A fluorescence-based assay for the apurinic/apyrimidinic-site cleavage activity of human tyrosyl-DNA phosphodiesterase 1

Graeme J. Thomson, Nicola S. Hamilton, Gemma V. Hopkins, Ian D. Waddell, Amanda J. Watson, Donald J. Ogilvie

Research output: Contribution to journalArticlepeer-review

Abstract

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) catalyzes the hydrolysis of phosphodiester bonds between the DNA 30-phosphate and tyrosine residues and plays a major role in the repair of stalled topoisomerase IDNA covalent complexes. Given this role, Tdp1 is of interest as a potential target for anticancer therapy. Inhibiting Tdp1 in combination with clinically used Top1 inhibitors may potentiate the effects of the latter and help to overcome some of the chemoresistance issues currently observed. In addition, Tdp1 can function during DNA repair to remove a variety of other 30 adducts from DNA such as phosphoglycolates and abasic or apurinic/apyrimidinic (AP) sites. Here we describe a new mix-and-read homogeneous fluorogenic assay for the measurement of the AP-site cleavage activity of Tdp1 that is compatible with highthroughput screening. The application of such an assay will open up further avenues for the discovery of novel Tdp1 inhibitors. © 2013 Elsevier Inc.
Original languageEnglish
Pages (from-to)1-5
Number of pages4
JournalAnalytical Biochemistry
Volume440
Issue number1
DOIs
Publication statusPublished - 2013

Keywords

  • Assay
  • DNA repair
  • High-throughput screen
  • Inhibitor
  • Tdp1
  • Topoisomerase

Research Beacons, Institutes and Platforms

  • Manchester Cancer Research Centre

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