ジャーナル: J Cell Biol / 年: 2022 タイトル: Autologous K63 deubiquitylation within the BRCA1-A complex licenses DNA damage recognition. 著者: Qinqin Jiang / Martina Foglizzo / Yaroslav I Morozov / Xuejiao Yang / Arindam Datta / Lei Tian / Vaughn Thada / Weihua Li / Elton Zeqiraj / Roger A Greenberg / 要旨: 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 ...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.
凍結剤: ETHANE / チャンバー内湿度: 100 % / チャンバー内温度: 277.15 K / 装置: FEI VITROBOT MARK IV 詳細: The sample was blotted for 6 seconds and with a blot force of 6 before plunging.
-
電子顕微鏡法
顕微鏡
FEI TITAN KRIOS
撮影
フィルム・検出器のモデル: FEI FALCON III (4k x 4k) 検出モード: COUNTING / 撮影したグリッド数: 1 / 実像数: 4392 / 平均露光時間: 70.0 sec. / 平均電子線量: 1.46 e/Å2
モデルのタイプ: INSILICO MODEL In silico モデル: Models for the five subunits constituting the complex (BRCC36, Abraxas1, BRCC45, MERIT40 and RAP80) were generated using SWISS-MODEL (Bertoni et al., 2018)