Energetics and affinity of the histone octamer for defined DNA sequences
Energetics and affinity of the histone octamer for defined DNA sequences
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DOI：
10.1021/bi0109966
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发表时间：
2001-09-18
期刊：
影响因子：
2.9
通讯作者：
Luger, K
中科院分区：
文献类型：
--
作者：
Gottesfeld, JM;Luger, K
以前的研究比较了组蛋白八聚体结合各种DNA序列的相对自由能，但是，没有报告的八聚体的平衡结合亲和力的独特序列已提交。已经表明，核小体核心颗粒（NCP）在稀释时解离成游离DNA和历史八聚体（或游离组蛋白），而不产生稳定的中间体。解离是可逆的，并且NCP和DNA的平衡分布迅速达到。在低离子强度条件下（< 400 mM NaCl），NCP解离遵循质量作用定律，使得可以计算在定义的DNA序列上重构的NCP的表观平衡解离常数（K（d）s）。我们已经使用了两个DNA序列，这两个DNA序列以前曾作为核小体重建研究的模型系统，即人α-卫星DNA和杂色石松5S DNA，并发现在50 mM NaCl下，八聚体对这些序列分别表现出0.03和0.06 nM的Kd（s）。这些DNA形成的NCP比从细胞染色质中分离的总NCP稳定约2kcal/mol。至于混合序列NCP，增加离子强度或温度促进解离。K（α）对温度的van 't霍夫图显示，与本体NCP相比，λ-卫星和5S NCP的结合自由能的差异几乎完全是由于与混合序列NCP相比，在独特序列上形成的NCP的更有利的熵分量。此外，我们通过使用重组无尾组蛋白来解决组蛋白H3和H4的氨基末端尾结构域对八聚体亲和力的贡献。
Previous studies have compared the relative free energies for histone octamer binding to various DNA sequences; however, no reports of the equilibrium binding affinity of the octamer for unique sequences have been presented. It has been shown that nucleosome core particles (NCPs) dissociate into free DNA and historic octamers (or free histones) on dilution without generation of stable intermediates. Dissociation is reversible, and an equilibrium distribution of NCPs and DNA is rapidly attained. Under low ionic strength conditions (< 400 mM NaCl), NCP dissociation obeys the law of mass action, making it possible to calculate apparent equilibrium dissociation constants (K(d)s) for NCPs reconstituted on defined DNA sequences. We have used two DNA sequences that have previously served as model systems for nucleosome reconstitution studies, human alpha -satellite DNA and Lytechinus variegatus 5S DNA, and find that the octamer exhibits Kd(s) of 0.03 and 0.06 nM, respectively, for these sequences at 50 mM NaCl. These DNAs form NCPs that are similar to2 kcal/mol more stable than total NCPs isolated from cellular chromatin. As for mixed sequence NCPs, increasing ionic strength or temperature promotes dissociation. van't Hoff plots of K(a)s versus temperature reveal that the difference in binding free energy for lambda -satellite and 5S NCPs compared to bulk NCPs is due almost entirely to a more favorable entropic component for NCPs formed on the unique sequences compared to mixed-sequence NCPs. Additionally, we address the contribution of the amino-terminal tail domains of histones H3 and H4 to octamer affinity through the use of recombinant tailless histones.