核小体 H2B 单泛素化在基于染色质的转录过程中起着至关重要的作用。一个RING 类型的 E3 连接酶（酵母 Bre1 或人类 RNF20/RNF40）和一个 E2 泛素连接酶（酵母 Rad6 或人类 hRAD6A）共同精确地在酵母中的 H2B K123 或人类中的 K120 上沉积泛素。本研究中，我们开发了一种化学诱捕策略，成功捕获了Bre1 或 RNF20/RNF40 介导的泛素从 Rad6 或 hRAD6A 转移至核小体 H2B 的瞬态结构。我们的结构显示，Bre1 和 RNF40 直接结合核小体 DNA，呈现出从酵母到人类的 H2B 单泛素化的保守 E3/E2/核小体相互作用模式。我们还发现了 Bre1 RING-Rad6 接口中的非疏水性的非规范性接触，将 Rad6 直接定位在目标 H2B 赖氨酸残基上方。我们的研究提供了关于 H2B 特异位置单泛素化的机制性洞见，揭示了核小体 DNA 在介导 E3 连接酶识别中的关键作用，并为理解 RNF20/RNF40 引起癌症突变提供了一个框架。版权所有 © 2023 Elsevier Inc. 保留所有权利。

Histone H2B monoubiquitylation plays essential roles in chromatin-based transcriptional processes. A RING-type E3 ligase (yeast Bre1 or human RNF20/RNF40) and an E2 ubiquitin-conjugating enzyme (yeast Rad6 or human hRAD6A), together, precisely deposit ubiquitin on H2B K123 in yeast or K120 in humans. Here, we developed a chemical trapping strategy and successfully captured the transient structures of Bre1- or RNF20/RNF40-mediated ubiquitin transfer from Rad6 or hRAD6A to nucleosomal H2B. Our structures show that Bre1 and RNF40 directly bind nucleosomal DNA, exhibiting a conserved E3/E2/nucleosome interaction pattern from yeast to humans for H2B monoubiquitylation. We also find an uncanonical non-hydrophobic contact in the Bre1 RING-Rad6 interface, which positions Rad6 directly above the target H2B lysine residue. Our study provides mechanistic insights into the site-specific monoubiquitylation of H2B, reveals a critical role of nucleosomal DNA in mediating E3 ligase recognition, and provides a framework for understanding the cancer-driving mutations of RNF20/RNF40.Copyright © 2023 Elsevier Inc. All rights reserved.