Preparation and purification of mono-ubiquitinated proteins using Avi-tagged ubiquitin.

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ID: 98371
2020
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Abstract
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.
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tan2020preparationplos Use this key to autocite in the manuscript while using SciMatic Manuscript Manager or Thesis Manager
Authors Tan, Winnie;Murphy, Vincent J;Charron, Aude;van Twest, Sylvie;Sharp, Michael;Constantinou, Angelos;Parker, Michael W;Crismani, Wayne;Bythell-Douglas, Rohan;Deans, Andrew J;
Journal PloS one
Year 2020
DOI
10.1371/journal.pone.0229000
URL
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