Biochemistry and Pharmacology - Research Publications

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Author: Crismani, Wayne × Author: Deans, Andrew × Author: Parker, Michael × End date: 2022 × Start date: 2020 × Type: Journal Article ×

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    Monoubiquitination by the human Fanconi anemia core complex clamps FANCI: FANCD2 on DNA in filamentous arrays
    Tan, W ; van Twest, S ; Leis, A ; Bythell-Douglas, R ; Murphy, VJ ; Sharp, M ; Parker, MW ; Crismani, W ; Deans, AJ (ELIFE SCIENCES PUBLICATIONS LTD, 2020-03-13)
    FANCI:FANCD2 monoubiquitination is a critical event for replication fork stabilization by the Fanconi anemia (FA) DNA repair pathway. It has been proposed that at stalled replication forks, monoubiquitinated-FANCD2 serves to recruit DNA repair proteins that contain ubiquitin-binding motifs. Here, we have reconstituted the FA pathway in vitro to study functional consequences of FANCI:FANCD2 monoubiquitination. We report that monoubiquitination does not promote any specific exogenous protein:protein interactions, but instead stabilizes FANCI:FANCD2 heterodimers on dsDNA. This clamping requires monoubiquitination of only the FANCD2 subunit. We further show using electron microscopy that purified monoubiquitinated FANCI:FANCD2 forms filament-like arrays on long dsDNA. Our results reveal how monoubiquitinated FANCI:FANCD2, defective in many cancer types and all cases of FA, is activated upon DNA binding.
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    Preparation and purification of mono-ubiquitinated proteins using Avi-tagged ubiquitin
    Tan, W ; Murphy, VJ ; Charron, A ; van Twest, S ; Sharp, M ; Constantinou, A ; Parker, MW ; Crismani, W ; Bythell-Douglas, R ; Deans, AJ ; Sobol, RW (PUBLIC LIBRARY SCIENCE, 2020-02-24)
    Site-specific conjugation of ubiquitin onto a range of DNA repair proteins regulates their critical functions in the DNA damage response. Biochemical and structural characterization of these functions are limited by an absence of tools for the purification of DNA repair proteins in purely the ubiquitinated form. To overcome this barrier, we designed a ubiquitin fusion protein that is N-terminally biotinylated and can be conjugated by E3 RING ligases onto various substrates. Biotin affinity purification of modified proteins, followed by cleavage of the affinity tag leads to release of natively-mono-ubiquitinated substrates. As proof-of-principle, we applied this method to several substrates of mono-ubiquitination in the Fanconi anemia (FA)-BRCA pathway of DNA interstrand crosslink repair. These include the FANCI:FANCD2 complex, the PCNA trimer and BRCA1 modified nucleosomes. This method provides a simple approach to study the role of mono-ubiquitination in DNA repair or any other mono-ubiquitination signaling pathways.