Martínez-García Belén, Díaz-Ingelmo Ofelia, Ayats-Fraile Alba, Roca Joaquim
DNA Topology Lab, Molecular Biology Institute of Barcelona (IBMB-CSIC), Barcelona, Spain.
Methods Mol Biol. 2025;2881:259-270. doi: 10.1007/978-1-0716-4280-1_13.
DNA supercoiling in biological systems can occur via three mechanisms. The first is by the activity of DNA topoisomerases, such as DNA gyrases, that can increase or reduce the linking number of relaxed DNA (Lk). The second is via DNA translocation motors, such as RNA and DNA polymerases, that produce twin supercoiled DNA domains: one positively supercoiled in front and one negatively supercoiled behind. The third is via molecular interactions that constrain DNA supercoils and thereby produce compensatory unconstrained ones. This chapter describes the use of agarose-gel electrophoresis to detect and quantify the DNA supercoils generated by these mechanisms. Particular emphasis is made on the preparation of a relaxed DNA plasmid as initial substrate that marks the position of Lk for calculating ΔLk.
生物系统中的DNA超螺旋可通过三种机制形成。第一种是通过DNA拓扑异构酶的活性，如DNA促旋酶，它可以增加或减少松弛DNA（Lk）的连环数。第二种是通过DNA易位马达，如RNA和DNA聚合酶，它们产生双超螺旋DNA结构域：一个在前面正向超螺旋，一个在后面负向超螺旋。第三种是通过分子相互作用来约束DNA超螺旋，从而产生补偿性的无约束超螺旋。本章介绍了使用琼脂糖凝胶电泳来检测和定量这些机制产生的DNA超螺旋。特别强调了制备松弛DNA质粒作为初始底物，该底物可标记Lk的位置以计算ΔLk。
