Fig. 5: Analysis of the role of steric hindrance by UAF1 in exo-cleavage preference.
A Left: USP1/UAF1 showing the subdomains of UAF1 (WD40, Ancillary domain, SLD) in different tints of blue. Right: Theoretical depiction of USP1/UAF1 binding the chain in an endo-cleavage mechanism shows how UAF1 SLD and Ancillary domains could obstruct binding the ubiquitin chain in the middle. B DUB assays on K63-PCNA-UbN shows that deletion variants USP1/UAF1ΔSLD (200 nM) and USP1/UAF1ΔAnc-SLD (200 nM) use a more flexible mixture between exo- and endo- cleavage. Furthermore, the reaction in USP1/UAF1ΔAnc-SLD is slower. Constructs used for deletion mutants are shown in lower panel. Please note that UAF1ΔAnc-SLD migrates at the same height as PCNA-Ub. Uncropped gels are provided as source data. Assays were repeated three times independently to similar results. C DUB assays of USP1 alone (1 µM) on K63-PCNA-UbN and K63-PCNA-Ub2 (left) show that USP1 employs a mix of exo- and endo-cleavage, but does not seem to cleave at the base as indicated by cleavage of K63-PCNA-Ub2. On K48-PCNA-UbN and K48-PCNA-Ub2 (right panel) USP1’s activity is even more reduced compared to K63-PCNA-UbN, but still uses exo-cleavage. Uncropped gels are provided as source data. Assays were repeated three times independently to similar results.
