Fig. 2: USP1/UAF1 strongly prefers exo-cleavage on K63- and K48-polyubiquitinated PCNA.
A Schematic of cleavage patterns on PCNA-UbN, PCNA-Ub2 and PCNA-Ub. Here we use terms as follows: Exo- and endo-cleavage both cleave a bond between two ubiquitins, whereas base-cleavage and mono-cleavage both cleave the bond between PCNA and ubiquitin. B−E SDS PAGE gels of USP1/UAF1 activity assays. B Conjugation of USP1/UAF1 to UbVAUb (complex of USP1 with a Ub2 chain). C Cleavage of K63-PCNA-UbVAUb (2 µM) results in conjugation of USP1/UAF1 (200 nM) to K63-PCNA-UbVAUb, seen as a large band shift. This indicates that USP1 performs exo-cleavage. D Fluorescence scan (left panel) and Coomassie-stained SDS PAGE gel (right panel) of K63-PCNA-UbVAUb-UbTMR (2 µM) and USP1/UAF1 (200 nM) reaction shows fast release of UbTMR. Subsequently, USP1 conjugates to PCNA-UbVAUb, only visible on the Coomassie stained gel. E DUB assay of USP1/UAF1 (200 nM) on K63-PCNA-UbN (2 µM, left panel) and K63-PCNA-Ub2 (2 µM, right panel). Assay shows rapid disappearance of chains, enrichment of PCNA-Ub and appearance of a free mono-ubiquitin, indicating that USP1/UAF1 prefers exo-cleavage on both longer K63-chains and diubiquitinated PCNA. F DUB assay of USP1/UAF1 (200 nM) on K48-PCNA-UbN (2 µM, left panel) and K48-PCNA-Ub2 (2 µM, right panel), shows that USP1 has an even stronger preference for exo-cleavage on K48-chains. BCDEF: Uncropped gels are provided as source data. All assays were repeated three times to similar results.
