BPS2026 – HMGB1 localization at ss-dsDNA junction prevents protamine-mediated extreme DNA compaction

In the sperm nucleus, protamine replaces histones to mediate extreme DNA compaction. The histone-to-protamine exchange involves the occurrence of double-strand breaks for histone release and protamine uptake, and is facilitated by transition proteins including those containing high-mobility-group (HMG) boxes. In previous work, we showed that protamine compacts double-stranded (ds) DNA into solid-like “tangles” that has extreme stability and can be initiated at ss-ds junctions. 1 We further suggested that DNA condensates induced by early protamine should be liquid-like to allow recruitment of the repair machinery to double-strand breaks. 2 To test whether HMG proteins can play an important role in maintaining condensate liquidity, here we used optical tweezers and confocal and brightfield imaging to study the interactions of HMGB1 and protamine with DNA. Confocal scans of HMGB1-GFP 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-ds junctions. Whereas the force-extension curves of protamine-bound λ-DNA show tangles that withstand forces > 60 pN along with bends and loops that rupture at 10–40 pN, 1 , premixing protamine with HMGB1 produces only bends and “weak” bridges (∼20 pN). The latter indicate cooperative binding of HMGB1 and protamine at ss-ds junctions. Cooperation involves the acidic C-terminal tail of HMGB1, as HMGB1-ΔC fails to prevent tangle formation. In line with these single-molecule results, brightfield 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 transition 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. 1 Ahlawat ⋯ Zhou, (2024). JACS 146, 30668. 2 Ahlawat ⋯ Zhou, bioRxiv .