O'Brian Addison K, Hardin Mattalyn R, Thomas Allison J, Latham Brooke D, Deweese Joseph
Department of Biological, Physical, and Human Sciences, Freed-Hardeman University, Henderson, TN, USA.
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, USA.
Methods Mol Biol. 2025;2928:197-204. doi: 10.1007/978-1-0716-4550-5_16.
The interlinking of DNA during replication and other DNA processes is resolved by type II DNA topoisomerases. In humans, topoisomerase IIα is the main enzyme involved in this process using a transient double-stranded DNA break to unlink sister chromatids during and after replication to allow for mitosis. To measure decatenation, a consistent catenated substrate called kinetoplast DNA (kDNA) is commonly used. kDNA is often purified from the trypanosome Crithidia fasciculata. kDNA represents catenated or interlinked circles of DNA. Early work on topoisomerases identified kDNA as a useful substrate for measuring decatenation by DNA topoisomerase II. This protocol will describe a common method for measuring decatenation of kDNA by eukaryotic type II topoisomerases.
DNA在复制及其他DNA过程中的相互连接由II型DNA拓扑异构酶解决。在人类中，拓扑异构酶IIα是参与此过程的主要酶，它利用瞬时双链DNA断裂在复制期间及之后解开姐妹染色单体，以便进行有丝分裂。为了测量解连环作用，通常使用一种称为动质体DNA（kDNA）的一致连环底物。kDNA通常从锥虫克氏锥虫中纯化得到。kDNA代表连环或相互连接的DNA环。早期关于拓扑异构酶的研究将kDNA鉴定为用于测量DNA拓扑异构酶II解连环作用的有用底物。本方案将描述一种用于测量真核生物II型拓扑异构酶对kDNA解连环作用的常用方法。
