Data_Sheet_1_Uncharged Components of Single-Stranded DNA Modulate Liquid–Liquid Phase Separation With Cationic Linker Histone H1.PDF

Liquid–liquid phase separation (LLPS) of proteins and DNAs has been recognized as a fundamental mechanism for the formation of intracellular biomolecular condensates. Here, we show the role of the constituent DNA components, i.e., the phosphate groups, deoxyribose sugars, and nucleobases, in LLPS with a polycationic peptide, linker histone H1, a known key regulator of chromatin condensation. A comparison of the phase behavior of mixtures of H1 and single-stranded DNA-based oligomers in which one or more of the constituent moieties of DNA were removed demonstrated that not only the electrostatic interactions between the anionic phosphate groups of the oligomers and the cationic residues of H1, but also the interactions involving nucleobases and deoxyriboses (i) promoted the generation of spherical liquid droplets via LLPS as well as (ii) increased the density of DNA and decreased its fluidity within the droplets under low-salt conditions. Furthermore, we found the formation of non-spherical assemblies with both mobile and immobile fractions at relatively higher concentrations of H1 for all the oligomers. The roles of the DNA components that promote phase separation and modulate droplet characteristics revealed in this study will facilitate our understanding of the formation processes of the various biomolecular condensates containing nucleic acids, such as chromatin organization.

蛋白质与DNA的液-液相分离（Liquid–liquid phase separation, LLPS）已被证实为细胞内生物分子凝聚体形成的基础机制。本研究以聚阳离子肽、连接组蛋白H1（已知的染色质凝聚关键调控因子）为研究对象，揭示了DNA组成组分——即磷酸基团、脱氧核糖与核碱基——在LLPS过程中的作用。我们对比了H1与去除了一个或多个DNA组成组分的单链DNA寡聚体混合体系的相行为，结果表明：不仅寡聚体带负电的磷酸基团与H1阳离子残基间的静电相互作用，涉及核碱基与脱氧核糖的相互作用同样具备双重效应：(i) 经由LLPS促进球形液滴的形成；(ii) 在低盐条件下提升液滴内DNA的密度并降低其流动性。此外，我们发现当所有寡聚体系的H1浓度相对较高时，会形成同时包含可流动与不可流动组分的非球形聚集体。本研究揭示的DNA组分促进相分离并调控液滴特性的作用机制，将有助于我们理解包括染色质组装在内的各类含核酸生物分子凝聚体的形成过程。