Extended Data Figure 1. E2_Ubc9-SUMO Thioester Mimic and Cross-Linking to Substrate PCNA for Reconstitution With E3_Siz1.

a, SDS-PAGE analysis of in vitro E2_Ubc9^A129K or E2_Ubc9^C93K charging with SUMO in the presence and absence of E3_Siz1^(167–465) at pH (7.5) (left) and purification of the E2_Ubc9^C93K-SUMO (middle) and E2_Ubc9^A129K-SUMO (right) thioester mimetics. b, Plots of rates for in vitro SUMO modification of PCNA in assays utilizing various concentrations of purified E2_Ubc9-SUMO^D68R-Alexa488 labeled thioester, 1 nM E3_Siz1^(167–465) and 32 µM PCNA with 0, 2, 5 or 20 µM of the E2_Ubc9^C93K-SUMO or E2_Ubc9^A129K-SUMO thioester mimic (left) with exemplary non-reducing SDS-PAGE for the 0.5 µM E2_Ubc9-SUMO^D68R-Alexa488 reactions (right). The calculated K_m and K_i from these fits are shown in Extended Data Tables 1a and 2 and the quantified data show mean ± s.d. (n=3 technical replicates). c, SDS-PAGE analysis (left) of numbered 0.5 ml fractions from Superose6 analytical gel-filtration analysis (right) of complex reconstitution between E2_Ubc9-SUMO-BMOE-PCNA and E3_Siz1^(167–465) (green) or the E3_Siz1^(167–465)-SUMO fusion (blue). Elution profiles for E2_Ubc9-SUMO-BMOE-PCNA (purple) and E3_Siz1^(167–465) (red) alone are shown. d, Plot of the normalized change in polarization observed upon addition of serially diluted E2_Ubc9 with Alexa488 labeled SUMO or SUMO^D68R. Data were fit to single-site binding model accounting for receptor depletion. Data show mean ± s.d. (n=3 technical replicates). For gel source data, see Supplementary Figure 1.