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A novel and unified two-metal mechanism for DNA cleavage by type II and IA topoisomerases

Nature volume 465, pages 641–644 (2010)Cite this article

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Abstract

Type II topoisomerases are required for the management of DNA tangles and supercoils1, and are targets of clinical antibiotics and anti-cancer agents2. These enzymes catalyse the ATP-dependent passage of one DNA duplex (the transport or T-segment) through a transient, double-stranded break in another (the gate or G-segment), navigating DNA through the protein using a set of dissociable internal interfaces, or ‘gates’3,4. For more than 20 years, it has been established that a pair of dimer-related tyrosines, together with divalent cations, catalyse G-segment cleavage5,6,7. Recent efforts have proposed that strand scission relies on a ‘two-metal mechanism’8,9,10, a ubiquitous biochemical strategy that supports vital cellular processes ranging from DNA synthesis to RNA self-splicing11,12. Here we present the structure of the DNA-binding and cleavage core of Saccharomyces cerevisiae topoisomerase II covalently linked to DNA through its active-site tyrosine at 2.5 Å resolution, revealing for the first time the organization of a cleavage-competent type II topoisomerase configuration. Unexpectedly, metal-soaking experiments indicate that cleavage is catalysed by a novel variation of the classic two-metal approach. Comparative analyses extend this scheme to explain how distantly-related type IA topoisomerases cleave single-stranded DNA, unifying the cleavage mechanisms for these two essential enzyme families. The structure also highlights a hitherto undiscovered allosteric relay that actuates a molecular ‘trapdoor’ to prevent subunit dissociation during cleavage. This connection illustrates how an indispensable chromosome-disentangling machine auto-regulates DNA breakage to prevent the aberrant formation of mutagenic and cytotoxic genomic lesions.

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Figure 1: Structure of a topo II–DNA cleavage complex.
Figure 2: A cleavage-competent active site.
Figure 3: DNA cleavage by type IA and II topoisomerases.
Figure 4: Cleavage-dependent control of C-gate dynamics.

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Accession codes

Primary accessions

Protein Data Bank

Data deposits

Coordinates for the apo and Zn-bound complexes have been deposited in the RSCB PDB under the accession numbers 3L4J and 3L4K.

Change history

  • 03 June 2010

    A correction was made to Fig. 1a.

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Acknowledgements

This work was supported the NIH (GM033944 and GM053960 for N.O.; T32CA09592 for J.E.D.; GM08295 for B.H.S. and CA077373 for J.M.B.).

Author information

Authors and Affiliations

  1. Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA,

    Bryan H. Schmidt & James M. Berger

  2. Emerald BioStructures, Bainbridge Island, Washington 98110, USA ,

    Alex B. Burgin

  3. Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA,

    Joseph E. Deweese & Neil Osheroff

  4. Department of Medicine (Hematology/Oncology), Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA,

    Neil Osheroff

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  1. Bryan H. Schmidt
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  2. Alex B. Burgin
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  4. Neil Osheroff
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  5. James M. Berger
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Contributions

B.H.S. purified the complex, grew the crystals, and solved the structures. J.M.B. assisted with refinement and inspection of the molecular models. A.B.B. synthesized the phosphorothiolate reagent. J.E.D. assisted the design of the DNA substrate and optimizing cleavage conditions. N.O. and J.M.B. designed the experiments. All authors contributed to the manuscript.

Corresponding author

Correspondence to James M. Berger.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Table S1, Supplementary Figures S1-S6 with legends, Supplementary Movie SM1 legend and References. (PDF 929 kb)

Supplementary Movie SM1

The movie shows that the transition between cleaved and uncleaved DNA states permits C-gate opening (see Supplementary Information file for full legend). (MOV 19109 kb)

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Schmidt, B., Burgin, A., Deweese, J. et al. A novel and unified two-metal mechanism for DNA cleavage by type II and IA topoisomerases. Nature 465, 641–644 (2010). https://doi.org/10.1038/nature08974

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