Generation of assays and antibodies to facilitate the study of human 5′-tyrosyl DNA phosphodiesterase
Generation of assays and antibodies to facilitate the study of human 5′-tyrosyl DNA phosphodiesterase
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DOI：
10.1016/j.ab.2013.02.001
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发表时间：
2013-05-15
影响因子：
2.9
通讯作者：
Ogilvie, Donald
中科院分区：
文献类型：
--
作者：
Thomson, Graeme;Watson, Amanda;Ogilvie, Donald
拓扑异构酶通过在转录和复制期间DNA的瞬时切割和重新连接来调节DNA拓扑结构。拓扑异构酶II（Topo II）毒物如依托泊苷可诱导失败的DNA链断裂，其中Topo II通过磷酸酪氨酰接头保持与5' DNA链末端共价结合。酪氨酰DNA磷酸二酯酶2（Tdp 2）是最近发现的人5 '-酪氨酰DNA磷酸二酯酶，其修复这种拓扑异构酶介导的DNA损伤，从而在维持细胞中正常DNA拓扑结构中发挥核心作用。Tdp 2的细胞耗竭已显示导致对Topo II诱导的DNA双链断裂的易感性和敏感性增加，从而揭示Tdp 2作为潜在的有吸引力的抗癌靶点。迄今为止，尚未鉴定出Tdp 2的药物样抑制剂，并且适用于高通量筛选（HTS）的测定尚未被广泛报道。在这里，我们已经确定了一个新的和有效的显色底物Tdp 2和开发一个均匀的和强大的HTS检测。还开发了第二种新的Tdp 2测定以交叉验证从HTS鉴定的命中物质。此外，还描述了一种新的特异性Tdp 2抗体。总之，这些新工具将有助于鉴定新型Tdp 2抑制剂和研究Tdp 2在癌症中的作用。(C)2013 Elsevier Inc. All rights reserved.
Topoisomerases regulate DNA topology by the transient cleavage and religation 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 that repairs this topoisomerase-mediated DNA damage, thereby playing a central role in maintaining normal DNA topology in cells. Cellular depletion of Tdp2 has been shown to result in increased susceptibility and sensitivity to Topo II-induced DNA double-strand breaks, thereby revealing Tdp2 as a potentially attractive anticancer 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 homogeneous and robust HTS assay. A second novel Tdp2 assay was also developed to cross-validate hit matter identified from an HTS. In addition, 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. (C) 2013 Elsevier Inc. All rights reserved.