Cleavage of plasmid DNA by eukaryotic topoisomerase II.
Cleavage of plasmid DNA by eukaryotic topoisomerase II.
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DOI：
10.1007/978-1-60761-340-4_4
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发表时间：
2009
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影响因子：
--
通讯作者：
Osheroff, Neil
中科院分区：
文献类型：
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作者：
Bandele, Omari J;Osheroff, Neil
拓扑异构酶II是一种必需的酶，是许多关键的核过程所需的。拓扑异构酶II的所有催化功能都需要该酶在双螺旋骨架中产生短暂的双链断裂。为了在切割过程中保持基因组的完整性，拓扑异构酶II在活性位点酪氨酰残基和新产生的5′-DNA末端之间形成共价键。除了II型酶的关键细胞功能外，几种重要的抗癌药物通过增加共价拓扑异构酶II-DNA切割复合物的水平来杀死细胞。由于拓扑异构酶II的生理重要性及其在癌症化疗中的作用，已经开发了几种方法来监测II型酶的体外DNA切割活性。本章描述的基于质粒的系统通过监测共价闭合的超螺旋DNA向线性分子的转化来定量酶介导的双链DNA切割。该测定方法简单、直接，不需要使用放射性标记底物。
Topoisomerase II is an essential enzyme that is required for a number of critical nuclear processes. All of the catalytic functions of topoisomerase II require the enzyme to generate a transient double-stranded break in the backbone of the double helix. To maintain genomic integrity during the cleavage event, topoisomerase II forms covalent bonds between active site tyrosyl residues and the newly generated 5′-DNA termini. In addition to the critical cellular functions of the type II enzyme, several important anticancer drugs kill cells by increasing levels of covalent topoisomerase II-DNA cleavage complexes. Due to the physiological importance of topoisomerase II and its role in cancer chemotherapy, several methods have been developed to monitor the in vitro DNA cleavage activity of the type II enzyme. The plasmid-based system described in this chapter quantifies enzyme-mediated double-stranded DNA cleavage by monitoring the conversion of covalently-closed supercoiled DNA to linear molecules. The assay is simple, straightforward, and does not require the use of radiolabeled substrates.