Journal: J Cell Biol / Year: 2022 Title: Autologous K63 deubiquitylation within the BRCA1-A complex licenses DNA damage recognition. Authors: Qinqin Jiang / Martina Foglizzo / Yaroslav I Morozov / Xuejiao Yang / Arindam Datta / Lei Tian / Vaughn Thada / Weihua Li / Elton Zeqiraj / Roger A Greenberg / Abstract: 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.
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV Details: The sample was blotted for 6 seconds and with a blot force of 6 before plunging.
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Electron microscopy
Microscope
FEI TITAN KRIOS
Image recording
Film or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 4392 / Average exposure time: 70.0 sec. / Average electron dose: 1.46 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Type of model: INSILICO MODEL In silico model: Models for the five subunits constituting the complex (BRCC36, Abraxas1, BRCC45, MERIT40 and RAP80) were generated using SWISS-MODEL (Bertoni et al., 2018)
Final reconstruction
Number classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 5.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 88975
Initial angle assignment
Type: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignment
Type: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final 3D classification
Number classes: 6 / Software - Name: RELION
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