Extended Data Figure 5. E2_Ubc9 and E3_Siz1 Determinants of Lysine Specificity.

a. Plots of the rates observed at different pH values for multiple turnover in vitro assays of SUMO modification of PCNA utilizing 0.1 µM purified E2_Ubc9-SUMO^D68R-Alexa488 thioester (or E2_Ubc9 mutant thioesters) with 5 nM E3_Siz1 and 4 µM PCNA at 4°C. b, SDS-PAGE analysis of multiple turnover assays of SUMO modification of PCNA utilizing in vitro reactions with coupled E1 (200 nM), E2 (100 nM), and E3 (50 nM) activities with 4 µM PCNA for the quantified data shown in Fig. 4d. c, SDS-PAGE analysis of multiple turnover assays of SUMO modification of PCNA utilizing in vitro reactions with coupled E1 (200 nM), E2 (100 nM), and E3 (50 nM) activities with 4 µM PCNA and quantified. d, Representative non-reducing SDS-PAGE analysis of the single turnover in vitro assays of SUMO modification of PCNA shown in Fig. 4e. These assays utilize 5 nM of the E2_Ubc9-SUMO^D68R-Alexa488 thioester (or E2_Ubc9 mutant thioesters) in reactions with 50 nM of the indicated E3_Siz1 and a titration of PCNA. Shown are typical SDS-PAGE analyses from the 10 µM PCNA reactions. The data were used to extract the kinetic constants for the reactions shown as histograms and in Extended Data Table 4. For panels a,c and d the quantified rate data show mean ± s.d. (n=3 technical replicates). For gel source data, see Supplementary Figure 1.