Development of a novel assay for human tyrosyl DNA phosphodiesterase 2

Sanjay Adhikari, Soumendra K. Karmahapatra, Hadi Elias, Priyanka Dhopeshwarkar, R. Scott Williams, Stephen Byers, Aykut Uren, Rabindra Roy

Research output: Contribution to journalArticle

16 Scopus citations

Abstract

Tyrosyl DNA phosphodiesterase 2 (TDP2), a newly discovered enzyme that cleaves 5′-phosphotyrosyl bonds, is a potential target for chemotherapy. TDP2 possesses both 3′- and 5′-tyrosyl-DNA phosphodiesterase activity, which is generally measured in a gel-based assay using 3′- and 5′-phosphotyrosyl linkage at the 3′ and 5′ ends of an oligonucleotide. To understand the enzymatic mechanism of this novel enzyme, the gel-based assay is useful, but this technique is cumbersome for TDP2 inhibitor screening. For this reason, we have designed a novel assay using p-nitrophenyl-thymidine-5′-phosphate (T5PNP) as a substrate. This assay can be used in continuous colorimetric assays in a 96-well format. We compared the salt and pH effect on product formation with the colorimetric and gel-based assays and showed that they behave similarly. Steady-state kinetic studies showed that the 5′ activity of TDP2 is 1000-fold more efficient than T5PNP. Tyrosyl DNA phosphodiesterase 1 (TDP1) and human AP-endonuclease 1 (APE1) could not hydrolyze T5PNP. Sodium orthovanadate, a known inhibitor of TDP2, inhibits product formation from T5PNP by TDP2 (IC50 = 40 mM). Our results suggest that this novel assay system with this new TDP2 substrate can be used for inhibitor screening in a high-throughput manner.

Original languageEnglish (US)
Pages (from-to)112-116
Number of pages5
JournalAnalytical Biochemistry
Volume416
Issue number1
DOIs
StatePublished - Sep 1 2011

Keywords

  • DNA repair
  • Enzyme assay
  • High-throughput screening
  • Kinetics
  • TDP2

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology
  • Cell Biology

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