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

• Published in: PloS one Vol. 15; no. 2; p. e0229000
• Main 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
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 24.02.2020; Public Library of Science (PLoS)
• Subjects: Affinity; Anemia; Animals; Avidin - chemistry; Biochemistry; Biology and Life Sciences; Biotin; BRCA1 protein; Characterization; Conjugation; Crosslinking; Deoxyribonucleic acid; DNA; DNA damage; DNA repair; E coli; EDTA; Enzymes; Fanconi Anemia Complementation Group D2 Protein - chemistry; Fanconi Anemia Complementation Group D2 Protein - isolation & purification; Fanconi Anemia Complementation Group Proteins - chemistry; Fanconi Anemia Complementation Group Proteins - isolation & purification; Fanconi syndrome; Fanconi's anemia; Fusion protein; Genetic research; Genomes; Humans; Life Sciences; Ligases; Medical research; Medicine; Medicine and Health Sciences; Nucleosomes; Physical Sciences; Plasmids; Proliferating cell nuclear antigen; Proliferating Cell Nuclear Antigen - chemistry; Proliferating Cell Nuclear Antigen - isolation & purification; Protein purification; Proteins; Purification; Repair; Sf9 Cells; Spodoptera; Structural analysis; Substrates; Trimers; Ubiquitin; Ubiquitin - chemistry; Ubiquitin - isolation & purification; Ubiquitin-protein ligase; Ubiquitin-Protein Ligases - chemistry; Ubiquitin-Protein Ligases - isolation & purification; Ubiquitinated Proteins - chemistry; Ubiquitinated Proteins - isolation & purification; Ubiquitination; France; Australia; Victoria Australia
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0229000

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.