title: Development of a novel assay for human tyrosyl DNA phosphodiesterase 2.
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 (IC(50)=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.
authors.affiliation: Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, USA. sa354@georgetown.edu
authors.forename: Sanjay
authors.initials: S
authors.lastname: Adhikari
authors.forename: Soumendra K
authors.initials: SK
authors.lastname: Karmahapatra
authors.forename: Hadi
authors.initials: H
authors.lastname: Elias
authors.forename: Priyanka
authors.initials: P
authors.lastname: Dhopeshwarkar
authors.forename: R Scott
authors.initials: RS
authors.lastname: Williams
authors.forename: Stephen
authors.initials: S
authors.lastname: Byers
authors.forename: Aykut
authors.initials: A
authors.lastname: Uren
authors.forename: Rabindra
authors.initials: R
authors.lastname: Roy
mesh_terms: D003124:Colorimetry / Q000379:methods*; D004268:DNA-Binding Proteins; D006801:Humans; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases; D013379:Substrate Specificity; D013938:Thymidine Monophosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014638:Vanadates / Q000494:pharmacology
chemical_list: D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D014157:Transcription Factors; D013938:Thymidine Monophosphate; D014638:Vanadates; D010727:Phosphoric Diester Hydrolases; C474866:TDP2 protein, human