Preparation and purification of mono-ubiquitinated proteins using Avi-tagged ubiquitin.
Animals
Avidin
/ chemistry
Fanconi Anemia Complementation Group D2 Protein
/ chemistry
Fanconi Anemia Complementation Group Proteins
/ chemistry
Humans
Proliferating Cell Nuclear Antigen
/ chemistry
Sf9 Cells
Spodoptera
Ubiquitin
/ chemistry
Ubiquitin-Protein Ligases
/ chemistry
Ubiquitinated Proteins
/ chemistry
Journal
PloS one
ISSN: 1932-6203
Titre abrégé: PLoS One
Pays: United States
ID NLM: 101285081
Informations de publication
Date de publication:
2020
2020
Historique:
received:
10
12
2019
accepted:
27
01
2020
entrez:
25
2
2020
pubmed:
25
2
2020
medline:
6
5
2020
Statut:
epublish
Résumé
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.
Identifiants
pubmed: 32092106
doi: 10.1371/journal.pone.0229000
pii: PONE-D-19-34194
pmc: PMC7039436
doi:
Substances chimiques
FANCD2 protein, human
0
FANCI protein, human
0
Fanconi Anemia Complementation Group D2 Protein
0
Fanconi Anemia Complementation Group Proteins
0
PCNA protein, human
0
Proliferating Cell Nuclear Antigen
0
Ubiquitin
0
Ubiquitinated Proteins
0
Avidin
1405-69-2
BRAP protein, human
EC 2.3.2.27
Ubiquitin-Protein Ligases
EC 2.3.2.27
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
e0229000Déclaration de conflit d'intérêts
The authors have declared that no competing interests exist.
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