Nucleosome dynamics IV.: Protein and DNA contributions in the chiral transition of the tetrasome, the histone (H3-H4)2 tetramer-DNA particle
Nucleosome dynamics IV.: Protein and DNA contributions in the chiral transition of the tetrasome, the histone (H3-H4)2 tetramer-DNA particle
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DOI：
10.1006/jmbi.1999.2988
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发表时间：
1999-08-27
影响因子：
5.6
通讯作者：
Prunell, A
中科院分区：
文献类型：
--
作者：
Alilat, M;Sivolob, A;Prunell, A
我们的实验室先前已经报道了DNA微环重构四体（由DNA包裹在组蛋白（H3-H4）（2）四聚体周围组成的颗粒）的手性转变。这种转变是由DNA的正扭转约束，产生的初始超螺旋的循环或拓扑异构酶松弛过程中的热波动。考虑到DNA在组蛋白周围的缠绕小于一圈，并且在入口-出口处负交叉，过渡被提出涉及围绕粒子二分体轴的环的360度旋转，以及正交叉的形成。八聚体内的四聚体马蹄形构象进一步表明，这一过程可以通过两个扇形H3-H4二聚体围绕其H3/H3界面的重新取向来介导，这将使蛋白质超螺旋的整体手性从左手变为右手。现在，我们提供了额外的证据，这样的贡献的蛋白质显示，通过凝胶电泳，拓扑异构酶松弛和电子显微镜，在H3/H3界面的空间位阻，通过巯基氧化H3半胱氨酸110的大体积加合物的共价连接引入，干扰过渡。这种干扰根据所用的特定SH试剂而变化;但最显著的效果是用5，5 '-二硫代双（2-硝基苯甲酸）（DTNB）获得的，其将四体的优选构象从左手置换为半右手，同时保留了显著程度的手性灵活性。DNA的贡献证明了一个特定的分馏的圆形四体在凝胶电泳，连同不同的定位控制和DTNB四体上的线性DNA，指出四体构象和位置之间的相互依赖性。此外，线性四体在盐依赖性平衡中在交叉和非交叉构象之间波动，这似乎随它们在DNA上的位置而变化。这些数据表明，在过渡中的二分体周围的DNA的可调节的作用，主要取决于其序列依赖的变形性。这种作用在H3-H4二聚体重定向和侧向开放两个水平上都起作用，通过这种机制，颗粒可以减轻其进入和退出DNA之间的冲突。这些特性使得四体成为体内核小体动力学中有吸引力的潜在中间体，特别是在X转录激活和延伸期间。(C)北京：科学出版社.
Our laboratory has previously reported the chiral transition of DNA minicircle-reconstituted tetrasomes (the particles made of DNA wrapped around the histone (H3-H4)(2) tetramer). This transition was induced by DNA positive torsional constraint, generated either by initial supercoiling of the loop or by its thermal fluctuations during topoisomerase relaxation. Taking into account the wrapping of the DNA around the histones into less than a turn, and its negative crossing at the entry-exit, the transition was proposed to involve a 360 degrees rotation of the loop around the particle dyad axis, and the formation of a positive crossing. The tetramer horseshoe-shaped conformation within the octamer further suggested that this process could be mediated by a reorientation of the two sector-like H3-H4 dimers about their H3/H3 interface, which would switch the overall handedness of the proteinaceous superhelix from left to right-handed. We now provide additional evidence for such a contribution of the protein by showing, through gel electrophoresis, topoisomerase relaxation and electron microscopy, that a sterical hindrance at the H3/H3 interface, introduced by covalent linking of bulky adducts through thiol oxidation of H3 cysteine 110, interferes with the transition. Such interference varies, depending on the particular SH-reagent used; but the most remarkable effect was obtained with 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB), which displaces the preferred conformation of the tetrasomes from left-handed to semi-right-handed, and at the same time preserves a significant degree of chiral flexibility. DNA contribution was evidenced by a specific fractionation of circular tetrasomes in gel electrophoresis which, together with a different positioning of control and DTNB tetrasomes on linear DNA, pointed to an interdependence between tetrasome conformation and positions. Moreover, linear tetrasomes fluctuate between crossed and uncrossed conformations in a salt-dependent equilibrium which appears to vary with their positions on the DNA. These data suggest a modulatable role of the DNA around the dyad in the transition, depending primarily on its sequence-dependent deformability. This role is played at both levels of H3-H4 dimer reorientation and lateral opening, a mechanism by which the particle may relieve the clash between its entering and exiting DNAs. These properties make the tetrasome an attractive potential intermediate in nucleosome dynamics in vivo, in particular duringX transcriptional activation and elongation. (C) 1999 Academic Press.