Edited by Ivan SadowskiKeywords: Yeast DNA damage response Proteasome Rpn4 RAD52 a b s t r a c t The 26S proteasome is an ATP-dependent multi-subunit protease complex and the major regulator of intracellular protein turnover and quality control. However, its role in the DNA damage response is controversial. We addressed this question in yeast by disrupting the transcriptional regulation of the PRE1 proteasomal gene. The mutant strain has decreased proteasome activity and is hyper-resistant to various DNA-damaging agents. We found that Rpn4-target genes MAG1, RAD23, and RAD52 are overexpressed in this strain due to Rpn4 stabilisation. These genes represent three different pathways of base excision, nucleotide excision and double strand break repair by homologous recombination (DSB-HR). Consistently, the proteasome mutant displays increased DSB-HR activity. Our data imply that the proteasome may have a negative role in DNA damage response.

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The marine yeast Debaryomyces hansenii is of high importance in the food, chemical, and medical industries. D. hansenii is also a popular model for studying molecular mechanisms of halo-and osmotolerance. The absence of genome editing technologies hampers D. hansenii research and limits its biotechnological application. We developed novel and efficient single-and dual-guide CRISPR systems for markerless genome editing of D. hansenii. The single-guide system allows highefficiency (up to 95%) mutation of genes or regulatory elements. The dual-guide system is applicable for efficient deletion of genomic loci. We used these tools to study transcriptional regulation of the 26S proteasome, an ATP-dependent protease complex whose proper function is vital for all cells and organisms. We developed a genetic approach to control the activity of the 26S proteasome by deregulation of its essential subunits. The mutant strains were sensitive to geno-and proteotoxic stresses as well as high salinity and osmolarity, suggesting a contribution of the proteasome to the extremophilic properties of D. hansenii. The developed CRISPR systems allow efficient D. hansenii genome engineering, providing a genetic way to control proteasome activity, and should advance applications of this yeast.

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Distilled spirits production using S. cerevisiae requires understanding the mechanisms of yeast cell response to alcohol stress. Reportedly, specific mutations in genes of the ubiquitinproteasome system, e.g. RPN4, may result in strains exhibiting hyper-resistance to different alcohols. To study Rpn4-dependent yeast response to short-term ethanol exposure, we performed comparative analysis of the wild-type strain (WT), strain with RPN4 gene deletion (rpn4-Δ), and mutant strain with decreased proteasome activity and consequent Rpn4 accumulation due to PRE1 deregulation (YPL). The stress resistance tests demonstrated an increased sensitivity of mutant strains to ethanol compared with WT. Comparative proteomics analysis revealed significant differences in molecular responses to ethanol between these strains.GO analysis of proteins upregulated in WT showed enrichments represented by oxidative and heat responses, protein folding/unfolding and protein degradation. Enrichment of at least one of these responses was not observed in the mutant strains. Moreover, activity of autophagy was not increased in RPN4 deletion strain upon ethanol stress which agrees with changes in mRNA levels of ATG7 and PRB1 genes of the autophagy system. Activity of the autophagic system was clearly induced and accompanied with PRB1 overexpression in the YPL strain upon ethanol stress.We demonstrated that Rpn4 stabilization contributes to the PRB1 upregulation. CRISPR-Cas9mediated repression of PACE-core Rpn4 binding sites in the PRB1 promoter inhibits PRB1 induction in the YPL strain upon ethanol treatment and results in YPL hypersensitivity to ethanol.Our data suggest that Rpn4 affects the autophagic system activity upon ethanol stress through the PRB1 regulation. These findings can be a basis for creating genetically modified yeast strains resistant to high levels of alcohol, being further used for fermentation in ethanol production.