Watson M A, Gowers D M, Halford S E
Department of Biochemistry School of Medical Sciences, University of Bristol, Bristol, BS8 1TD, UK.
J Mol Biol. 2000 May 5;298(3):461-75. doi: 10.1006/jmbi.2000.3676.
Many processes are governed by proteins that bind to separate sites in DNA and loop out the intervening DNA, but the geometries of the loops have seldom been determined. The SfiI endonuclease cleaves DNA after interacting with two recognition sites, and is a favourable system for the analysis of DNA looping. A gel-shift assay was used here to examine the binding of SfiI to a series of linear DNA molecules containing two SfiI sites separated by 109-170 base-pairs. The complexes in which SfiI trapped a loop by binding to two sites in the same DNA were separated from the complexes containing SfiI bound to separate DNA molecules. Step-wise changes in the inter-site spacing generated two forms of the looped complex with different electrophoretic mobilities. The yields of each looped complex and the complexes from intermolecular synapses all varied cyclically with the inter-site spacing, with similar periodicities ( approximately 10.5 base-pairs) but with different phases. One looped complex predominated whenever the DNA between the sites needed to be underwound in order to produce the correct helical orientation of the binding sites. The other looped complex predominated whenever the intervening DNA needed to be overwound. We conclude that the former has trapped a right-handed loop with a negative node and the latter a left-handed loop with a positive node.
许多过程由与DNA中不同位点结合并使中间DNA形成环的蛋白质所调控，但环的几何形状很少被确定。SfiI核酸内切酶在与两个识别位点相互作用后切割DNA，是分析DNA环化的一个有利系统。本文使用凝胶迁移实验来检测SfiI与一系列线性DNA分子的结合情况，这些分子含有两个相距109 - 170个碱基对的SfiI位点。通过结合同一DNA上两个位点而捕获环的SfiI复合物，与含有结合在不同DNA分子上的SfiI的复合物分离开来。位点间距的逐步变化产生了两种具有不同电泳迁移率的环化复合物形式。每种环化复合物以及分子间突触形成的复合物的产量都随位点间距呈周期性变化，具有相似的周期（约10.5个碱基对）但相位不同。每当位点之间的DNA需要负超螺旋以产生正确的结合位点螺旋方向时，一种环化复合物占主导。每当中间DNA需要正超螺旋时，另一种环化复合物占主导。我们得出结论，前者捕获了一个带有负节点的右手环，后者捕获了一个带有正节点的左手环。
