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Structure of a topoisomerase II–DNA–nucleotide complex reveals a new control mechanism for ATPase activity

Nature Structural & Molecular Biology volume 19, pages 1147–1154 (2012)Cite this article

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Abstract

Type IIA topoisomerases control DNA supercoiling and disentangle chromosomes through a complex ATP-dependent strand-passage mechanism. Although a general framework exists for type IIA topoisomerase function, the architecture of the full-length enzyme has remained undefined. Here we present the structure of a fully catalytic Saccharomyces cerevisiae topoisomerase II homodimer complexed with DNA and a nonhydrolyzable ATP analog. The enzyme adopts a domain-swapped configuration wherein the ATPase domain of one protomer sits atop the nucleolytic region of its partner subunit. This organization produces an unexpected interaction between bound DNA and a conformational transducing element in the ATPase domain, which we show is critical for both DNA-stimulated ATP hydrolysis and global topoisomerase activity. Our data indicate that the ATPase domains pivot about each other to ensure unidirectional strand passage and that this state senses bound DNA to promote ATP turnover and enzyme reset.

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Figure 1: Structure of topo II bound to DNA and AMPPNP.
Figure 2: The topo II ATPase domain appears to engage bound DNA.
Figure 3: K-loop mutants are deficient at relaxing negatively supercoiled DNA.
Figure 4: K-loop mutants are defective for decatenation but not for DNA cleavage.
Figure 5: K-loop mutants maintain basal ATPase rates but lose DNA stimulation of ATP hydrolysis.
Figure 6: Oligonucleotides stimulate ATP hydrolysis in a length-dependent manner.
Figure 7: Unexpected complexities in the type IIA topoisomerase catalytic cycle.

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Acknowledgements

The authors thank A. Burgin, at Emerald BioStructures, Bainbridge Island, Washington, USA, for synthesizing the phosphorothioamidite reagent, as well as members of the Berger Lab for helpful discussions. This work was supported by a US National Institutes of Health training grant (GM08295 to B.H.S.) and the US National Cancer Institute (CA077373 to J.M.B.) and the US National Institutes of Health (GM33944 to N.O.).

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Authors and Affiliations

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

    Bryan H Schmidt & James M Berger

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

    Neil Osheroff

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

    Neil Osheroff

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B.H.S., J.M.B. and N.O. designed the experiments. B.H.S. performed all of the experiments. B.H.S. and J.M.B. wrote the paper.

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Correspondence to James M Berger.

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Schmidt, B., Osheroff, N. & Berger, J. Structure of a topoisomerase II–DNA–nucleotide complex reveals a new control mechanism for ATPase activity. Nat Struct Mol Biol 19, 1147–1154 (2012). https://doi.org/10.1038/nsmb.2388

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