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

Abstract

Topoisomerases 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.