A UBIQUITIN MUTANT WITH SPECIFIC DEFECTS IN DNA-REPAIR AND MULTIUBIQUITINATION

The degradation of many proteins involves the sequential ligation of u biquitin molecules to the substrate to form a multiubiquitin chain lin ked through Lys-48 of ubiquitin. To test for the existence of alternat e forms of multiubiquitin chains, we examined the effects of individua lly substituting each of six other Lys residues in ubiquitin,vith Arg. Substitution of Lys-63 resulted in the disappearance of a family of a bundant multiubiquitin-protein conjugates. The UbK63R mutants were not generally impaired in ubiquitination, because they grew at a wild-typ e rate, were fully proficient in the turnover of a variety of short-li ved proteins, and exhibited normal levels of many ubiquitin-protein co njugates. The UbK63R mutation also conferred sensitivity to the DNA-da maging agents methyl methanesulfonate and UV as well as a deficiency i n DNA damage-induced mutagenesis. Induced mutagenesis is mediated by a repair pathway that requires Rad6 (Ubc2), a ubiquitin-conjugating enz yme. Thus, the UbK63R mutant appears to be deficient in the Rad6 pathw ay of DNA repair. However, the UbK63R mutation behaves as a partial su ppressor of a rad6 deletion mutation, indicating that an effect of UbK 63R on repair can be manifest in the absence of the Rad6 gene product. The U6K63R mutation may therefore define a new role of ubiquitin in D NA repair. The results of this study suggest that Lys-63 is used as a linkage site in the formation of novel multiubiquitin chain structures that play an important role in DNA repair.