FIGURE 1.

Rad5 accelerates unanchored Lys⁶³-linked polyUb synthesis by Ubc13-Mms2. A, standard free chain conjugation reactions were performed in the presence of Ubc13-Mms2, E1, wild-type Ub, and either no Rad5 (lanes 1–3), wild-type Rad5 (lanes 4–6), or Rad5(I916A) (lanes 7–9). Reaction products were resolved by SDS-PAGE and visualized with Coomassie Blue. B, reactions were as in A but used Ub(K63R) and Ub-D77 (lanes 1–9). The reactions containing wild-type Rad5 were also done with Ub(K63R) as the sole Ub source (lanes 10–12). C, conjugation assays employed Ubc13-Mms2, E1, Ub-D77, ¹²⁵I-labeled Ub(K63R), and increasing amounts of Rad5. Following SDS-PAGE and autoradiography, ¹²⁵I-Ub₂ products were quantified and are plotted with respect to Rad5 concentration.