HMGB1在ss-dsDNA结合位点的反作用维持了鱼精蛋白-DNA共凝聚物的流动性

摘要(中文译文)

※ 中文译文由 AI 辅助生成,仅供学术参考,请以英文原文为准。

在精子细胞核中，鱼精蛋白取代组蛋白，介导DNA的极度压缩。组蛋白向鱼精蛋白的转换涉及双链断裂的发生，并由过渡蛋白（包括含高迁移率族框的蛋白）促进。本研究利用光镊和显微镜技术，探究HMGB1和鱼精蛋白对DNA的作用。GFP-HMGB1在过度拉伸的λ-DNA上的共聚焦扫描显示2–3个焦点，在回缩时沿DNA扩散。焦点的扩散与单链DNA区域的重新退火同时发生，证实其定位于单链-双链DNA交界处。鱼精蛋白浸泡的λ-DNA的力-伸长曲线显示出能承受大于60 pN力的缠结，而将鱼精蛋白与HMGB1预混合仅产生弯曲和桥接（约20 pN）。HMGB1的拮抗作用涉及其酸性C端尾部，因为HMGB1-ΔC无法阻止缠结的形成。与这些单分子结果一致，明场和共聚焦成像显示，在HMGB1存在下，鱼精蛋白-dsDNA聚集体转变为液滴，而HMGB1-ΔC则无此效果。与HMGB1类似，人过渡蛋白1也能拮抗鱼精蛋白对DNA浓缩的影响。p53作为修复因子的招募者，与HMGB1和鱼精蛋白共定位于单链-双链DNA交界处。综上所述，这些观察结果支持我们的假说：HMGB1等染色质相关蛋白有助于维持早期鱼精蛋白介导的DNA凝聚物处于液态，从而促进双链断裂的修复。

Abstract

In the sperm nucleus, protamine replaces histones to mediate extreme DNA compaction. The histone-to-protamine transition involves the occurrence of double-strand breaks, and is facilitated by transition proteins including those containing high-mobility-group boxes. Here we use optical tweezers and microscopy to study the actions of HMGB1 and protamine on DNA. Confocal scans of GFP-HMGB1 on overstretched λ-DNA show 2-3 foci that spread on the DNA upon retraction. Spreading of foci coincides with reannealing of ssDNA tracts, confirming their localization at ss-dsDNA junctions. Whereas the force-extension curves of protamine-soaked λ-DNA show tangles that withstand forces > 60 pN, premixing protamine with HMGB1 produces only bends and bridges ( ~ 20 pN). The counteraction of HMGB1 involves its acidic C-terminal tail, as HMGB1-ΔC fails to prevent tangle formation. In line with these single-molecule results, brightfield and confocal imaging show that protamine-dsDNA aggregates change to liquid droplets in the presence of HMGB1 but not HMGB1-ΔC. Similar to HMGB1, human transition protein 1 counteracts the effects of protamine on DNA condensation. p53, a recruiter of repair factors, colocalizes with HMGB1 and protamine at ss-dsDNA junctions. Together, these observations support our hypothesis that chromatin-associated proteins like HMGB1 help maintain early protamine-mediated DNA condensates in a liquid state to facilitate the repair of double-strand breaks.

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