Studying TDP1 Function in DNA Repair

  • Shih-Chieh Chiang
  • Kirsty Liversidge
  • Sherif F. El-Khamisy
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1703)

Abstract

Topoisomerase poisons act by inducing abortive topoisomerase reactions, which generate stable protein–DNA breaks (PDBs) that interfere with transcription elongation and progression of replication forks. In vertebrates, Tyrosyl-DNA phosphodiesterase 1 (TDP1) plays a major role in removal of topoisomerase 1-associated PDBs in the nucleus and mitochondria by hydrolyzing the 3′-phosphotyrosine bond. Depletion of TDP1 sensitizes tumor cells with defective DNA repair capacity to the genotoxic effect of TOP1 poisons, while homozygous mutation of the catalytic residue of TDP1 is associated with cerebellar degeneration and ataxia. We describe here two fluorescence based biochemical assays for measuring TDP1 phosphodiesterase activity in cellular lysates. The Gyrasol assay is sensitive, high-throughput, and useful for screening potential TDP1 inhibitors or cell lines that are likely to develop resistance to TOP1 poisons. The gel-shift assay is low cost and simple to set up, and is also suitable for screening cell lines that are likely to develop resistance to TOP1 poisons, as well as for diagnostic screening for individuals with hereditary ataxias.

Key words

TDP1 Topoisomerase Assay High-throughput screen Cell-based Drug discovery 

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Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  • Shih-Chieh Chiang
    • 1
  • Kirsty Liversidge
    • 1
  • Sherif F. El-Khamisy
    • 1
  1. 1.Department of Molecular Biology and BiotechnologyKrebs Institute, University of SheffieldSheffieldUK

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