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.
Cytoprotective functions of a 20S proteasome activator were investigated. Saccharomyces cerevisiae Blm10 and human 20S proteasome activator 200 (PA200) are homologs. Comparative genome-wide analyses of untreated diploid cells lacking Blm10 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. Blm10 loss or truncation of the Ubp3/Blm3 deubiquitinating enzyme caused massive chromosomal damage and cell death in homozygous diploids after phleomycin treatments, indicating that Blm10 and Ubp3/Blm3 function to stabilize the genome and protect against cell death. Diploids lacking Blm10 also were sensitized to doxorubicin, hydroxyurea, 5-fluorouracil, rapamycin, hydrogen peroxide, methyl methanesulfonate, and calcofluor. Fluorescently tagged Blm10 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 Blm10 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 Blm10 can interact with the proteasome apart from this region. Without its carboxyl-terminus, Blm10((-339aa)) localized to nuclei in untreated, nonproliferating (G(0)) cells, but not during G(1) S, G(2), and M. The results indicate Blm10 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 Blm10/PA200 together with one or more of the upregulated chaperones or a conventional treatment could be efficacious.
Department of Microbiology and Immunology, City University of New York Sophie Davis School of Biomedical Education, City College, New York, NY 10031-9101, USA.
Kevin M
KM
Doherty
Leah D
LD
Pride
James
J
Lukose
Brian E
BE
Snydsman
Ronald
R
Charles
Ajay
A
Pramanik
Eric G
EG
Muller
David
D
Botstein
Carol Wood
CW
Moore
D000970:Antineoplastic Agents / Q000633:toxicity; D002467:Cell Nucleus / Q000378:metabolism; D004249:DNA Damage / Q000235:genetics; D004171:Diploidy; D015536:Down-Regulation; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism; D059565:G2 Phase Cell Cycle Checkpoints / Q000187:drug effects; D042822:Genomic Instability; D059566:M Phase Cell Cycle Checkpoints / Q000187:drug effects; D018832:Molecular Chaperones / Q000378:metabolism; D009154:Mutation; D016877:Oxidants / Q000633:toxicity; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation / Q000235:genetics
D000970:Antineoplastic Agents; C500597:Blm10 protein, S cerevisiae; D018832:Molecular Chaperones; D016877:Oxidants; D029701:Saccharomyces cerevisiae Proteins; D010450:Endopeptidases; D046988:Proteasome Endopeptidase Complex; C101111:UBP3 protein, S cerevisiae