Tanaka H, Yasuzawa K, Kohno K, Goshima N, Kano Y, Saiki T, Imamoto F
Department of Molecular Genetics, Kyoto Pharmaceutical University, Japan.
Mol Gen Genet. 1995 Sep 20;248(5):518-26. doi: 10.1007/BF02423446.
Induction of supercoiling in plasmid DNA by HU heterotypic and homotypic dimers, a mutant HU-2 (HupAN12), HBs and HB1 proteins with different DNA-binding affinities was investigated in vitro. The abilities of these proteins to induce supercoiling in DNA correlated with their affinities for DNA. Stoichiometrical analysis of HU heterodimers bound to DNA in the complex restraining the negative torsional tension of DNA showed that 12-13 dimers account for a single superhelical turn. The number of supercoils in the plasmid in vivo decreased on inhibition of DNA gyrase with coumermycin, reaching a steady-state level that indicated the existence of a compartment of restrained supercoils. The size of the restrained compartment was reduced in the absence of HU, indicating the participation of HU in constituting this fraction, and was larger on overproduction of HU-2 in the cells. An increased level of DNA gyrase, expressed from a plasmid carrying both gyr genes, in the cells did not compensate for the deficit of the restrained supercoils caused by HU deficiency, indicating seeming distinct and unrelated action of HU and DNA gyrase in introducing and constraining supercoiling of intracellular DNA.
在体外研究了HU异型和同型二聚体、突变体HU-2（HupAN12）、HBs和具有不同DNA结合亲和力的HB1蛋白对质粒DNA超螺旋的诱导作用。这些蛋白诱导DNA超螺旋的能力与其对DNA的亲和力相关。对结合在抑制DNA负扭转张力的复合物中的DNA上的HU异二聚体进行化学计量分析表明，12 - 13个二聚体构成一个超螺旋圈。用香豆霉素抑制DNA回旋酶后，体内质粒中的超螺旋数量减少，达到一个稳态水平，这表明存在一个受抑制超螺旋的区室。在没有HU的情况下，受抑制区室的大小减小，表明HU参与构成这一部分，并且在细胞中过量表达HU-2时该区域更大。从携带两个回旋酶基因的质粒表达的细胞中DNA回旋酶水平升高，不能弥补由HU缺乏引起的受抑制超螺旋的不足，这表明在引入和限制细胞内DNA超螺旋方面，HU和DNA回旋酶的作用似乎是不同且不相关的。
