Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4255, USA.
Nucleic Acids Res. 2010 Apr;38(7):2444-52. doi: 10.1093/nar/gkp1206. Epub 2010 Jan 21.
Human tyrosyl-DNA phosphodiesterase (Tdp1) processes 3'-blocking lesions, predominantly 3'-phosphotyrosyl bonds resulting from the trapping of topoisomerase I (Top1) cleavage complexes. The controversial ability of yeast Tdp1 to hydrolyze 5'-phosphotyrosyl linkage between topoisomerase II (Top2) and DNA raises the question whether human Tdp1 possesses 5'-end processing activity. Here we characterize the end-binding and cleavage preference of human Tdp1 using single-stranded 5'- and 3'-fluorescein-labeled oligonucleotides. We establish 3'-fluorescein as an efficient surrogate substrate for human Tdp1, provided it is attached to the DNA by a phosphodiester (but not a phosphorothioate) linkage. We demonstrate that human Tdp1 lacks the ability to hydrolyze a phosphodiester linked 5'-fluorescein. Using both fluorescence anisotropy and time-resolved fluorescence quenching techniques, we also show the preferential binding of human Tdp1 to the 3'-end. However, DNA binding competition experiments indicate that human Tdp1 binding is dependent on DNA length rather than number of DNA ends. Lastly, using surface plasmon resonance, we show that human Tdp1 selectively binds the 3'-end of DNA. Together, our results suggest human Tdp1 may act using a scanning mechanism, in which Tdp1 bind non-specifically upstream of a 3'-blocking lesion and is preferentially stabilized at 3'-DNA ends corresponding to its site of action.
人类酪氨酸-DNA 磷酸二酯酶（Tdp1）可处理 3'-阻断性损伤，主要是拓扑异构酶 I（Top1）切割复合物捕获导致的 3'-磷酸酪氨酸键。酵母 Tdp1 水解拓扑异构酶 II（Top2）与 DNA 之间 5'-磷酸酪氨酸键的争议性能力引发了一个问题，即人类 Tdp1 是否具有 5'-末端加工活性。在这里，我们使用单链 5'-和 3'-荧光标记的寡核苷酸来表征人类 Tdp1 的末端结合和切割偏好性。我们建立了 3'-荧光作为人 Tdp1 的有效替代底物，前提是它通过磷酸二酯（而不是硫代磷酸酯）键连接到 DNA 上。我们证明人 Tdp1 缺乏水解磷酸二酯连接的 5'-荧光的能力。使用荧光各向异性和时间分辨荧光猝灭技术，我们还表明人 Tdp1 优先结合 3'-末端。然而，DNA 结合竞争实验表明，人 Tdp1 的结合依赖于 DNA 长度而不是 DNA 末端的数量。最后，使用表面等离子体共振，我们表明人 Tdp1 选择性地结合 DNA 的 3'-末端。总之，我们的结果表明，人 Tdp1 可能使用扫描机制发挥作用，其中 Tdp1 在 3'-阻断性损伤的上游非特异性结合，并优先稳定在与其作用位点相对应的 3'-DNA 末端。
