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DNA cleavage assay for the identification of topoisomerase I inhibitors

Nature Protocols volume 3, pages 1736–1750 (2008)Cite this article

Abstract

The inhibition of DNA topoisomerase I (Top1) has proven to be a successful approach in the design of anticancer agents. However, despite the clinical successes of the camptothecin derivatives, a significant need for less toxic and more chemically stable Top1 inhibitors still persists. Here, we describe one of the most frequently used protocols to identify novel Top1 inhibitors. These methods use uniquely 3′-radiolabeled DNA substrates and denaturing polyacrylamide gel electrophoresis to provide evidence for the Top1-mediated DNA cleaving activity of potential Top1 inhibitors. These assays allow comparison of the effectiveness of different drugs in stabilizing the Top1-DNA intermediate or cleavage (cleavable) complex. A variation on these assays is also presented, which provides a suitable system for determining whether the inhibitor blocks the forward cleavage or religation reactions by measuring the reversibility of the drug-induced Top1–DNA cleavage complexes. This entire protocol can be completed in ∼2 d.

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Figure 1
Figure 2: Structure and mechanism of the Top1 cleavage complex (Top1cc) trapping by a specific Top1 inhibitor (indenoisoquinoline derivative)4,5,55.
Figure 3: Structures of camptothecin derivatives.
Figure 4: Structures of the three chemical families of noncamptothecin Top1 inhibitors.
Figure 5: Cartoon illustrating the advantages of using a 3′-end-labeled DNA substrate.
Figure 6: Top1-mediated DNA cleavage assay.
Figure 7: Top1–DNA cleavage complex reversal assay.

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Acknowledgements

Our studies are supported by the Intramural Program of the Center for Cancer Research, NCI, NIH. We thank all members of the Laboratory of Molecular Pharmacology, past and present, for their contributions. Special thanks to Dr Kurt W. Kohn who pioneered studies on DNA topoisomerases and first proposed the trapping of topoisomerases by anticancer drugs (Ross, W.E., Glaubiger, D. & Kohn, K.W. Qualitative and quantitative aspects of intercalator-induced DNA strand breaks. Biochim. Biophys. Acta 562, 41–50 (1979)).

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  1. Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, 20892, Maryland, USA

    Thomas S Dexheimer & Yves Pommier

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Correspondence to Yves Pommier.

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Dexheimer, T., Pommier, Y. DNA cleavage assay for the identification of topoisomerase I inhibitors. Nat Protoc 3, 1736–1750 (2008). https://doi.org/10.1038/nprot.2008.174

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