Polyubiquitin (polyUb) chains linked through lysine-48 (K48) of Ub represent the predominant targeting signal in the Ub-proteasome proteolytic pathway, whereas polyUb chains linked through K63 function in postreplicative DNA repair by a mechanism which does not appear to involve proteolysis. These observations suggest that the assembly of Ub into distinct types of polymers may be a mechanism to diversify the signaling functions of Ub. To test this hypothesis we will apply methods of biochemistry and yeast molecular genetics to characterize the assembly and recognition of K63-linked polyUb chains, and elucidate the signaling function of these polymers in DNA repair. Our discovery of yeast enzymes which assemble homopolymeric K63-linked polyUb chains in vitro, and function in DNA repair in vivo, provides the starting point for this work. These proteins are Ubcl3p (a Ub conjugating enzyme) and Mms2p (a Ub E2 variant or UEV protein). By characterizing the mechanism and function of the Mms2p/Ubc13p complex, we will explicate the process of signal generation; by identifying cellular proteins that are linked to K63-linked chains, we will gain insight into the functional consequences of signal recognition; finally, by using an in vitro-assembled chain as a tool, we will directly address the proteolytic signaling competence of K63-linked polyUb chains, and isolate chain-recognizing factors that may represent downstream elements in the proposed DNA repair signaling pathway. The results of these studies will provide new insights into the mechanistic role of Ub in DNA repair, and may ultimately lead to improved strategies for the treatment of cancer and other diseases. Moreover, if our results support the hypothesis that polyUb chains serve to diversify the signaling functions of Ub, it will help to explain how this small molecule is able to act as a distinct signal in multiple cellular processes. Finally, our results will provide the first biochemical insight into the functioning of UEV protein family, which in mammals includes the tumor suppressor Tsgl0l, and other proteins implicated in differention and cell transformation.