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 (HMG) boxes. Here we used 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 tracks, confirming their localization at ss-dsDNA junctions. Whereas the force-extension curves of protamine-bound 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 shows that HMGB1 converts protamine-dsDNA aggregates into liquid droplets whereas HMGB1-ΔC fails to do so. Together, these observations support our hypothesis that chromatin-associated proteins like HMGB1 help maintain early protamine-mediated DNA condensates in a liquid state, enabling the recruitment of the repair machinery to restore the duplex structure.