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PLoS One
2020 Feb 24;152:e0229000. doi: 10.1371/journal.pone.0229000.
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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
.
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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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32092106
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Fig 1. Purification of recombinant Avi-ubiquitin.
(A) Schematic of the Avi-ubiquitin construct used for expression and purification of recombinant Avi-ubiquitin from E. coli cells. #1–4 refers to features of Avi-ubiquitin required for biotinylation and purification. (B) Coomassie stained SDS-PAGE gel showing purification of recombinant Avi-ubiquitin using Ni-NTA resin. (C) Coomassie stained SDS-PAGE gel (top), anti-Streptavidin (middle) and anti-ubiquitin (bottom) western blots showing elution of Avi-ubiquitin by using 5 mM biotin or by cleavage of bound ubiquitin using 3C protease. Experiments in (B-C) were performed in triplicate with similar results. Note: cleaved ubiquitin is below the 10 kDa lower limit for optimal binding to PVDF membranes, so only weakly observed by western blot.
https://doi.org/10.1371/journal.pone.0229000.g001
Fig 2. Reconstitution of human FANCI:FANCD2, PCNA and nucleosome in vitro ubiquitination assay using Avi-ubiquitin.
(A) Coomassie stained 4–12% Bis-Tris gel run in 1X MES buffer showing purified human FANCI:FANCD2, BRCA1Δexon11:BARD1, reconstituted nucleosome, PCNA, UBCH5C (S22R), Avi-ubiquitin, FANCB-FANCL-FAAP100 (BL100), FANCC-FANCE-FANCF (CEF), UBE1 and UBE2T (lanes 2–11). (B) Western blot of the time course ubiquitination reaction of human FANCI:FANCD2 at 25 °C, 30 °C and 37 °C. The percentage of mono-ubiquitinated FANCD2 or FANCI were calculated and showed under each western blot panel. (C) PCNA mono-ubiquitination time course experiments using UBCH5C as an E2 enzyme. (D) Western blots of time course experiments revealing that BRCA1Δexon11 and BARD1 were auto-ubiquitinated at multiple lysine residues in the presence of UBCH5C. (E) Mono-ubiquitination timecourse of nucleosomes as substrates in the presence of BRCA1Δexon11:BARD1 and UBCH5C. Experiments in (B-E) were performed in triplicate with similar results.
https://doi.org/10.1371/journal.pone.0229000.g002
Fig 3. Purification of mono-ubiquitinated proteins using the 3C protease enzyme.
(A) Coomassie of 4–12% Bis-Tris gel run in 1X MES buffer showing the purification Avi-ubiquitin. (B) Coomassie of 3–8% Tris-acetate gel showing the purification of mono-ubiquitinated FANCI:FANCD2 complex. (C) Coomassie stained SDS-PAGE gel showing purification of mono-ubiquitinated PCNA. (D) Silver stained SDS-PAGE gel showing purification of mono-ubiquitinated nucleosomes. Experiments in (A-D) were performed in triplicate with similar results.
https://doi.org/10.1371/journal.pone.0229000.g003
