A ubiquitin mutant with specific defects in DNA repair and multiubiquitination.
The degradation of many proteins involves the sequential ligation of ubiquitin molecules to the substrate to form a multiubiquitin chain linked through Lys-48 of ubiquitin. To test for the existence of alternate forms of multiubiquitin chains, we examined the effects of individually substituting each of six other Lys residues in ubiquitin with Arg. Substitution of Lys-63 resulted in the disappearance of a family of abundant multiubiquitin-protein conjugates. The UbK63R mutants were not generally impaired in ubiquitination, because they grew at a wild-type rate, were fully proficient in the turnover of a variety of short-lived proteins, and exhibited normal levels of many ubiquitin-protein conjugates. The UbK63R mutation also conferred sensitivity to the DNA-damaging agents methyl methanesulfonate and UV as well as a deficiency in DNA damage-induced mutagenesis. Induced mutagenesis is mediated by a repair pathway that requires Rad6 (Ubc2), a ubiquitin-conjugating enzyme. Thus, the UbK63R mutant appears to be deficient in the Rad6 pathway of DNA repair. However, the UbK63R mutation behaves as a partial suppressor of a rad6 deletion mutation, indicating that an effect of UbK63R on repair can be manifest in the absence of the Rad6 gene product. The UbK63R mutation may therefore define a new role of ubiquitin in DNA 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.
Spence
J
J
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115.
Sadis
S
S
Haas
A L
AL
Finley
D
D
D000595:Amino Acid Sequence; D004260:DNA Repair / Q000187:drug effects / Q000528:radiation effects; D004307:Dose-Response Relationship, Radiation; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000378:metabolism; D005800:Genes, Fungal; D015151:Immunoblotting; D007700:Kinetics; D008025:Ligases / Q000378:metabolism; D008239:Lysine; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D044763:Ubiquitin-Conjugating Enzymes; D014452:Ubiquitins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D014466:Ultraviolet Rays
D005656:Fungal Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D014452:Ubiquitins; D008741:Methyl Methanesulfonate; C471606:RAD6 protein, S cerevisiae; D044763:Ubiquitin-Conjugating Enzymes; D008025:Ligases; D008239:Lysine