Peterson Sharon, Danowit Rachel, Wunsch Ann, Jackson Vaughn
Department of Biochemistry, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, Wisconsin 53226, USA.
Biochemistry. 2007 Jul 24;46(29):8634-46. doi: 10.1021/bi6025215. Epub 2007 Jun 27.
We have studied the tetramer-dimer equilibrium of histones H3/H4 and its effect on DNA supercoiling. Two approaches were found to shift the equilibrium toward dimer. In both instances, when deposited on DNA, the dimers formed positively coiled DNA. The first approach was to modify cysteine 110 of H3 with 5,5'-dithio-bis(2-nitrobenzoic acid (DTNB) and to directly add the histones to DNA at physiological ionic strength. The second approach involved adding an excess of the histone chaperone, nucleosome assembly protein 1 (NAP1) to the H3/H4 prior to deposition on the DNA. It was also observed that when H3/H4 were deposited in the tetrameric state, negatively coiled DNA was formed. The topological state of the DNA prior to deposition was also found to influence the final conformational state of H3/H4. It is proposed that in the tetrameric state, the H3-H3 interface has a left-handed pitch prior to binding DNA. In the dimeric state, the H3-H3 interface is not established until bound to DNA, at which point either the left or right-handed pitch will form on the basis of the initial topology of the DNA. Formaldehyde cross-linking and reversal were applied to identify the histone-histone interactions that facilitate the formation of positive stress. Higher-order interactions between multiple H3/H4 dimers were required to propagate this specific conformation. Changes in the conformational state of H3/H4 were also observed when the histones were bound to DNA prior to treatment with NAP1. It is proposed that these conformational changes in H3/H4 are involved in promoter activation and transcription elongation through nucleosomes.
我们研究了组蛋白H3/H4的四聚体-二聚体平衡及其对DNA超螺旋的影响。发现有两种方法可使平衡向二聚体方向移动。在这两种情况下，当沉积在DNA上时，二聚体形成正超螺旋DNA。第一种方法是用5,5'-二硫代双(2-硝基苯甲酸)(DTNB)修饰H3的半胱氨酸110，并在生理离子强度下将组蛋白直接添加到DNA中。第二种方法是在沉积到DNA之前，向H3/H4中加入过量的组蛋白伴侣核小体组装蛋白1(NAP1)。还观察到，当H3/H4以四聚体状态沉积时，会形成负超螺旋DNA。还发现沉积前DNA的拓扑状态会影响H3/H4的最终构象状态。有人提出，在四聚体状态下，H3-H3界面在结合DNA之前具有左手螺距。在二聚体状态下，H3-H3界面直到与DNA结合时才形成，此时根据DNA的初始拓扑结构会形成左手或右手螺距。应用甲醛交联和逆转来鉴定促进正应力形成的组蛋白-组蛋白相互作用。多个H3/H4二聚体之间的高阶相互作用是传播这种特定构象所必需的。在用NAP1处理之前，当组蛋白与DNA结合时，也观察到了H3/H4构象状态的变化。有人提出，H3/H4的这些构象变化参与了通过核小体的启动子激活和转录延伸。
