Krämer H, Amouyal M, Nordheim A, Müller-Hill B
Institut für Genetik der Universität zu Köln, FRG.
EMBO J. 1988 Feb;7(2):547-56. doi: 10.1002/j.1460-2075.1988.tb02844.x.
We have used a gel retardation assay to investigate the influence of DNA supercoiling on loop formation between lac repressor and two lac operators. A series of 15 DNA minicircles of identical size (452 bp) was constructed carrying two lac operators at distances ranging from 153 to 168 bp. Low positive or negative supercoiling (sigma = +/- 0.023) changed the spacing between the two lac operators required for the formation of the most stable loops. This reveals the presence of altered double helical repeats (ranging from 10.3 to 10.7 bp) in supercoiled DNA minicircles. At elevated negative supercoiling (sigma = -0.046) extremely stable loops were formed at all operator distances tested, with a slight spacing periodicity remaining. After relaxation of minicircle-repressor complexes with topoisomerase I one superhelical turn was found to be constrained in those minicircles which carry operators at distances corresponding to a non-integral number of helical turns. This indicates that DNA loop formation can define local DNA domains with altered topological properties of the DNA helix.
我们使用凝胶阻滞分析法研究了DNA超螺旋对乳糖阻遏物与两个乳糖操纵基因之间环形成的影响。构建了一系列大小相同（452 bp）的15个DNA微环，其中两个乳糖操纵基因的间距在153至168 bp之间。低正超螺旋或负超螺旋（σ = +/- 0.023）改变了形成最稳定环所需的两个乳糖操纵基因之间的间距。这揭示了超螺旋DNA微环中存在改变的双螺旋重复序列（范围从10.3至10.7 bp）。在较高的负超螺旋（σ = -0.046）下，在所有测试的操纵基因间距处都形成了极其稳定的环，仍保留有轻微的间距周期性。用拓扑异构酶I使微环 - 阻遏物复合物松弛后，发现在那些携带操纵基因间距对应于非整数螺旋匝数的微环中，有一个超螺旋匝数受到限制。这表明DNA环的形成可以定义具有改变的DNA螺旋拓扑性质的局部DNA结构域。
