BackgroundThe maintenance of genomic integrity is essential for cell viability. Complex signalling pathways (DNA integrity checkpoints) mediate the response to genotoxic stresses. Identifying new functions involved in the cellular response to DNA-damage is crucial. The Saccharomyces cerevisiae SLT2 gene encodes a member of the mitogen-activated protein kinase (MAPK) cascade whose main function is the maintenance of the cell wall integrity. However, different observations suggest that SLT2 may also have a role related to DNA metabolism.ResultsThis work consisted in a comprehensive study to connect the Slt2 protein to genome integrity maintenance in response to genotoxic stresses. The slt2 mutant strain was hypersensitive to a variety of genotoxic treatments, including incubation with hydroxyurea (HU), methylmetanosulfonate (MMS), phleomycin or UV irradiation. Furthermore, Slt2 was activated by all these treatments, which suggests that Slt2 plays a central role in the cellular response to genotoxic stresses. Activation of Slt2 was not dependent on the DNA integrity checkpoint. For MMS and UV, Slt2 activation required progression through the cell cycle. In contrast, HU also activated Slt2 in nocodazol-arrested cells, which suggests that Slt2 may respond to dNTP pools alterations. However, neither the protein level of the distinct ribonucleotide reductase subunits nor the dNTP pools were affected in a slt2 mutant strain. An analysis of the checkpoint function revealed that Slt2 was not required for either cell cycle arrest or the activation of the Rad53 checkpoint kinase in response to DNA damage. However, slt2 mutant cells showed an elongated bud and partially impaired Swe1 degradation after replicative stress, indicating that Slt2 could contribute, in parallel with Rad53, to bud morphogenesis control after genotoxic stresses.ConclusionsSlt2 is activated by several genotoxic treatments and is required to properly cope with DNA damage. Slt2 function is important for bud morphogenesis and optimal Swe1 degradation under replicative stress. The MAPK Slt2 appears as a new player in the cellular response to genotoxic stresses.

The protein kinase C (PKC) superfamily plays key regulatory roles in numerous cellular processes. Saccharomyces cerevisiae contains a single PKC, Pkc1, whose main function is cell wall integrity maintenance. In this work, we connect the Pkc1 protein to the maintenance of genome integrity in response to genotoxic stresses. Pkc1 and its kinase activity are necessary for the phosphorylation of checkpoint kinase Rad53, histone H2A and Xrs2 protein after deoxyribonucleic acid (DNA) damage, indicating that Pkc1 is required for activation of checkpoint kinases Mec1 and Tel1. Furthermore, Pkc1 electrophoretic mobility is delayed after inducing DNA damage, which reflects that Pkc1 is post-translationally modified. This modification is a phosphorylation event mediated by Tel1. The expression of different mammalian PKC isoforms at the endogenous level in yeast pkc1 mutant cells revealed that PKCδ is able to activate the DNA integrity checkpoint. Finally, downregulation of PKCδ activity in HeLa cells caused a defective activation of checkpoint kinase Chk2 when DNA damage was induced. Our results indicate that the control of the DNA integrity checkpoint by PKC is a mechanism conserved from yeast to humans.

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The budding yeast, Saccharomyces cerevisiae, has been widely used as a model organism to study the molecular mechanisms that regulate gene expression in eukaryotic cells. In the yeast Cell Wall Integrity Pathway (CWI), the protein Kinase C, Pkc1, activates the MAP Kinase Slt2, which in turn targets the transcription factors Rlm1 and SBF (Swi4-Swi6) and the transcriptional complex Paf1C, to modulate and control the expression of cell wall integrity genes. To better describe the connection between the CWI components and the transcriptional regulation of the cell integrity genes, a series of Chromatin Immunoprecipitation (ChIP) assays were performed. Our results reveal that the MAPK Slt2, associates to the promoter of several cell wall housekeeping genes like FKS1, MNN1 and GAS1. The expression of these genes is reduced in slt2 and pkc1 mutant strains. However, neither the recruitment of the transcription factors Rlm1 and Swi6 to the promoter, nor the binding of the RNApol II or Paf1 to the initiation site is affected. When the association to the 3´ end of FKS1, MNN1 and GAS1 was analyzed, the RNApol II occupancy is not altered but, remarkably, the Paf1 association is importantly reduced in slt2 and pkc1 mutant strains. This result suggests that Slt2 is required for a stable association of Paf1C to the RNApol II along the cell wall genes and that in its absence, Paf1 dissociates from RNApol II causing a defect in RNA 3' end formation, which in turn leads to a reduced mRNA levels.