Figure 5
USP1 autocleavage: assessing cis/trans mode of cleavage and the effect of cancer-associated mutations. A. Representation of USP1 showing the catalytic triad (C90/H593/D751, green spheres) and the autocleavage site (G670/G671, blue spheres). Below, potential cis/trans autocleavage modes. B. Experiment used to assess cleavage mode. The C90S and GG/AA mutants (mutated residues in red) are unable to undergo cis autocleavage. If autocleavage occurs in trans, co-expression of both mutants could lead to cleavage of GFP-USP1C90S by GFP-USP1GG/AA. C. Immunoblot analysis of cells expressing Xpress-UAF1 and GFP-USP1 constructs. Autocleavage was detected in cells transfected with wild type GFP-USP1, but not with GFP-USP1C90S or GFP-USP1GG/AA. No cleavage was observed upon co-expression of GFP-USP1C90S and GFP-USP1GG/AA, suggesting that trans cleavage does not occur. D. Location of cancer mutations [24] near USP1 autocleavage site. E. Immunoblot analysis of cells expressing Xpress-UAF1 and different USP1 mutants. GFP-USP1G667A, GFP-USP1K673T and GFP-USP1A676T are cleaved as efficiently as wild type USP1, whereas GFP-USP1L669P is less efficiently cleaved. F. PCNA ubiquitination in 293T cells treated with 4 mM HU for 24 h. An example of immunoblot results is shown (left). The dotted line indicates that the panel is a composite of two images from a single exposure of the same gel. UbPCNA/PCNA ratio was determined by densitometry and the results (mean and SEM of 3 independent experiments) are shown in the graph (right). Cancer-associated mutations did not alter the ubPCNA/PCNA ratio. G. The C90S mutation (S90) was introduced into each cancer-related mutant construct, rendering these mutants unable to undergo cis autocleavage (Additional Figure 5C). These double mutants were expressed together with GFP-USP1GG/AA to evaluate if they undergo trans cleavage. No autocleavage band was observed with any of the cancer-related/(S90) mutants upon co-expression with GFP-USP1GG/AA, suggesting that the cancer-related mutations tested do not alter the cis/trans mode of cleavage.