Duband-Goulet I, Carot V, Ulyanov A V, Douc-Rasy S, Prunell A
Institut Jaques Monod, Centre National de la Recherche Scientifique and Université, Paris, France.
J Mol Biol. 1992 Apr 20;224(4):981-1001. doi: 10.1016/0022-2836(92)90464-u.
Nucleosome formation on inverted repeats or on some alternations of purines and pyrimidines can be inhibited in vitro by DNA supercoiling through their supercoiling-induced structural transitions to cruciforms or Z-form DNA, respectively. We report here, as a result of study of single nucleosome reconstitutions on a DNA minicircle, that a physiological level of DNA supercoiling can also enhance nucleosome sequence preference. The 357 base-pair minicircle was composed of a promoter of phage SP6 RNA polymerase joined to a 256 base-pair fragment containing a sea urchin 5 S RNA gene. Nucleosome formation on the promoter was found to be enhanced on a topoisomer with in vivo superhelix density when compared to topoisomers of lower or higher superhelical densities, to the nicked circle, or to the linear DNA. In contrast, nucleosomes at other positions appeared to be insensitive to supercoiling. This observation relied on a novel procedure for the investigation of nucleosome positioning. The reconstituted circular chromatin was first linearized using a restriction endonuclease, and the linear chromatin so obtained was electrophoresed as nucleoprotein in a polyacrylamide gel. The gel showed well-fractionated bands whose mobilities were a V-like function of nucleosome positions, with the nucleosome near the middle migrating less. This behavior is similar to that previously observed for complexes of sequence-specific DNA-bending proteins with circularly permuted DNA fragments, and presumably reflects the change in the direction of the DNA axis between the entrance and the exit of the particle. Possible mechanisms for such supercoiling-induced modulation of nucleosome formation are discussed in the light of the supercoiling-dependent susceptibility to cleavage of the naked minicircle with S1 and Bal31 nucleases; and a comparison between DNase I cleavage patterns of the modulated nucleosome and of another, non-modulated, overlapping nucleosome.
在体外，通过超螺旋诱导的结构转变分别形成十字形或Z型DNA，反向重复序列或嘌呤与嘧啶的某些交替序列上的核小体形成可被DNA超螺旋抑制。我们在此报告，通过对DNA微环上的单核小体重组进行研究发现，生理水平的DNA超螺旋也可增强核小体序列偏好性。这个357个碱基对的微环由噬菌体SP6 RNA聚合酶的启动子与一个包含海胆5S RNA基因的256个碱基对的片段连接而成。与超螺旋密度较低或较高的拓扑异构体、切口环或线性DNA相比，发现在具有体内超螺旋密度的拓扑异构体上，启动子上的核小体形成增强。相反，其他位置的核小体似乎对超螺旋不敏感。这一观察结果依赖于一种研究核小体定位的新方法。首先使用限制性内切酶将重组的环状染色质线性化，然后将如此获得的线性染色质作为核蛋白在聚丙烯酰胺凝胶中进行电泳。凝胶显示出分离良好的条带，其迁移率是核小体位置的V型函数，中间附近的核小体迁移较少。这种行为类似于先前观察到的序列特异性DNA弯曲蛋白与环形排列的DNA片段的复合物的行为，大概反映了颗粒入口和出口之间DNA轴方向的变化。根据超螺旋依赖性对用S1和Bal31核酸酶切割裸微环的敏感性，以及对受调控的核小体和另一个未受调控的重叠核小体的DNase I切割模式的比较，讨论了这种超螺旋诱导的核小体形成调节的可能机制。
