The H1 linker histone family is the most abundant group of eukaryotic chromatin-binding proteins. However, their contribution to chromosome structure and function remains incompletely understood. Here we use single-molecule fluorescence and force microscopy to directly visualize the behavior of H1 on various nucleic acid and nucleosome substrates. We observe that H1 coalesces around single-stranded DNA generated from tension-induced DNA duplex melting. Using a droplet fusion assay controlled by optical tweezers, we find that single-stranded nucleic acids mediate the formation of gel-like H1 droplets, whereas H1–double-stranded DNA and H1–nucleosome droplets are more liquid-like. Molecular dynamics simulations reveal that multivalent and transient engagement of H1 with unpaired DNA strands drives their enhanced phase separation. Using eGFP-tagged H1, we demonstrate that inducing single-stranded DNA accumulation in cells causes an increase in H1 puncta that are able to fuse. We further show that H1 and Replication Protein A occupy separate nuclear regions, but that H1 colocalizes with the replication factor Proliferating Cell Nuclear Antigen, particularly after DNA damage. Overall, our results provide a refined perspective on the diverse roles of H1 in genome organization and maintenance, and indicate its involvement at stalled replication forks.

H1 连接组蛋白家族是最丰富的真核染色质结合蛋白组。然而，它们对染色体结构和功能的贡献仍未完全了解。在这里，我们使用单分子荧光和力显微镜直接可视化 H1 在各种核酸和核小体底物上的行为。我们观察到 H1 围绕由张力诱导的 DNA 双链体熔化产生的单链 DNA 聚结。使用光镊控制的液滴融合测定，我们发现单链核酸介导凝胶状 H1 液滴的形成，而 H1-双链 DNA 和 H1-核小体液滴则更像液体。分子动力学模拟表明，H1 与未配对 DNA 链的多价和瞬时结合可促进其增强的相分离。使用 eGFP 标记的 H1，我们证明诱导细胞中单链 DNA 积累会导致能够融合的 H1 斑点增加。我们进一步表明，H1 和复制蛋白 A 占据不同的核区域，但 H1 与复制因子增殖细胞核抗原共定位，特别是在 DNA 损伤后。总的来说，我们的结果为 H1 在基因组组织和维护中的不同作用提供了一个精确的视角，并表明它参与了停滞的复制叉。