Counteraction of HMGB1 at ss-dsDNA junctions maintains liquidity of protamine-DNA co-condensates.

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.