Wasserman S A, Cozzarelli N R
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235.
J Biol Chem. 1991 Oct 25;266(30):20567-73.
The mechanism by which the type 2 topoisomerase from bacteriophage T4 mediates knotting of negatively supercoiled DNA was deduced from an analysis of product topology. The knotted products were nicked and then subjected to electrophoresis in order to separate species on the basis of the minimum number of crossings in the knotted form. Knots with defined numbers of crossings were purified and the configuration of these crossings determined in the electron microscope by the RecA coating method. The product knots were exclusively of the twist form, in which an interwound region is entrapped by a single interlock of two looped ends. The interwound region was of negative sign in greater than 98% of the molecules examined, whereas the single interlock was equally likely to be positive or negative. These results are interpreted in terms of a model for knot formation in which random strand passage mediated by the topoisomerase links bent or branched portions of a superhelix that has a specific interwound geometry. Superhelix interwinding and DNA contacts stabilized by excess enzyme molecules explain the very high frequency of knotting.
通过对产物拓扑结构的分析，推导了噬菌体T4的II型拓扑异构酶介导负超螺旋DNA打结的机制。将打结产物切开，然后进行电泳，以便根据打结形式中的最少交叉数分离不同种类。纯化具有确定交叉数的结，并通过RecA包被法在电子显微镜下确定这些交叉的构型。产物结均为扭曲形式，其中一个相互缠绕的区域被两个环状末端的单个互锁所包围。在所检测的分子中，超过98%的分子其相互缠绕区域为负号，而单个互锁为正号或负号的可能性相同。这些结果根据一个打结形成模型进行了解释，在该模型中，由拓扑异构酶介导的随机链通过连接了具有特定相互缠绕几何结构的超螺旋的弯曲或分支部分。由过量酶分子稳定的超螺旋相互缠绕和DNA接触解释了非常高的打结频率。
