The BRCA1–BARD1 tumour suppressor is an E3 ubiquitin ligase necessary for the repair of DNA double-strand breaks by homologous recombination^{1,2,3,4,5,6,7,8,9,10}. The BRCA1–BARD1 complex localizes to damaged chromatin after DNA replication and catalyses the ubiquitylation of histone H2A and other cellular targets^11,12,13,14. The molecular bases for the recruitment to double-strand breaks and target recognition of BRCA1–BARD1 remain unknown. Here we use cryo-electron microscopy to show that the ankyrin repeat and tandem BRCT domains in BARD1 adopt a compact fold and bind to nucleosomal histones, DNA and monoubiquitin attached to H2A amino-terminal K13 or K15, two signals known to be specific for double-strand breaks^15,16. We further show that RING domains¹⁷ in BRCA1–BARD1 orient an E2 ubiquitin-conjugating enzyme atop the nucleosome in a dynamic conformation, primed for ubiquitin transfer to the flexible carboxy-terminal tails of H2A and variant H2AX. Our work reveals a regulatory crosstalk in which recognition of monoubiquitin by BRCA1–BARD1 at the N terminus of H2A blocks the formation of polyubiquitin chains and cooperatively promotes ubiquitylation at the C terminus of H2A. These findings elucidate the mechanisms of BRCA1–BARD1 chromatin recruitment and ubiquitylation specificity, highlight key functions of BARD1 in both processes and explain how BRCA1–BARD1 promotes homologous recombination by opposing the DNA repair protein 53BP1 in post-replicative chromatin^{18,19,20,21,22}. These data provide a structural framework to evaluate BARD1 variants and help to identify mutations that drive the development of cancer.

^{BRCA1-BARD1 肿瘤抑制因子是一种 E3 泛素连接酶，通过同源重组1,2,3,4,5,6,7,8,9,10}修复 DNA 双链断裂是必需的。BRCA1-BARD1 复合物在 DNA 复制后定位于受损的染色质，并催化组蛋白 H2A 和其他细胞靶标的泛素化^11,12,13,14。BRCA1-BARD1 的双链断裂募集和靶标识别的分子基础仍然未知。在这里，我们使用冷冻电子显微镜显示 BARD1 中的锚蛋白重复和串联 BRCT 结构域采用紧凑折叠并与核小体组蛋白、DNA 和连接到 H2A 氨基末端 K13 或 K15 的单泛素结合，这两个信号已知对双- 链断裂^15,16。我们进一步表明 RING 域^{BRCA1-BARD1 中的17}以动态构象将 E2 泛素结合酶定向到核小体顶部，准备将泛素转移到 H2A 和变体 H2AX 的柔性羧基末端尾部。我们的工作揭示了一种调节性串扰，其中 BRCA1-BARD1 在 H2A 的 N 端识别单泛素会阻止多泛素链的形成，并协同促进 H2A 的 C 端的泛素化。这些发现阐明了 BRCA1-BARD1 染色质募集和泛素化特异性的机制，突出了 BARD1 在这两个过程中的关键功能，并解释了 BRCA1-BARD1 如何通过对抗复制后染色质中的 DNA 修复蛋白 53BP1 来促进同源重组^{18,19,20, 21,22}. 这些数据提供了一个结构框架来评估 BARD1 变体并帮助识别驱动癌症发展的突变。