A Ubiquitin Mutant with Specific Defects in DNA Repair and Multiubiquitination

Abstract

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 ubiquitinconjugating 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.

SUMO-1 conjugation to topoisomerase I: A possible repair response to topoisomerase-mediated DNA damage

“…The possible role of the UBC9 pathway in DNA repair has been suggested from studies of ubc9 mutants (21). Indeed, our results have also demonstrated that a ubc9 ts mutant is hypersensitive to hTOP1-mediated DNA damage induced by CPT (Fig.…”

“…UBC9 was originally identified to play an essential function in G 2 -M cell cycle progression in yeast (20). However, UBC9 is also known to be important for DNA repair (21). It has been shown that SUMO-1 can be conjugated to a number of proteins such as PML (22), RanGAP1 (23,24), I B␣ (25), FAS͞apolipoprotein-1 (26), p53 (27,28), RAD51, and RAD52 (29).…”

Solution Conformation of Lys63-linked Di-ubiquitin Chain Provides Clues to Functional Diversity of Polyubiquitin Signaling

“…PolyUb chains assembled via Lys 29 have been implicated in degradation of certain model proteasome substrates (10). Lys 63 -linked polyUb chains, on the other hand, function in post-replicative DNA repair in the RAD6 pathway (11,12), I B␣ kinase activation (13), translational regulation (6), and some instances of endocytosis (14), although their mechanistic role in these processes is still unclear. Mono-Ub, like Lys 63 -linked polyUb chains, is not involved in proteasome proteolysis and appears to act as a trafficking signal in a host of processes, including endocytosis, endosome internalization, and viral budding (for reviews, see Refs.…”

Proteomic techniques to probe the ubiquitin landscape

“…The preparation of ubiquitin-specific antibodies has been hampered by low affinity and high background, so the introduction of N-terminally epitope-tagged ubiquitin as a probe was quickly identified as a useful alternative for ubiquitination studies [74]. Tagged ubiquitin allows for a more robust purification [75], as it can be performed under strongly denaturing conditions [76]. Cells transformed with tagged-ubiquitin formed the basis for several studies in different organisms that have been undertaken since, with the first large-scale study performed in yeast [54].…”