Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, van der Maasweg 9, 2629 HZ Delft, The Netherlands.
Institute of Molecular Biology, Biocenter, Medical University of Innsbruck, Innrain 80-82, 6020 Innsbruck, Austria.
Biophys J. 2019 Dec 3;117(11):2217-2227. doi: 10.1016/j.bpj.2019.07.055. Epub 2019 Aug 21.
Eukaryotic genomes are hierarchically organized into protein-DNA assemblies for compaction into the nucleus. Nucleosomes, with the (H3-H4) tetrasome as a likely intermediate, are highly dynamic in nature by way of several different mechanisms. We have recently shown that tetrasomes spontaneously change the direction of their DNA wrapping between left- and right-handed conformations, which may prevent torque buildup in chromatin during active transcription or replication. DNA sequence has been shown to strongly affect nucleosome positioning throughout chromatin. It is not known, however, whether DNA sequence also impacts the dynamic properties of tetrasomes. To address this question, we examined tetrasomes assembled on a high-affinity DNA sequence using freely orbiting magnetic tweezers. In this context, we also studied the effects of mono- and divalent salts on the flipping dynamics. We found that neither DNA sequence nor altered buffer conditions affect overall tetrasome structure. In contrast, tetrasomes bound to high-affinity DNA sequences showed significantly altered flipping kinetics, predominantly via a reduction in the lifetime of the canonical state of left-handed wrapping. Increased mono- and divalent salt concentrations counteracted this behavior. Thus, our study indicates that high-affinity DNA sequences impact not only the positioning of the nucleosome but that they also endow the subnucleosomal tetrasome with enhanced conformational plasticity. This may provide a means to prevent histone loss upon exposure to torsional stress, thereby contributing to the integrity of chromatin at high-affinity sites.
真核生物基因组通过多种不同的机制，在蛋白质-DNA 复合物的水平上被高度组织化，以便压缩进细胞核内。核小体及其（H3-H4）四聚体可能作为中间体，具有高度的动态性。我们最近发现，四聚体可以自发地在左手和右手构象之间改变其 DNA 缠绕的方向，这可能有助于在活跃的转录或复制过程中防止染色质中产生扭矩。已有研究表明，DNA 序列强烈影响整个染色质中核小体的定位。然而，目前尚不清楚 DNA 序列是否也会影响四聚体的动态特性。为了解决这个问题，我们使用自由旋转的磁镊研究了在高亲和力 DNA 序列上组装的四聚体。在这种情况下，我们还研究了单离子和二价盐对翻转动力学的影响。我们发现，无论是 DNA 序列还是改变的缓冲条件都不会影响四聚体的整体结构。相比之下，与高亲和力 DNA 序列结合的四聚体显示出明显改变的翻转动力学，主要是通过减少左手缠绕的典型状态的寿命来实现。增加单离子和二价盐浓度可以抵消这种行为。因此，我们的研究表明，高亲和力 DNA 序列不仅影响核小体的定位，而且赋予亚核小体四聚体增强的构象可塑性。这可能为防止暴露于扭转应力时组蛋白丢失提供了一种手段，从而有助于高亲和力位点处染色质的完整性。
