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Crystal structure of a complex of a type IA DNA topoisomerase with a single-stranded DNA molecule

Nature volume 411, pages 1077–1081 (2001)Cite this article

Abstract

A variety of cellular processes, including DNA replication, transcription, and chromosome condensation, require enzymes that can regulate the ensuing topological changes occurring in DNA. Such enzymes—DNA topoisomerases—alter DNA topology by catalysing the cleavage of single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA), the passage of DNA through the resulting break, and the rejoining of the broken phosphodiester backbone1. DNA topoisomerase III from Escherichia coli belongs to the type IA family of DNA topoisomerases, which transiently cleave ssDNA via formation of a covalent 5′ phosphotyrosine intermediate. Here we report the crystal structure, at 2.05 Å resolution, of an inactive mutant of E. coli DNA topoisomerase III in a non-covalent complex with an 8-base ssDNA molecule. The enzyme undergoes a conformational change that allows the oligonucleotide to bind within a groove leading to the active site. We note that the ssDNA molecule adopts a conformation like that of B-DNA while bound to the enzyme. The position of the DNA within the realigned active site provides insight into the role of several highly conserved residues during catalysis. These findings confirm various aspects of the type IA topoisomerase mechanism while suggesting functional implications for other topoisomerases and proteins that perform DNA rearrangements.

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Figure 1: Overall structure of the E. coli DNA topoisomerase III–ssDNA complex.
Figure 2: Comparison of the bound ssDNA structure to B-DNA.
Figure 3: Conformational changes on ssDNA binding.
Figure 4: Active site and catalytic mechanism.

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References

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

    Article  CAS  Google Scholar 

  2. Zhang, H. L., Malpure, S. & DiGate, R. J. Escherichia coli DNA topoisomerase III is a site-specific DNA binding protein that binds asymmetrically to its cleavage site. J. Biol. Chem. 270, 23700–23705 (1995).

    Article  CAS  Google Scholar 

  3. Mondragon, A. & DiGate, R. The structure of Escherichia coli DNA topoisomerase III. Struct. Fold Des. 7, 1373–1383 (1999).

    Article  CAS  Google Scholar 

  4. Zhang, H. L. & DiGate, R. J. The carboxyl-terminal residues of Escherichia coli DNA topoisomerase III are involved in substrate binding. J. Biol. Chem. 269, 9052–9059 (1994).

    CAS  PubMed  Google Scholar 

  5. Li, Z., Mondragon, A., Hiasa, H., Marians, K. J. & DiGate, R. J. Identification of a unique domain essential for Escherichia coli DNA topoisomerase III-catalysed decatenation of replication intermediates. Mol. Microbiol. 35, 888–895 (2000).

    Article  CAS  Google Scholar 

  6. Lima, C. D., Wang, J. C. & Mondragon, A. Three-dimensional structure of the 67K N-terminal fragment of E. coli DNA topoisomerase I. Nature 367, 138–146 (1994).

    Article  ADS  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  8. Redinbo, M. R., Champoux, J. J. & Hol, W. G. Novel insights into catalytic mechanism from a crystal structure of human topoisomerase I in complex with DNA. Biochemistry 39, 6832–6840 (2000).

    Article  CAS  Google Scholar 

  9. 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 

  10. Zhu, C. X., Roche, C. J., Papanicolaou, N., DiPietrantonio, A. & Tse-Dinh, Y. C. Site-directed mutagenesis of conserved aspartates, glutamates and arginines in the active site region of Escherichia coli DNA topoisomerase I. J. Biol. Chem. 273, 8783–8789 (1998).

    Article  CAS  Google Scholar 

  11. Wang, J. C. Interaction between DNA and an Escherichia coli protein ω. J. Mol. Biol. 55, 523–533 (1971).

    Article  CAS  Google Scholar 

  12. Domanico, P. L. & Tse-Dinh, Y. C. Mechanistic studies on Escherichia coli DNA topoisomerase I: Divalent ion effects. J. Inorg. Biochem. 42, 87–96 (1991).

    Article  CAS  Google Scholar 

  13. Zhu, C. X. & Tse-Dinh, Y. C. The acidic triad conserved in type IA DNA topoisomerases is required for binding of Mg(II) and subsequent conformational change. J. Biol. Chem. 275, 5318–5322 (2000).

    Article  CAS  Google Scholar 

  14. Nichols, M. D., DeAngelis, K., Keck, J. L. & Berger, J. M. Structure and function of an archaeal topoisomerase VI subunit with homology to the meiotic recombination factor Spo11. EMBO J. 18, 6177–6188 (1999).

    Article  CAS  Google Scholar 

  15. Berger, J. M., Fass, D., Wang, J. C. & Harrison, S. C. Structural similarities between topoisomerases that cleave one or both DNA strands. Proc. Natl Acad. Sci. USA 95, 7876–7881 (1998).

    Article  ADS  CAS  Google Scholar 

  16. Aravind, L., Leipe, D. D. & Koonin, E. V. Toprim-a conserved catalytic domain in type IA and II topoisomerases, DnaG-type primases, OLD family nucleases and RecR proteins. Nucleic Acids Res. 26, 4205–4213 (1998).

    Article  CAS  Google Scholar 

  17. Keck, J. L., Roche, D. D., Lynch, A. S. & Berger, J. M. Structure of the RNA polymerase domain of E. coli primase. Science 287, 2482–2486 (2000).

    Article  ADS  CAS  Google Scholar 

  18. Podobnik, M., McInerney, P., O'Donnell, M. & Kuriyan, J. A TOPRIM domain in the crystal structure of the catalytic core of Escherichia coli primase confirms a structural link to DNA topoisomerases. J. Mol. Biol. 300, 353–362 (2000).

    Article  CAS  Google Scholar 

  19. 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  ADS  CAS  Google Scholar 

  20. Berger, J. M., Gamblin, S. J., Harrison, S. C. & Wang, J. C. Structure and mechanism of DNA topoisomerase II. Nature 379, 255–232 (1996).

    Article  ADS  Google Scholar 

  21. Fass, D., Bogden, C. E. & Berger, J. M. Quaternary changes in topoisomerase II may direct orthogonal movement of two DNA strands. Nature Struct. Biol. 6, 322–326 (1999).

    Article  CAS  Google Scholar 

  22. Harp, J. M., Timm, D. E. & Bunick, G. J. Macromolecular crystal annealing: overcoming increased mosaicity associated with cryocrystallography. Acta Crystallogr. D 54, 622–628 (1998).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  26. Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991).

    Article  Google Scholar 

  27. Nicholls, A., Sharp, K. A. & Honig, B. H. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins Struct. Funct. Genet. 11, 281–286 (1991).

    Article  CAS  Google Scholar 

  28. Kraulis, P. J. MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallogr. 24, 946–950 (1991).

    Article  Google Scholar 

  29. Merritt, E. A. & Murphy, M. E. P. Raster 3D version 2.0. A program for photorealistic molecular graphics. Acta Crystallogr. D 50, 869–873 (1994).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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Acknowledgements

We acknowledge the contributions of H. Feinberg to the early stages of this project, and thank W. Niu for technical assistance. We thank K. Perry, E. Sontheimer and J. Widom for comments and suggestions. Research was supported by the NIH (A.M.) and an NRSA Institutional Training Grant in Molecular Biophysics (A.C.). We acknowledge the use of instruments in the Keck Biophysics Facility at Northwestern University. Portions of this work were performed at the DuPont-Northwestern-Dow collaborative Access Team (DND-CAT) Synchrotron Research Center at the Advanced Photon Source and at the Stanford Synchrotron Radiation Laboratory (SSRL). DND-CAT is supported by DuPont, Dow and the NSF, and use of the APS is supported by the DOE. SSRL is operated by the DOE, Office of Basic Energy Sciences. The SSRL Biotechnology Program is supported by the NIH and the DOE.

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Authors and Affiliations

  1. Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, 60208, Illinois, USA

    Anita Changela & Alfonso Mondragón

  2. Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, 21201, Maryland, USA

    Russell J. DiGate

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  1. Anita Changela
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Correspondence to Alfonso Mondragón.

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Changela, A., DiGate, R. & Mondragón, A. Crystal structure of a complex of a type IA DNA topoisomerase with a single-stranded DNA molecule. Nature 411, 1077–1081 (2001). https://doi.org/10.1038/35082615

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