Department of Biological Sciences, Ulsan National Institute of Science and Technology.
Department of Biological Sciences, Ulsan National Institute of Science and Technology; Center for Genomic Integrity, Institute for Basic Science (IBS);
J Vis Exp. 2022 Mar 9(181). doi: 10.3791/63501.
Chromatin is a higher-order structure that packages eukaryotic DNA. Chromatin undergoes dynamic alterations according to the cell cycle phase and in response to environmental stimuli. These changes are essential for genomic integrity, epigenetic regulation, and DNA metabolic reactions such as replication, transcription, and repair. Chromatin assembly is crucial for chromatin dynamics and is catalyzed by histone chaperones. Despite extensive studies, the mechanisms by which histone chaperones enable chromatin assembly remains elusive. Moreover, the global features of nucleosomes organized by histone chaperones are poorly understood. To address these problems, this work describes a unique single-molecule imaging technique named DNA curtain, which facilitates the investigation of the molecular details of nucleosome assembly by histone chaperones. DNA curtain is a hybrid technique that combines lipid fluidity, microfluidics, and total internal reflection fluorescence microscopy (TIRFM) to provide a universal platform for real-time imaging of diverse protein-DNA interactions.Using DNA curtain, the histone chaperone function of Abo1, the Schizosaccharomyces pombe bromodomain-containing AAA+ ATPase, is investigated, and the molecular mechanism underlying histone assembly of Abo1 is revealed. DNA curtain provides a unique approach for studying chromatin dynamics.
染色质是一种高级结构，用于包装真核生物的 DNA。染色质根据细胞周期阶段和环境刺激发生动态变化。这些变化对于基因组完整性、表观遗传调控以及 DNA 代谢反应（如复制、转录和修复）至关重要。染色质组装对于染色质动力学至关重要，由组蛋白伴侣催化。尽管进行了广泛的研究，但组蛋白伴侣如何促进染色质组装的机制仍然难以捉摸。此外，组蛋白伴侣组织的核小体的全局特征了解甚少。为了解决这些问题，这项工作描述了一种名为 DNA 幕帘的独特单分子成像技术，它促进了对组蛋白伴侣组装核小体的分子细节的研究。DNA 幕帘是一种混合技术，结合了脂质流动性、微流控和全内反射荧光显微镜（TIRFM），为实时研究各种蛋白-DNA 相互作用提供了通用平台。利用 DNA 幕帘，研究了裂殖酵母的含有溴结构域的 AAA+ ATP 酶 Abo1 的组蛋白伴侣功能，并揭示了 Abo1 组装组蛋白的分子机制。DNA 幕帘为研究染色质动力学提供了一种独特的方法。
