Interaction between the reductase Tah18 and highly conserved Fe-S containing Dre2 C-terminus is essential for yeast viability.

Tah18-Dre2 is a recently identified yeast protein complex, which is highly conserved in human and has been implicated in the regulation of oxidative stress induced cell death and in cytosolic Fe-S proteins synthesis. Tah18 is a diflavin oxido-reductase with binding sites for flavin mononucleotide, flavin adenine dinucleotide and nicotinamide adenine dinucleotide phosphate, which is able to transfer electrons to Dre2 Fe-S clusters. In this work we characterized in details the interaction between Tah18 and Dre2, and analysed how it conditions yeast viability. We show that Dre2 C-terminus interacts in vivo and in vitro with the flavin mononucleotide- and flavin adenine dinucleotide-binding sites of Tah18. Neither the absence of the electron donor nicotinamide adenine dinucleotide phosphate-binding domain in purified Tah18 nor the absence of Fe-S in aerobically purified Dre2 prevents the binding in vitro. In vivo, when this interaction is affected in a dre2 mutant, yeast viability is reduced. Conversely, enhancing artificially the interaction between mutated Dre2 and Tah18 restores cellular viability despite still reduced cytosolic Fe-S cluster biosynthesis. We conclude that Tah18-Dre2 interaction in vivo is essential for yeast viability. Our study may provide new insight into the survival/death switch involving this complex in yeast and in human cells.

DNA replication inhibitor hydroxyurea alters Fe-S centers by producing reactive oxygen species in vivo.

Redox homeostasis is tightly controlled in cells as it is critical for most cellular functions. Iron-Sulfur centers (Fe-S) are metallic cofactors with electronic properties that are associated with proteins and allow fine redox tuning. Following the observation that altered Fe-S biosynthesis is correlated with a high sensitivity to hydroxyurea (HU), a potent DNA replication blocking agent, we identified that oxidative stress response pathway under the control of the main regulator Yap1 attenuates HU deleterious effects, as it significantly increases resistance to HU, Fe-S biosynthesis and DNA replication kinetics in the presence of HU. Yap1 effect is mediated at least in part through up-regulation of two highly conserved genes controlling cytosolic Fe-S biosynthesis and oxidative stress, Dre2 and Tah18. We next observed that HU produces deleterious effects on cytosolic Fe-S clusters in proteins in vivo but not in vitro, suggesting that HU's impact on Fe-S in vivo is mediated by cellular metabolism. Finally, we evidenced that HU exposure was accompanied by production of reactive oxygen species intracellularly. Altogether, this study provides mechanistic insight on the initial observation that mutants with altered Fe-S biosynthesis are highly sensitive to HU and uncovers a novel mechanism of action of this widely used DNA replication inhibitor.

Yeast as a model system to screen purine derivatives against human CDK1 and CDK2 kinases.

Cyclin-dependent kinases (Cdk) play crucial roles in cell cycle progression. Aberrant activation of Cdk1 has been observed in a number of primary tumors and Cdk2 is deregulated in various malignancies. The therapeutic value of targeting Cdk1 and Cdk2 has been explored in a number of experimental systems. In the present study, taking advantage of the fact that deletion of the yeast CDC28 gene is functionally complemented by human CDK1 or CDK2, we set up an in vivo screen system to evaluate the inhibitory potency of purine derivatives against these two human Cdks. We constructed three isogenic strains highly sensitive to small molecules and harboring genes CDK1, CDK2 or CDC28, under the control of the CDC28 promoter. In a proof of principle assay, we determined the inhibitory effect of 82 purine derivatives on the growth rate of these strains. Thirty-three of them were revealed to be able to inhibit the Cdk1- or Cdk2-harboring strains but not the Cdc28-harboring strain, suggesting a specific inhibitory effect on human Cdks. Our data demonstrate that the yeast-based assay is an efficient system to identify potential specific inhibitors that should be preferentially selected for further investigation in cultured human cell lines.

A newly identified essential complex, Dre2-Tah18, controls mitochondria integrity and cell death after oxidative stress in yeast.

A mutated allele of the essential gene TAH18 was previously identified in our laboratory in a genetic screen for new proteins interacting with the DNA polymerase delta in yeast [1]. The present work shows that Tah18 plays a role in response to oxidative stress. After exposure to lethal doses of H(2)O(2), GFP-Tah18 relocalizes to the mitochondria and controls mitochondria integrity and cell death. Dre2, an essential Fe/S cluster protein and homologue of human anti-apoptotic Ciapin1, was identified as a molecular partner of Tah18 in the absence of stress. Moreover, Ciapin1 is able to replace yeast Dre2 in vivo and physically interacts with Tah18. Our results are in favour of an oxidative stress-induced cell death in yeast that involves mitochondria and is controlled by the newly identified Dre2-Tah18 complex.

Kinase Cak1 functionally interacts with the PAF1 complex and phosphatase Ssu72 via kinases Ctk1 and Bur1.

Protein kinases orthologous with Cak1 of Saccharomyces cerevisiae (ScCak1) appear specific to ascomycetes. ScCak1 phosphorylates Cdc28, the cyclin-dependent kinase (CDK) governing the cell cycle, as well as Kin28, Bur1 and Ctk1, CDKs required for the transcription process performed by RNA polymerase II (RNA Pol II). Using genetic methods, we found that Cak1 genetically interacts with Paf1 and Ctr9, two components belonging to the PAF1 elongation complex needed for histone modifications, and with Ssu72, a protein phosphatase that dephosphorylates serine-5 phosphate in the RNA Pol II C-terminal domain. We present evidence suggesting that the interactions linking Cak1 with the PAF1 complex and with Ssu72 are not direct but mediated via Ctk1 and Bur1. We discuss the possibility that Ssu72 intervenes at the capping checkpoint step of the transcription cycle.

Ssu72 is a phosphatase essential for transcription termination of snoRNAs and specific mRNAs in yeast.

Ssu72 is an essential yeast protein that is involved in transcription. It physically interacts with transcription initiation and termination complexes. In this report, we provide evidence that Ssu72 is a phosphatase that physically interacts with the CTD kinase Kin28 and functionally interacts with the CTD phosphatase Fcp1. A genome-wide expression analysis of mutant ssu72-ts69 during growth in complete medium revealed a number of defects, including the accumulation of a limited number of mRNAs and the read-through transcription of small nucleolar RNAs and of some mRNAs. We hypothesize that Ssu72 plays a key role in the transcription termination of certain transcripts, possibly by promoting RNA polymerase pausing and release. The possibility that the CTD of the largest subunit of RNA polymerase II is a substrate of Ssu72 is discussed.