Alilat M, Sivolob A, Révet B, Prunell A
Institut Jacques Monod, Centre National de la Recherche Scientifique, et Université Denis Diderot Paris 7, 2 place Jussieu, Paris, Cédex 05, 75251, France.
J Mol Biol. 1999 Aug 27;291(4):815-41. doi: 10.1006/jmbi.1999.2988.
Our laboratory has previously reported the chiral transition of DNA minicircle-reconstituted tetrasomes (the particles made of DNA wrapped around the histone (H3-H4)2tetramer). 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.
我们实验室之前报道过DNA微环重构四聚体（由缠绕在组蛋白(H3-H4)2四聚体上的DNA构成的颗粒）的手性转变。这种转变是由DNA正扭转约束诱导的，该约束可通过环的初始超螺旋或拓扑异构酶松弛过程中的热波动产生。考虑到DNA缠绕在组蛋白上不足一圈，且在进出口处有负交叉，有人提出这种转变涉及环围绕颗粒二分轴旋转360度，并形成一个正交叉。八聚体内的四聚体马蹄形构象进一步表明，这一过程可能由两个扇形H3-H4二聚体围绕其H3/H3界面重新定向介导，这将把蛋白质超螺旋的整体手性从左旋切换为右旋。我们现在通过凝胶电泳、拓扑异构酶松弛和电子显微镜显示，通过H3半胱氨酸110的硫醇氧化将庞大加合物共价连接引入H3/H3界面的空间位阻会干扰这种转变，从而为蛋白质的这种作用提供了额外证据。这种干扰因所用的特定硫醇试剂而异；但使用5,5'-二硫代双(2-硝基苯甲酸)(DTNB)获得了最显著的效果，它将四聚体的首选构象从左旋转变为半右旋，同时保留了相当程度的手性灵活性。凝胶电泳中环状四聚体的特定分级分离证明了DNA的作用，这与对照和DTNB四聚体在线性DNA上的不同定位一起，表明四聚体构象和位置之间存在相互依赖性。此外，线性四聚体在交叉和非交叉构象之间以盐依赖性平衡波动，这种平衡似乎随它们在DNA上的位置而变化。这些数据表明，二分体周围的DNA在转变中起可调节的作用，主要取决于其序列依赖性可变形性。这种作用在H3-H4二聚体重新定向和侧向打开这两个层面上发挥，通过这种机制颗粒可以缓解其进出DNA之间的冲突。这些特性使四聚体成为体内核小体动力学中一个有吸引力的潜在中间体，特别是在X转录激活和延伸过程中。
