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Gcn2p regulates a G1/S cell cycle checkpoint in response to DNA damage

ABSTRACT: In Saccharomyces cerevisiae, the Gcn2p kinase controls a gene expression program in response to nutrient deprivation. Upon amino acid deprivation Gcn2p phosphorylates the translation initiation factor 2a subunit (eIF2a). Phosphorylation of eIF2a transiently inhibits translation initiation, and favours selective translation of the GCN4 transcription factor mRNA. High levels of Gcn4p then activate the expression of genes encoding amino acid biosynthesis pathways. Besides nutrient deprivation, Gcn2p can also be activated by other stresses such as DNA damage. Previous studies in our lab suggested that Gcn2p regulated other cellular pathways apart from translation initiation. To gain insights into these new regulatory roles of Gcn2p, we generated a strain called (GCN2c) expressing a constitutively active Gcn2p kinase mutant. In the GCN2c strain, phosphorylation of eIF2a and translation of a GCN4-lacz reporter was constitutively high in the absence of amino acid starvation. We investigated the transcriptional profile of the GCN2c mutant strain by cDNA microarrays. As expected, numerous amino acid biosynthetic genes were upregulated, being dependent of Gcn4p. Most interestingly, expression of genes encoding proteins involved in DNA replication/repair was decreased, suggesting that Gcn2p could regulate cell cycle progression from G1 to S phase. Accordingly, the Gcn2c strain exhibited, i) slow growth, ii) a delayed entry into S phase, and iii) hypersensitivity to hydroxyurea. In addition, MMS treatment induced a G1 checkpoint and a growth defect which was dependent on Gcn2p and Gcn3p. Considering that MMS delays cell cycle progression by generating DNA lesions, the findings suggest that DNA damage induces a G1/S checkpoint by a novel pathway involving the protein kinase Gcn2p. Keywords: mutant analysis

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE8111 | GEO | 2007/10/22

SECONDARY ACCESSION(S): PRJNA100979

REPOSITORIES: GEO

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Project description:Two nutrient sensing and regulatory pathways, the general amino acid control (GAAC) and the target of rapamycin (TOR), control yeast growth and metabolism in response to changes in nutrient availability. Starvation for amino acids activates the GAAC pathway, involving Gcn2p phosphorylation of eIF2 and preferential translation of GCN4, a transcription activator of genes involved in amino acid metabolism. TOR senses nitrogen availability and regulates gene expression through transcription factors, such as Gln3p. We used microarray analyses to address the integration of the GAAC and TOR pathways in directing the yeast transcriptome in response to amino acid starvation and rapamycin treatment. Of the ~2500 genes whose expression was changed by 2-fold or greater, Gcn4p and Gln3p were required for 542 and 657 genes, respectively. While Gcn4p activates a common core of 57 genes in response to amino acid starvation or rapamycin treatment, the different stress arrangements allow for variations in Gcn4p-directed transcription. With few exceptions, genes requiring Gcn2p eIF2 kinase for induced expression also required Gcn4p, emphasizing the role of Gcn2p as an upstream activator of Gcn4p-directed transcription. There is also significant coordination between the GAAC and TOR pathways, with Gcn4p being required for activation of more genes during rapamycin treatment than Gln3p. Importantly, TOR regulates the GAAC-directed transcription of genes required for assimilation of nitrogen sources, such as Î³-amino-butyric acid. Therefore, yeast has integrated gene expression responses to amino acid abundance and nitrogen source quality through the control of Gcn2p phosphorylation of eIF2 and GCN4 translation. Keywords: gene expression In this study, we carried out microarray analyses in a collection of yeast strains deleted for GCN2, GCN4, and GLN3, individually or in combinations, to explore the importance of the TOR and GAAC pathways in directing the transcriptome in response to amino acid starvation and rapamycin treatment.

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Gcn2p regulates a G1/S cell cycle checkpoint in response to DNA damage

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