The BRCA1-A complex contains matching lysine-63 ubiquitin (K63-Ub) binding and deubiquitylating activities. How these functionalities are coordinated to effectively respond to DNA damage remains unknown. We generated Brcc36 deubiquitylating enzyme (DUB) inactive mice to address this gap in knowledge in a physiologic system. DUB inactivation impaired BRCA1-A complex damage localization and repair activities while causing early lethality when combined with Brca2 mutation. Damage response dysfunction in DUB-inactive cells corresponded to increased K63-Ub on RAP80 and BRCC36. Chemical cross-linking coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) and cryogenic-electron microscopy (cryo-EM) analyses of isolated BRCA1-A complexes demonstrated the RAP80 ubiquitin interaction motifs are occupied by ubiquitin exclusively in the DUB-inactive complex, linking auto-inhibition by internal K63-Ub chains to loss of damage site ubiquitin recognition. These findings identify RAP80 and BRCC36 as autologous DUB substrates in the BRCA1-A complex, thus explaining the evolution of matching ubiquitin-binding and hydrolysis activities within a single macromolecular assembly.

中文翻译：

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BRCA1-A 复合物内的自体 K63 去泛素化许可 DNA 损伤识别

BRCA1-A 复合物包含匹配的赖氨酸 63 泛素 (K63-Ub) 结合和去泛素化活性。如何协调这些功能以有效应对 DNA 损伤仍然未知。我们培育了 Brcc36 去泛素化酶 (DUB) 失活小鼠，以解决生理系统中的这一知识空白。 DUB 失活会损害 BRCA1-A 复合物损伤定位和修复活性，同时与 Brca2 突变结合时会导致早期致死。 DUB 失活细胞中的损伤反应功能障碍与 RAP80 和 BRCC36 上 K63-Ub 的增加相对应。对分离的 BRCA1-A 复合物进行化学交联，结合液相色谱-串联质谱 (LC-MS/MS) 和低温电子显微镜 (cryo-EM) 分析，证明 RAP80 泛素相互作用基序仅被 DUB 中的泛素占据-非活性复合物，将内部 K63-Ub 链的自动抑制与损伤位点泛素识别的丧失联系起来。这些发现将 RAP80 和 BRCC36 确定为 BRCA1-A 复合物中的自体 DUB 底物，从而解释了单个大分子组装体中匹配泛素结合和水解活性的演变。