Yeast Rpn4 Links the Proteasome and DNA Repair via RAD52 Regulation

Abstract

Environmental and intracellular factors often damage DNA, but multiple DNA repair pathways maintain genome integrity. In yeast, the 26S proteasome and its transcriptional regulator and substrate Rpn4 are involved in DNA damage resistance. Paradoxically, while proteasome dysfunction may induce hyper-resistance to DNA-damaging agents, Rpn4 malfunction sensitizes yeasts to these agents. Previously, we proposed that proteasome inhibition causes Rpn4 stabilization followed by the upregulation of Rpn4-dependent DNA repair genes and pathways. Here, we aimed to elucidate the key Rpn4 targets responsible for DNA damage hyper-resistance in proteasome mutants. We impaired the Rpn4-mediated regulation of candidate genes using the CRISPR/Cas9 system and tested the sensitivity of mutant strains to 4-NQO, MMS and zeocin. We found that the separate or simultaneous deregulation of 19S or 20S proteasome subcomplexes induced MAG1, DDI1, RAD23 and RAD52 in an Rpn4-dependent manner. Deregulation of RAD23, DDI1 and RAD52 sensitized yeast to DNA damage. Genetic, epigenetic or dihydrocoumarin-mediated RAD52 repression restored the sensitivity of the proteasome mutants to DNA damage. Our results suggest that the Rpn4-mediated overexpression of DNA repair genes, especially RAD52, defines the DNA damage hyper-resistant phenotype of proteasome mutants. The developed yeast model is useful for characterizing drugs that reverse the DNA damage hyper-resistance phenotypes of cancers.

Yeast Ribonucleotide Reductase Is a Direct Target of the Proteasome and Provides Hyper Resistance to the Carcinogen 4-NQO

“…Therefore, this experiment can be considered as a model study of the effect of RNR inhibitors on the phenotype of cancer hyper resistance to DNA-damaging drugs. As previously shown, YPL cells are more viable when chronically exposed to 4-NQO [ 11 , 13 ]. Thus, if increased RNR activity is involved in cell resistance to 4-NQO, its inhibition may cause reduced YPL growth under 4-NQO stress.…”

“…Despite this, we and other researchers have previously shown that proteasome mutant strains of yeast are hyper-resistant to several DNA-damaging agents, including 4-NQO [ 11 , 12 ]. Previously, we showed that increased levels of Rpn4-dependent DNA repair genes cause hyper resistance to 4-NQO in proteasome mutants [ 11 , 13 ]. Acquiring resistance should be accompanied by increased levels of proteins managing DNA reparation/replication, as well as degradation of damaged proteins.…”

“…The complex cellular response to 4-NQO also includes activation of the DNA damage response [ 8 ]. We have previously shown that several DNA repair pathways [ 11 , 13 ] are upregulated in the YPL strain. Accordingly, the MCODE algorithm identified a complex involved in DNA replication and consisting of RNR1, MCM3, and MCM6 ( Figure 1 b).…”

“…To study proteasome-dependent mechanisms of yeast responses to stress, including DNA damage, we previously created mutant strains with decreased expression of the essential proteasome subunits [ 11 , 13 , 14 ] due to mutation of binding sites (Proteasome Associated Control Element, PACE) for Rpn4, the main regulator of proteasome biogenesis [ 15 , 16 ]. In particular, the YPL strain carries a pre1–8 PACE mutation in the promoter region of PRE1 , which encodes an essential structural subunit of the 20S proteolytic proteasome subcomplex [ 11 ].…”

“…We and other researchers have shown that deregulation of PRE1 decreases proteasome activity and leads to the phenotypes of impaired proteasome function seen in other strains carrying mutations in the coding region of PRE1 and other genes encoding proteasome subunits [ 11 , 17 ]. Using a set of these mutants, we showed that disruption of proteasome function leads to an increase in Rpn4-dependent upregulation of key genes of DNA repair systems [ 11 , 13 ]. Thus, we gain insight into how disruption of proteasome function can lead to increased resistance to DNA damage.…”