Panessa, G. M. | Search bioRxiv

bioRxiv2026

Kcs1-dependent inositol pyrophosphates promote DNA double-strand break repair via Pho80-Pho85 in budding yeast

The cleavage of membrane-bound phosphoinositides produces inositol polyphosphates (IPs) and pyrophosphates (PP-IPs), small water-soluble signaling molecules that are highly conserved in eukaryotes and play a critical role in cell metabolism. Loss of the inositol pyrophosphate kinase (PP-IPk) Kcs1 and its mammalian ortholog IP6K1 was previously shown to cause hypersensitivity to genotoxic stress and reduced homologous recombination (HR) repair. To identify the mechanism linking PP-IPs to DNA double-strand break (DSB) repair, we combined quantitative proteomics with biochemical, genetic, and cell-cycle analyses in budding yeast. We found that loss of Kcs1, but not the PP-IPk Vip1, caused increased sensitivity to the radiomimetic drug zeocin. kcs1{Delta} cells displayed persistent Rad53 signaling and elevated {gamma}-H2A levels, remaining arrested in G1/S during treatment and accumulating in G2/M for hours after damage removal. Unexpectedly, Kcs1-dependent PP-IPs promoted DSB repair independently of the core HR machinery and of cell-cycle stage. Quantitative proteomics revealed increased levels of PHO pathway proteins in zeocin treated kcs{Delta} cells, and deletion of the CDK inhibitor PHO81 substantially rescued zeocin hypersensitivity, indicating that constitutive inhibition of the Pho80-Pho85 cyclin-dependent kinase underlies the DNA repair defect caused by Kcs1 loss. This function of Pho80-Pho85 was mechanistically distinct from its canonical role in phosphate homeostasis, and partially independent of its connections to Ku80 regulation. Together, our findings identify Kcs1-dependent PP-IP signaling as a new upstream input controlling Pho80-Pho85 activity during the DNA damage response, establishing an HR-independent axis for genome maintenance and linking PP-IP metabolism to the DNA damage response in eukaryotic cells.

cell biology↗

bioRxiv2022

The transcriptional repressor Opi1 modulates the DNA Damage Response by downregulation of inositol pyrophosphates in Saccharomyces cerevisiae

In budding yeast, the transcriptional repressor Opi1 regulates phospholipid biosynthesis by repressing expression of genes containing inositol-sensitive upstream activation sequences (UASINO). Upon genotoxic stress, cells activate the DNA Damage Response (DDR) to coordinate a complex network of signaling pathways aimed at preserving genomic integrity. Here, we reveal that Opi1 is important to modulate transcription in response to genotoxic stress. We find that cells lacking Opi1 exhibit hypersensitivity to genotoxins, along with a delayed G1 to S-phase transition and decreased gamma-H2A levels. Transcriptome analysis using RNA-seq reveals that Opi1 plays a central role in modulating essential biological processes during genotoxic stress induced by methyl methanesulfonate, including repression of phospholipid biosynthesis and transduction of mating signaling. Moreover, Opi1 induces sulfate assimilation and amino acid metabolic processes, such as arginine and histidine biosynthesis and glycine catabolism. Furthermore, we observe increased mitochondrial DNA instability in opi1{Delta} cells upon MMS treatment. Notably, we show that constitutive activation of the transcription factors Ino2-Ino4 is responsible for genotoxin sensitivity in Opi1-deficient cells, and the production of inositol pyrophosphates by Kcs1 counteracts Opi1 function specifically during MMS-induced genotoxic stress. Overall, our findings highlight Opi1 as a critical sensor of genotoxic stress in budding yeast, orchestrating gene expression to facilitate appropriate DNA damage response.

cell biology↗