Abstract
Most mutants affected either in the proteasome biogenesis or function accumulate polyubiquitylated proteins and display growth defects at 37°C or in the presence of canavanine, an arginine analog that impairs protein synthesis. We uncovered a new striking phenotype related to DNA damage for some proteasome mutants: mutant strains grew better than the wild type in the presence of specific genotoxic agents (4NQO, Cpt, and MMS). Hyperresistance to 4NQO or Cpt is a new sensitive tool to detect proteasomal defects. Here, we describe simple methods that can be used to show and quantitatively measure this phenotype in budding yeast.
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References
Le Tallec B, Barrault MB, Courbeyrette R, et al (2007) 20S proteasome assembly is orchestrated by two distinct pairs of chaperones in yeast and in mammals, Mol Cell 27, 660–674.
Le Tallec B, Barrault MB, Guerois R, et al (2009) Hsm3/S5b participates in the assembly pathway of the 19S regulatory particle of the proteasome. Mol Cell 33:389–399.
Ornstein RL, Rein R (1980) Molecular models of induced DNA premutational damage and mutational pathways for the carcinogen 4-nitroquinoline 1-oxide and its metabolites. Chem Biol Interact 30:87–103.
Pommier Y (2006) Topoisomerase I inhibitors: camptothecins and beyond. Nat Rev Cancer 6:789–802.
Wyatt MD, Pittman DL (2006) Methylating agents and DNA repair responses: Methylated bases and sources of strand breaks. Chem Res Toxicol 19:1580–1594.
London MK, Keck BI, Ramos PC, Dohmen RJ (2004) Regulatory mechanisms controlling biogenesis of ubiquitin and the proteasome. FEBS Lett 567:259–264.
Ramos PC, Hockendorff J, Johnson ES, et al (1998) Ump1p is required for proper maturation of the 20S proteasome and becomes its substrate upon completion of the assembly. Cell 92:489–499.
Acknowledgments
This work was supported by the Commisariat à l’Energie Atomique (CEA), the Agence Nationale de la Recherche (ANR), and the Association pour la Recherche sur le Cancer (ARC).
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Tallec, B.L., Peyroche, A. (2012). Using DNA Damage Sensitivity Phenotypes to Characterize Mutations Affecting Proteasome Function. In: Dohmen, R., Scheffner, M. (eds) Ubiquitin Family Modifiers and the Proteasome. Methods in Molecular Biology, vol 832. Humana Press. https://doi.org/10.1007/978-1-61779-474-2_25
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DOI: https://doi.org/10.1007/978-1-61779-474-2_25
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