Synthesis of free and PCNA-bound polyubiquitin chains by the E2 heterodimer, Ubc13-Mms2 and the ring E3 ubiquitin ligase, Rad5

Ubiquitination is a diverse posttranslational modification that executes regulatory functions in virtually every eukaryotic cellular pathway. A substrate can be modified by different ubiquitin (Ub) signals, which are translated into distinct downstream effects. These downstream effects are thought to be directed, at least in part, by the number of Ubs attached to a substrate and, in the case of polyubiquitination, the types of Ub-Ub linkages within the Ub signal. The specific enzymatic mechanisms that promote ubiquitination, especially polyubiquitination, are poorly understood.;Free K63-linked polyubiquitin chains can be assembled in vitro by a heterodimeric Ub conjugating (E2) enzyme from yeast comprised of Ubc13 and Mms2. This property of Ubc 13-Mms2 allowed investigation of the mechanisms mediating polyubiquitination. From structural studies, Ubc13-Mms2 facilitates free chain synthesis primarily by positioning of the K63 residue of the acceptor Ub in the active site of Ubc 13.;Together with the Rad5 Ub ligase (E3), Ubc13-Mms2 assists in the polyubiquitination of PCNA in vivo to promote error-free DNA damage tolerance. In biochemical studies, we found that Rad5 stimulated the synthesis of free polyubiquitin (polyUb) by Ubc13-Mms2 by increasing the reactivity of the Ub-Ubc13 thiolester bond. In addition to this general activation of catalysis, the N-terminus of Rad5 interacts with PCNA and therefore Rad5 also promotes polyubiquitination of monoubiquitinated PCNA by facilitating an interaction between the Ubc13-Mms2 E2 and monoubiquitinated PCNA. Rad5 therefore acts both to align the substrate with Ub-charged Ubc13 and to stimulate Ub transfer from Ubc13-Mms2 onto the Ub moiety of monoubiquitinated PCNA.