Viknesh Sivanathan in:

  2. Search for Mark D Allen in:

  3. Search for Charissa de Bekker in:

  4. Search for Rachel Baker in:

  5. Search for Lidia K Arciszewska in:

  6. Search for Stefan M Freund in:

  7. Search for Mark Bycroft in:

  8. Search for Jan Löwe in:

  9. Search for David J Sherratt in:

Contributions

V.S., molecular biology and manuscript preparation. M.D.A., biochemistry, NMR and manuscript preparation. C.d.B., molecular biology. R.B., molecular biology. L.K.A., project direction and manuscript preparation. S.M.F., NMR. M.B., NMR. J.L., molecular biology, structural biology, project direction and manuscript preparation. D.J.S., project conception, project direction and manuscript preparation.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to David J Sherratt.

Supplementary information

PDF files

  1. 1.

    Supplementary Fig. 1

    HSQC spectra of KOPS-FtsKγ interaction.

  2. 2.

    Supplementary Fig. 2

    13C HSQC of E. coli FtsKγ.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nsmb1158

Further reading