Schieferstein U, Thoma F
Institut für Zellbiologie, ETH-Hönggerberg, Zürich, Switzerland.
Biochemistry. 1996 Jun 18;35(24):7705-14. doi: 10.1021/bi953011r.
We have used a defined-sequence nucleosome to concomitantly investigate the generation and location of DNA lesions in nucleosomes and their influence on nucleosome positioning (translational and rotational setting). A 134 bp HISAT sequence from the yeast DED1 promoter, containing a polypyrimidine region (40 bp) with a T6-tract, two T5-tracts, and a T9-tract, was reconstituted in nucleosomes with a defined rotational setting. T-tracts adopt unusually rigid DNA structures in solution ("T-tract structure") and are hot spots of cyclobutane pyrimidine dimer (CPD) formation by UV light (254 nm). DNA was irradiated with UV light before or after reconstitution. The CPD yields and distribution were analyzed by cleavage with T4 endonuclease V. The rotational setting of nucleosomal DNA was characterized by DNase I digestion. With the exception of one T5-tract (1T5), the T6-, the 2T5-, and the T9-tracts formed T-tract structure in solution. T-tract structure was lost upon folding in nucleosomes, demonstrating a dominant constraint of DNA folding in nucleosomes over that of T-tract structure. CPD formation was strongly modulated by the nucleosome structure, but the CPD distribution differed from that reported for mixed-sequence DNA. CPD formation in the nucleosome had no effect on the rotational setting of nucleosomal DNA, but the rotational setting was affected when nucleosomes were assembled on damaged DNA. The toleration of DNA distortions imposed by CPDs in nucleosomes may have important implications for the recognition and repair of these damages in chromatin.
我们使用了一个定义序列的核小体来同时研究核小体中DNA损伤的产生和位置及其对核小体定位（平移和旋转设置）的影响。来自酵母DED1启动子的134 bp HISAT序列，包含一个带有T6序列、两个T5序列和一个T9序列的聚嘧啶区域（40 bp），在具有确定旋转设置的核小体中进行了重构。T序列在溶液中呈现出异常刚性的DNA结构（“T序列结构”），并且是紫外线（254 nm）诱导环丁烷嘧啶二聚体（CPD）形成的热点。DNA在重构之前或之后用紫外线照射。通过用T4内切核酸酶V切割来分析CPD的产量和分布。通过DNase I消化来表征核小体DNA的旋转设置。除了一个T5序列（1T5）外，T6序列、2T5序列和T9序列在溶液中形成了T序列结构。在折叠成核小体时T序列结构消失，这表明核小体中DNA折叠对T序列结构具有主导性限制。CPD的形成受到核小体结构的强烈调节，但CPD的分布与混合序列DNA的报道不同。核小体中CPD的形成对核小体DNA的旋转设置没有影响，但当在受损DNA上组装核小体时旋转设置会受到影响。核小体中CPD对DNA扭曲的耐受性可能对染色质中这些损伤的识别和修复具有重要意义。
