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Holoenzyme assembly and ATP-mediated conformational dynamics of topoisomerase VI

Nature Structural & Molecular Biology volume 14, pages 611–619 (2007)Cite this article

Abstract

Type II topoisomerases help disentangle chromosomes to facilitate cell division. To advance understanding of the structure and dynamics of these essential enzymes, we have determined the crystal structure of an archaeal type IIB topoisomerase, topo VI, at 4.0-Å resolution. The 220-kDa heterotetramer adopts a 'twin-gate' architecture, in which a pair of ATPase domains at one end of the enzyme is poised to coordinate DNA movements into the enzyme and through a set of DNA-cleaving domains at the other end. Small-angle X-ray scattering studies show that nucleotide binding elicits a major structural reorganization that is propagated to the enzyme's DNA-cleavage center, explaining how ATP is coupled to DNA capture and strand scission. These data afford important insights into the mechanisms of topo VI and related proteins, including type IIA topoisomerases and the Spo11 meiotic recombination endonuclease.

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Figure 1: General features of type IIA and IIB topoisomerases.
Figure 2: Structure of topo VI.
Figure 3: Structure of the B subunit CTD.
Figure 4: SAXS profiles of S. shibatae topo VI in apo and ATP-bound states.
Figure 5: Modeling apo– and ATP-bound S. shibatae topo VI on the basis of SAXS and EM.
Figure 6: Proposed ATPase–strand passage cycle of topo VI.

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Acknowledgements

This work is dedicated to the memory of Nicholas R. Cozzarelli, who provided invaluable advice and criticism to both K.D.C. and J.M.B. over many years. The authors thank J. Holton, G. Meigs and J. Tanamachi for assistance at ALS beamline 8.3.1, G. Hura and D. S. Classen for assistance at ALS beamline 12.3.1, A. Bergerat and J. Wang (Harvard University) for the gift of S. shibatae topo VI subunits, M. Nollmann for advice on SAXS methodology, and members of the Berger laboratory for helpful advice. J.M.B. acknowledges support from the US National Cancer Institute (CA077373), and P.B. acknowledges support from the Ministero dell'Instruzione, dell'Università e della Ricerca Cofinanziamento, Fondo Investimenti Ricerca di Base, Ministero della Salute and Genomica Funzionale Consiglio Nazionale delle Ricerche.

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Author notes

  1. Kevin D Corbett

    Present address: Current address: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.,

Authors and Affiliations

  1. Department of Molecular and Cellular Biology, QB3 Institute, Stanley Hall #3220, University of California, Berkeley, 94720-3220, California, USA

    Kevin D Corbett & James M Berger

  2. Department of Biology, University of Padua, Via U. Bassi 58/B Padua, PD, 35131, Italy

    Piero Benedetti

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Contributions

K.D.C. performed all experiments, with the exception of the EM, which was performed by P.B. K.D.C. and J.M.B. devised all experiments and prepared the manuscript together, with editorial assistance from P.B.

Note: Supplementary information is available on the Nature Structural & Molecular Biology website.

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Correspondence to James M Berger.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Biochemical characterization of M. mazei topo VI. (PDF 620 kb)

Supplementary Fig. 2

Experimental and refined electron density maps. (PDF 1560 kb)

Supplementary Fig. 3

Sequence alignments. (PDF 208 kb)

Supplementary Figs. 4

Cross-linking of SsT6S424C. (PDF 597 kb)

Supplementary Fig. 5

Nucleotide addition to SsT6 results in a mixture of conformational states. (PDF 87 kb)

Supplementary Fig. 6

DAMMIN models of ATP-SsT6S424C. (PDF 1122 kb)

Supplementary Fig. 7

Manual modeling of ATP-SsT6S424C. (PDF 630 kb)

Supplementary Fig. 8

MmT6 does not simplify DNA topology below thermodynamic equilibrium. (PDF 250 kb)

Supplementary Video 1

Different conformational states of topo VI and proposed strand passage mechanism. The movie begins with the partially closed conformation of MmT6 observed in the crystal structure, transitions to the open state observed in the apo-SsT6 SAXS sample, then transitions to the fully-closed state observed in the ATP-SsT6S424C SAXS sample and also based on the structure of the nucleotide-mediated S. shibatae topo VI B-subunit dimer1. Subsequently, a proposed strand passage reaction is modeled based on these states and on the nucleotide-mediated transducer domain motion observed in previous structures of the S. shibatae topo VI B-subunit2. In the movie, the B-subunit GHKL and H2TH domains are colored yellow, the transducer domain orange, the A-subunit CAP domains green, Toprim domains blue, and G- and T-segment DNAs magenta and cyan, respectively. (MOV 6222 kb)

Corbett, K.D. & Berger, J.M. Structure of the topoisomerase VI B subunit: implications for type II topoisomerase mechanism and evolution. EMBO J. 22, 151-163 (2003).

Corbett, K.D. & Berger, J.M. Structural dissection of ATP turnover in the prototypical GHL ATPase topo VI. Structure 13, 873-882 (2005).

Supplementary Methods (PDF 112 kb)

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Corbett, K., Benedetti, P. & Berger, J. Holoenzyme assembly and ATP-mediated conformational dynamics of topoisomerase VI. Nat Struct Mol Biol 14, 611–619 (2007). https://doi.org/10.1038/nsmb1264

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