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 assembled through Lys-63 play a mechanistically undefined role in post-replicative 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 polyubiquitin chains in vitro. Here we confirm that the Mms2.Ubc13 complex functions as a high affinity heterodimer in the chain assembly reaction in vitro and report the results of a kinetic characterization of the polyubiquitin chain assembly reaction. To test whether a Lys-63-linked polyubiquitin chain can signal degradation, we conjugated Lys-63-linked tetra-ubiquitin to a model substrate of 26 S proteasomes. Although the noncanonical chain effectively signaled substrate degradation, the results of new genetic epistasis studies agree with previous genetic data in suggesting that the proteolytic activity of proteasomes is not required for error-free post-replicative repair.
Hofmann
R M
RM
Department of Biochemistry and Molecular Biology, School of Public Health, Johns Hopkins University, Baltimore, Maryland 21205, USA.
Pickart
C M
CM
D001667:Binding, Competitive; D001704:Biopolymers / Q000737:chemistry*; D002384:Catalysis; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004260:DNA Repair; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004307:Dose-Response Relationship, Radiation; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008025:Ligases / Q000737:chemistry* / Q000378:metabolism; D008239:Lysine / Q000737:chemistry*; D009097:Multienzyme Complexes / Q000378:metabolism; D009154:Mutation; D010447:Peptide Hydrolases / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D025821:Polyubiquitin; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D015398:Signal Transduction; D013997:Time Factors; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000737:chemistry*; D014466:Ultraviolet Rays
D001704:Biopolymers; D005656:Fungal Proteins; C112520:MMS2 protein, S cerevisiae; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D014452:Ubiquitins; D025821:Polyubiquitin; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D010447:Peptide Hydrolases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; D008025:Ligases; D008239:Lysine