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 kinetics 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.

Involvement of the host DNA-repair enzyme TDP2 in formation of the covalently closed circular DNA persistence reservoir of hepatitis B viruses

“…3 F and G), in accord with recent reports (20,26,35). Given the interest in TDP2 inhibitors for cancer therapy (13), we note that our fluorescent TDP2 assay should be easily adaptable to high-throughput applications with potential advantages over assays using low-affinity chromogenic substrates (42,50).…”

Generation of assays and antibodies to facilitate the study of human 5′-tyrosyl DNA phosphodiesterase

“…An additional advantage of NPPP over T5PNP is that it is approximately 10-fold cheaper, thereby making an assay with this substrate much more cost-effective. The apparent K m for NPPP was determined to be 130 mM, which is similar to the value reported for the K m of T5PNP of 54 mM [10]. All three substrates were also tested against the closely related enzymes tyrosyl DNA phosphodiesterase 1 (Tdp1) and AP endonuclease 1 (APE1), and neither could be hydrolyzed by these enzymes (data not shown), indicating that NPPP was a relatively specific substrate for Tdp2.…”

“…Such an approach is unsuitable for an HTS campaign for the identification of novel inhibitors of Tdp2. Recently, a new 96-well plate colorimetric assay for the measurement of Tdp2 enzyme activity that helps to overcome some of these limitations was described [10]. The authors of that study described an assay that uses the chromogenic substrate thymidine 5 0 -monophosphate p-nitrophenyl (T5PNP).…”

An RNA virus hijacks an incognito function of a DNA repair enzyme

“…Second, mass spectrometry analysis of fractions containing VPg unlinkase generated by previous purification protocols had not detected TDP2 (7). Considering that TDP2 is a fast (20) and low abundance enzyme (21), it is likely that protein purity in relation to TDP2 abundance was insufficient for identification in these fractions. Third, the molecular weight of full-length TDP2 did not correlate with any of the molecular weights previously reported for VPg unlinkase [∼27 kDa (3) and 24-30 kDa (22)].…”