A Site‐Specific Click Chemistry Approach to Di‐Ubiquitylate H1 Variants Reveals Position‐Dependent Stimulation of the DNA Repair Protein RNF168

Ubiquitylation of histone H2A at lysines 13 and 15 by the E3 ligase RNF168 plays a key role in orchestrating DNA double‐strand break (DSB) repair, which is often deregulated in cancer. RNF168 activity is triggered by DSB signaling cascades, reportedly through K63‐linked poly‐ubiquitylation of linker histone H1. However, direct experimental evidence of this mechanism has been elusive, primarily due to the lack of methods to specifically poly‐ubiquitylate H1. Here, we developed a versatile click chemistry approach to covalently link multiple proteins in a site‐specific, controlled, and stepwise manner. Applying this method, we synthesized H1 constructs bearing triazole‐linked di‐ubiquitin on four DNA repair‐associated ubiquitylation hotspots (H1KxUb2, at K17, 46, 64 and 96). Integrated into nucleosome arrays, the H1KxUb2 variants stimulated H2A ubiquitylation by RNF168 in a position‐dependent manner, with H1K17Ub2 showing the strongest RNF168 activation effect. Moreover, we show that di‐ubiquitin binding is the driving force underlying RNF168 recruitment, introducing H1K17Ub2 into living U‐2 OS cells. Together, our results support the hypothesis of poly‐ubiquitylated H1 guiding RNF168 recruitment to DSB sites. Moreover, we demonstrate how the streamlined synthesis of H1KxUb2 variants enables mechanistic studies into RNF168 regulation, with potential implications for its inhibition in susceptible cancers. We developed a click chemistry approach to di‐ubiquitylate histone H1 at relevant DNA repair‐associated positions (H1KxUb2) to probe its effect on the E3 ligase RNF168. We demonstrate that H1KxUb2 stimulate RNF168 activity in vitro depending on the Ub2N3 attachment site on H1. Moreover, we show that H1K17Ub2 can recruit RNF168 to chromatin in cells, underlining the hypothesis that poly‐ubiquitylated H1 directs RNF168 to DNA damaged sites.