Generation of assays and antibodies to facilitate the study of human 5'-tyrosyl DNA phosphodiesterase.
AuthorsThomson, Graeme J
Watson, Amanda J
Hamilton, Nicola S
Hopkins, Gemma V
Jordan, Allan M
Morrow, Christopher J
Waddell, Ian D
Ogilvie, Donald J
AffiliationCancer Research UK Drug Discovery Unit, Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester, M20 4BX, UK.
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AbstractTopoisomerases regulate DNA topology by the transient cleavage and re-ligation of DNA during transcription and replication. Topoisomerase II (Topo II) poisons such as etoposide can induce abortive DNA strand breaks in which Topo II remains covalently bound to a 5' DNA strand terminus via a phosphotyrosyl linker. Tyrosyl DNA phosphodiesterase 2 (Tdp2) is a recently discovered human 5'-tyrosyl DNA phosphodiesterase which repairs this topoisomerase-mediated DNA damage, thus playing a central role in maintaining normal DNA topology in cells. Cellular depletion of Tdp2 has been shown to result in an increased susceptibility and sensitivity to Topo II-induced DNA double strand breaks, thereby revealing Tdp2 as a potentially attractive anti-cancer target. No drug-like inhibitors of Tdp2 have been identified to date and assays suitable for high throughput screening (HTS) have not been widely reported. Here we have identified a new and effective chromogenic substrate for Tdp2 and developed a homogenous and robust HTS assay. A second novel Tdp2 assay was also developed to cross-validate hit matter identified from an HTS. Additionally, a new and specific Tdp2 antibody is described. Together these new tools will aid in the identification of novel Tdp2 inhibitors and the investigation of the role of Tdp2 in cancer.
CitationGeneration of assays and antibodies to facilitate the study of human 5'-tyrosyl DNA phosphodiesterase. 2013: Anal Biochem
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