The DNA damage repair regulatory protein RNF168, a monomeric RING-type E3 ligase, has a crucial role in regulating cell fate and DNA repair by specific and efficient ubiquitination of the adjacent K13 and K15 (K13/15) sites at the H2A N-terminal tail. However, understanding how RNF168 coordinates with its cognate E2 enzyme UbcH5c to site-specifically ubiquitinate H2A K13/15 has long been hampered by the lack of high-resolution structures of RNF168 and UbcH5c~Ub (ubiquitin) in complex with nucleosomes. Here we developed chemical strategies and determined the cryo-electron microscopy structures of the RNF168–UbcH5c~Ub–nucleosome complex captured in transient H2A K13/15 monoubiquitination and adjacent dual monoubiquitination reactions, providing a ‘helix-anchoring’ mode for monomeric E3 ligase RNF168 on nucleosome in contrast to the ‘compass-binding’ mode of dimeric E3 ligases. Our work not only provides structural snapshots of H2A K13/15 site-specific monoubiquitination and adjacent dual monoubiquitination but also offers a near-atomic-resolution structural framework for understanding pathogenic amino acid substitutions and physiological modifications of RNF168.

DNA 损伤修复调节蛋白 RNF168 是一种单体 RING 型 E3 连接酶，通过对 H2A N 端尾部相邻的 K13 和 K15 （K13/15） 位点进行特异性和有效的泛素化，在调节细胞命运和 DNA 修复中起着至关重要的作用。然而，长期以来，由于 RNF168 和 UbcH5c~Ub（泛素）与核小体复合物中缺乏高分辨率结构，因此了解 RNF168 如何与其同源 E2 酶 UbcH5c 与 UbcH5c 协调以位点特异性泛素化 H2A K13/15 一直受到阻碍。在这里，我们开发了化学策略并确定了在瞬时 H2A K13/15 单泛素化和相邻的双单泛素化反应中捕获的 RNF168-UbcH5c~Ub-核小体复合物的冷冻电子显微镜结构，为核小体上的单体 E3 连接酶 RNF168 提供了“螺旋锚定”模式，与二聚体 E3 连接酶的“指南针结合”模式相反。我们的工作不仅提供了 H2A K13/15 位点特异性单泛素化和相邻双单泛素化的结构快照，还为理解 RNF168 的致病性氨基酸取代和生理修饰提供了一个近原子分辨率的结构框架。