Promotion of RNF168‐Mediated Nucleosomal H2A Ubiquitylation by Structurally defined K63‐Polyubiquitylated Linker Histone H1

The chemical synthesis of histones with homogeneous modifications is a powerful approach for quantitatively deciphering the functional crosstalk between different post-translational modifications (PTMs). In this study, we developed an expedient site-specific (poly)ubiquitylation strategy (CAEPL, Cysteine Aminoethylation coupled with Enzymatic Protein Ligation), which integrates the Cys-aminoethylation reaction with the process of ubiquitin-activating enzyme UBA1-assisted native chemical ligation. Using this strategy, we successfully prepared monoubiquitylated and K63-linked di- and tri-ubiquitylated linker histone H1.0 proteins, which were incorporated into individual chromatosomes. Quantitative biochemical analysis of different RNF168 constructs on H1 ubiquitylated chromatosomes with different ubiquitin chain lengths demonstrated that K63-linked polyubiquitylated H1.0 could directly stimulate RNF168 ubiquitylation activity by enhancing the affinity between RNF168 and the chromatosome. Subsequent cryo-EM structural analysis of the RNF168/UbcH5c-Ub/H1.0-K63-Ub