Development of a novel assay for human tyrosyl DNA phosphodiesterase 2.
Development of a novel assay for human tyrosyl DNA phosphodiesterase 2.
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DOI：
10.1016/j.ab.2011.05.008
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发表时间：
2011-09-01
影响因子：
2.9
通讯作者：
Roy R
中科院分区：
文献类型：
--
作者：
Adhikari S;Karmahapatra SK;Elias H;Dhopeshwarkar P;Williams RS;Byers S;Uren A;Roy R
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.
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DOI：
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发表时间：
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期刊：
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影响因子：
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影响因子：
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DOI：
10.1073/pnas.0500986102
发表时间：
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影响因子：
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作者：
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