Abstract
The topoisomerase II (topo II) DNA incision-and-ligation cycle can be poisoned (for example following treatment with cancer chemotherapeutics) to generate cytotoxic DNA double-strand breaks (DSBs) with topo II covalently conjugated to DNA. Tyrosyl-DNA phosphodiesterase 2 (Tdp2) protects genomic integrity by reversing 5′-phosphotyrosyl–linked topo II–DNA adducts. Here, X-ray structures of mouse Tdp2–DNA complexes reveal that Tdp2 β–2-helix–β DNA damage–binding 'grasp', helical 'cap' and DNA lesion–binding elements fuse to form an elongated protein-DNA conjugate substrate-interaction groove. The Tdp2 DNA-binding surface is highly tailored for engagement of 5′-adducted single-stranded DNA ends and restricts nonspecific endonucleolytic or exonucleolytic processing. Structural, mutational and functional analyses support a single–metal ion catalytic mechanism for the exonuclease-endonuclease-phosphatase (EEP) nuclease superfamily and establish a molecular framework for targeted small-molecule blockade of Tdp2-mediated resistance to anticancer topoisomerase drugs.
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Acknowledgements
This research was supported by the intramural research program of the US National Institutes of Health (NIH), National Institute of Environmental Health Sciences (NIEHS) grant 1Z01ES102765-02 (R.S.W.) and by NIH grant CA 084442 (D.A.R.). We thank T. Kunkel and members of the Williams lab for discussions and critical reading of the manuscript, L. Pedersen of the NIEHS Collaborative crystallography group, the Advanced Light Source beamline 12.3.1 (SIBYLS) staff and the Advanced Photon Source (APS) Southeast Regional Collaborative Access Team (SER-CAT) staff for assistance with SAXS and crystallographic data collection and J. Williams of the NIEHS Protein Microcharacterization Core Facility for mass spectrometry analysis.
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M.J.S., C.D.A., P.D.R. and S.A. characterized the Tdp2 protein and enzymatic activity. C.D.A. and M.J.S. performed mutagenesis and analyzed mutant Tdp2 proteins. M.J.S. crystallized Tdp2 and Tdp2–DNA complexes. M.J.S. and R.S.W. collected SAXS and X-ray diffraction data and solved the X-ray crystal structures. M.J.S. and D.A.R. designed experiments, analyzed results and helped prepare the manuscript. R.S.W. conceived of and managed the study, designed experiments, analyzed results and wrote the manuscript.
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Schellenberg, M., Appel, C., Adhikari, S. et al. Mechanism of repair of 5′-topoisomerase II–DNA adducts by mammalian tyrosyl-DNA phosphodiesterase 2. Nat Struct Mol Biol 19, 1363–1371 (2012). https://doi.org/10.1038/nsmb.2418
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DOI: https://doi.org/10.1038/nsmb.2418
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