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首页> 外文期刊>Journal of Molecular Biology >Nucleosome dynamics V. Ethidium bromide versus histone tails in modulating ethidium bromide-driven tetrasome chiral transition. A fluorescence study of tetrasomes on DNA minicircles.
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Nucleosome dynamics V. Ethidium bromide versus histone tails in modulating ethidium bromide-driven tetrasome chiral transition. A fluorescence study of tetrasomes on DNA minicircles.

机译：核仁动力学V.溴乙锭与组蛋白尾巴在调节溴乙锭驱动的四体手性过渡中的作用。 DNA小环上的四体体的荧光研究。

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Protein and DNA contributions in the chiral transition of DNA minicircle-reconstituted tetrasomes (the particles made of DNA wrapped around the histone (H3-H4)(2) tetramer) to a right-handed conformation have been investigated in a recent article from this laboratory. As the evidence for a protein contribution, a sterical hindrance introduced at the H3/H3 interface of the two constituent H3-H4 dimers by oxidation of H3 cysteine 110 blocked the tetramer in a half-left-handed or semi-right-handed conformation, depending on the SH-reagent used. The DNA contributed at the level of the dyad region, which appeared to act through its sequence-dependent deformability in modulating both the loop threshold positive constraint required to trigger the transition, and the tetrasome lateral opening. This opening, which electron microscopic visualizations directly showed to be associated with the transition, is expected to help remove the clash between the entering and exiting DNAs. In this work, the transition mechanism was further investigated by applying a positive constraint in the loop through ethidium bromide (EtBr) intercalation. This technique, including the determination of binding isotherms, has first been used with mononucleosomes on DNA minicircles, and has revealed that these particles could tolerate large positive supercoilings without disruption, owing to the loop ability to cross positively in a histone tail-dependent manner. The transition of 359 bp tetrasomes was found to go to completion in lower salt (10 mM), but not in higher salt (100 mM), whereas the transition of 256 bp tetrasomes was already hindered in lower salt. Histone acetylation relieved that lower salt hindrance but enhanced the higher salt hindrances. These data again pointed to the DNA in the dyad region as a regulator of the transition. The block was indeed expected to originate from a local EtBr intercalation in that DNA, which opposed its overtwisting during the transition. The occurrence of the block, or its relief, then depended on the outcome of the competition between the tails and EtBr for binding to that region, that is, on whether the tails could prevent EtBr intercalation before the ongoing transition hampered both bindings. Destabilization of the tails in the course of the transition is documented in an accompanying article through a relaxation study of a 351-366 bp tetrasome series. Copyright 2000 Academic Press.
机译：该实验室在最近的一篇文章中研究了蛋白质和DNA在DNA微小环重构四体（由包裹在组蛋白（H3-H4）（2）四聚体上的DNA制成的颗粒）向右手构型的手性过渡中的贡献。 。作为蛋白质贡献的证据，由于H3半胱氨酸110的氧化而在两个H3-H4组成二聚体的H3 / H3界面处引入了空间位阻，从而阻碍了四聚体的半左手或半右手构型，取决于所用的SH试剂。 DNA在二分体区域的水平上起作用，该二分体区域似乎通过其依赖序列的可变形性来调节触发转变所需的环阈正向约束和四倍体侧向开放。电子显微镜可视化直接显示出与过渡有关的这一开口，有望帮助消除进入和退出DNA之间的冲突。在这项工作中，通过在溴化乙锭（EtBr）插层的环中施加正约束进一步研究了过渡机制。这项技术，包括确定结合等温线，已首先与DNA小环上的单核小体一起使用，并已揭示出这些颗粒由于环能够以组蛋白尾巴依赖性方式阳性交叉，因此可以耐受较大的正超螺旋而不破坏。在低盐（10 mM）中发现359 bp四体的转变已经完成，而在高盐（100 mM）中没有完成，而在低盐中256 bp四体的转变已经受到阻碍。组蛋白乙酰化减轻了较低的盐障碍，但增加了较高的盐障碍。这些数据再次指出，二分体区域中的DNA是过渡的调节剂。确实预期该嵌段来自该DNA中的局部EtBr插入，其在过渡期间反对其过度扭曲。然后，阻滞的发生或其缓解取决于尾巴与EtBr之间竞争与该区域结合的结果，即取决于尾巴是否能够阻止EtBr嵌入，然后正在进行的过渡阻碍了两种结合。通过对351-366 bp四体序列的松弛研究，在随附的文章中记录了过渡过程中尾巴的失稳。版权所有2000学术出版社。

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