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Studying TDP1 Function in DNA Repair.

Shih-Chieh Chiang1, Kirsty Liversidge1, Sherif F El-Khamisy2

  • 1Department of Molecular Biology and Biotechnology, Krebs Institute, University of Sheffield, Firth Court, S10 2TN, Sheffield, UK.

Methods in Molecular Biology (Clifton, N.J.)
|November 28, 2017
PubMed
Summary
This summary is machine-generated.

We developed two biochemical assays to measure Tyrosyl-DNA phosphodiesterase 1 (TDP1) activity. These assays can screen for TDP1 inhibitors and identify individuals with hereditary ataxias or tumor cells resistant to topoisomerase poisons.

Keywords:
AssayCell-basedDrug discoveryHigh-throughput screenTDP1Topoisomerase

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Genetics

Background:

  • Topoisomerase poisons induce protein-DNA breaks (PDBs), interfering with DNA replication and transcription.
  • Tyrosyl-DNA phosphodiesterase 1 (TDP1) repairs these breaks by hydrolyzing the 3'-phosphotyrosine bond.
  • TDP1 deficiency is linked to increased sensitivity to topoisomerase poisons and hereditary ataxias.

Purpose of the Study:

  • To develop and validate novel biochemical assays for measuring TDP1 phosphodiesterase activity.
  • To enable screening for TDP1 inhibitors and identification of cells resistant to topoisomerase poisons.
  • To facilitate diagnostic screening for hereditary ataxias associated with TDP1 mutations.

Main Methods:

  • Development of two fluorescence-based biochemical assays: Gyrasol and gel-shift assays.
  • Assays measure TDP1 phosphodiesterase activity in cellular lysates.
  • Validation of assays for high-throughput screening and diagnostic purposes.

Main Results:

  • The Gyrasol assay is sensitive and high-throughput, suitable for screening TDP1 inhibitors and resistant cell lines.
  • The gel-shift assay is cost-effective and simple, useful for screening resistant cell lines and diagnostic purposes.
  • Both assays effectively measure TDP1 activity, supporting their utility in various research and clinical settings.

Conclusions:

  • The developed Gyrasol and gel-shift assays provide valuable tools for studying TDP1 function and its clinical relevance.
  • These assays can accelerate the discovery of new cancer therapeutics and improve the diagnosis of TDP1-related disorders.
  • The assays are applicable to screening for TDP1 inhibitors, identifying drug-resistant tumor cells, and diagnosing hereditary ataxias.