In vitro assembly and recognition of Lys-63 polyubiquitin chains

Polyubiquitin chains assembled through lysine 48 (Lys-48) of ubiquitin act as a signal for substrate proteolysis by 26 S proteasomes, whereas chains a ssembled through Lys-63 play a mechanistically undefined role in post-repli cative DNA repair. We showed previously that the products of the UBC13 and MMS2 genes function in error-free post-replicative DNA repair in the yeast Saccharomyces cerevisiae and form a complex that assembles Lys-63-linked po lyubiquitin chains in vitro. Here we confirm that the Mms2.Ubc13 complex fu nctions as a high affinity heterodimer in the chain assembly reaction in vi tro and report the results of a kinetic characterization of the polyubiquit in chain assembly reaction. To test whether a Lys-63-linked polyubiquitin c hain can signal degradation, we conjugated Lys-63-linked tetra-ubiquitin to a model substrate of 26 S proteasomes. Although the noncanonical chain eff ectively signaled substrate degradation, the results of new genetic epistas is studies agree with previous genetic data in suggesting that the proteoly tic activity of proteasomes is not required for error-free post-replicative repair.