Loss of a 20S Proteasome Activator in Saccharomyces cerevisiae Downregulates Genes Important for Genomic Integrity Increases DNA Damage and Selectively Sensitizes Cells to Agents With Diverse Mechanisms of Action

摘要

Cytoprotective functions of a 20S proteasome activator were investigated. Saccharomyces cerevisiae and human 20S proteasome activator 200 (PA200) are homologs. Comparative genome-wide analyses of untreated diploid cells lacking and growing at steady state at defined growth rates revealed downregulation of numerous genes required for accurate chromosome structure, assembly and repair, and upregulation of a specific subset of genes encoding protein-folding chaperones. loss or truncation of the / deubiquitinating enzyme caused massive chromosomal damage and cell death in homozygous diploids after phleomycin treatments, indicating that and / function to stabilize the genome and protect against cell death. Diploids lacking also were sensitized to doxorubicin, hydroxyurea, 5-fluorouracil, rapamycin, hydrogen peroxide, methyl methanesulfonate, and calcofluor. Fluorescently tagged localized in nuclei, with enhanced fluorescence after DNA replication. After DNA damage that caused a classic G2/M arrest, fluorescence remained diffuse, with evidence of nuclear fragmentation in some cells. Protective functions of did not require the carboxyl-terminal region that makes close contact with 20S proteasomes, indicating that protection does not require this contact or the truncated can interact with the proteasome apart from this region. Without its carboxyl-terminus, (−339aa) localized to nuclei in untreated, nonproliferating (G0) cells, but not during G1 S, G2, and M. The results indicate functions in protective mechanisms that include the machinery that assures proper assembly of chromosomes. These essential guardian functions have implications for ubiquitin-independent targeting in anticancer therapy. Targeting /PA200 together with one or more of the upregulated chaperones or a conventional treatment could be efficacious.