RESUMO
The Saccharomyces cerevisiae protein kinase Rad53 plays a key role in maintaining genomic integrity after DNA damage and is an essential component of the 'intra-S-phase checkpoint'. In budding yeast, alkylating chemicals, such as methyl methanesulfonate (MMS), or depletion of nucleotides by hydroxyurea (HU) stall DNA replication forks and thus activate Rad53 during S-phase. This stabilizes stalled DNA replication forks and prevents the activation of later origins of DNA replication. Here, we report that a reduction in the level of Rad53 kinase causes cells to behave very differently in response to DNA alkylation or to nucleotide depletion. While cells lacking Rad53 are unable to activate the checkpoint response to HU or MMS, so that they rapidly lose viability, a reduction in Rad53 enhances cell survival only after DNA alkylation. This reduction in the level of Rad53 allows S-phase cells to maintain the stability of DNA replication forks upon MMS treatment, but does not prevent the collapse of forks in HU. Our results may have important implications for cancer therapies, as they suggest that partial impairment of the S-phase checkpoint Rad53/Chk2 kinase provides cells with a growth advantage in the presence of drugs that damage DNA.
Assuntos
Antineoplásicos Alquilantes/toxicidade , Proteínas de Ciclo Celular/metabolismo , Dano ao DNA , Proteínas Serina-Treonina Quinases/metabolismo , Fase S/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Alelos , Proteínas de Ciclo Celular/genética , Quinase do Ponto de Checagem 2 , Replicação do DNA , Resistência a Medicamentos , Deleção de Genes , Hidroxiureia/toxicidade , Metanossulfonato de Metila/toxicidade , Mutação , Proteínas Serina-Treonina Quinases/genética , Fase S/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transcrição GênicaRESUMO
Swm1p, a subunit of the APC cyclosome, was originally identified for its role in the later stages of the sporulation process and is required for spore wall assembly. In addition, this protein is required to maintain cell wall integrity in vegetative cells during growth at high temperature. Electron microscopy analyses of mutant cells grown at the restrictive temperature in the absence of osmotic support show that the cell wall is clearly abnormal, with large number of discontinuities that may be responsible for the observed lysis. The mutant cells show a 7-fold reduction in glucan synthase activity during growth at 38 degrees C and a 3.5-fold increase in the chitin content of the cell wall. The chitin is deposited in a delocalized manner all over the cell wall, where it accumulates in patches in abnormal regions. The excess chitin is mainly synthesized by the action of chitin synthase III (Chs3p), since it disappears in the swm1 chs3 double-mutant.
Assuntos
Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/genética , Complexos Ubiquitina-Proteína Ligase/genética , Ciclossomo-Complexo Promotor de Anáfase , Temperatura Alta , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas de Saccharomyces cerevisiae/genética , TermodinâmicaRESUMO
Checkpoint response, tolerance and repair are three major pathways that eukaryotic cells evolved independently to maintain genome stability and integrity. Here, we studied the sensitivity to DNA damage in checkpoint-deficient budding yeast cells and found that checkpoint kinases Mec1 and Rad53 may modulate the balance between error-free and error-prone branches of the tolerance pathway. We have consistently observed that mutation of the RAD53 counterbalances error-free and error-prone branches upon exposure of cells to DNA damage induced either by MMS alkylation or by UV-radiation. We have also found that the potential Mec1/Rad53 balance modulation is independent from Rad6/Rad18-mediated PCNA ubiquitylation, as mec1Δ or rad53Δ mutants show no defects in the modification of the sliding clamp, therefore, we infer that it is likely exerted by acting on TLS polymerases and/or template switching targets.
Assuntos
Alquilantes/farmacologia , Proteínas de Ciclo Celular/genética , Quinase do Ponto de Checagem 2/genética , Regulação Fúngica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Metanossulfonato de Metila/farmacologia , Proteínas Serina-Treonina Quinases/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mutação , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efeitos da radiação , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Raios UltravioletaRESUMO
Borna disease virus (BDV) is a nonsegmented, negative-stranded RNA virus that causes neurological diseases in a variety of warm-blooded animal species. Recently, we showed that the nucleoside analog 1-beta-D-arabinofuranosylcytosine (Ara-C) was a potent inhibitor of BDV. This finding was surprising for an RNA virus, since Ara-C is a DNA polymerase inhibitor. Thus, we sought to better define the mechanism of action of Ara-C on BDV. Here, we show that (i) this effect is specific for an arabinoside ring carrying a cytosine base, (ii) it requires phosphorylation of the nucleotide, and (iii) it can be reversed by an excess of cytidine. Using the recently described minigenome assay for BDV, we provide evidence suggesting that Ara-C may act as a competitive inhibitor of the BDV replication complex.
Assuntos
Antivirais/farmacologia , Vírus da Doença de Borna/efeitos dos fármacos , Citarabina/farmacologia , Inibidores Enzimáticos/farmacologia , Replicação Viral/efeitos dos fármacos , Vírus da Doença de Borna/fisiologiaRESUMO
ENG1 (YNR067c), a gene encoding a new endo-1,3-beta-glucanase, was cloned by screening a genomic library with a DNA probe obtained by PCR with synthetic oligonucleotides designed according to conserved regions found between yeast exo-1,3-beta-glucanases (Exglp, Exg2p, and Ssglp). Eng1p shows strong sequence similarity to the product of the Saccharomyces cerevisiae ACF2 gene, involved in actin assembly "in vitro," and to proteins present in other yeast and fungal species. It is also related to plant glucan-binding elicitor proteins, which trigger the onset of a defense response upon fungal infection. Eng1p and Acf2p/Eng2p are glucan-hydrolyzing proteins that specifically act on 1,3-beta linkages, with an endolytic mode of action. Eng1p is an extracellular, heavily glycosylated protein, while Acf2p/Eng2p is an intracellular protein with no carbohydrate linked by N-glycosidic bonds. ENG1 transcription fluctuates periodically during the cell cycle; maximal accumulation occurs during the M/G1 transition and is dependent on the transcription factor Ace2p. Interestingly, eng1 deletion mutants show defects in cell separation, and Eng1p localizes asymmetrically to the daughter side of the septum, suggesting that this protein is involved, together with chitinase, in the dissolution of the mother-daughter septum.
Assuntos
Glucana Endo-1,3-beta-D-Glucosidase/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Divisão Celular , Clonagem Molecular , Regulação Fúngica da Expressão Gênica , Glucana Endo-1,3-beta-D-Glucosidase/genética , Glucanos/metabolismo , Dados de Sequência Molecular , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Análise de Sequência de DNA , Esporos Fúngicos/fisiologia , Especificidade por SubstratoRESUMO
SWM1 was originally identified for its role in the late steps of the sporulation process, being required for spore wall assembly. This protein, recently identified as one of the core subunits of the anaphase-promoting complex (APC) is also required to complete cell separation in vegetative cells during growth at high temperature. Mutants lacking SWM1 show a thermosensitive growth defect that is suppressed by osmotic support in the culture medium. At the restrictive temperature, swm1 mutants are unable to complete separation, forming chains of cells that remain associated and, with prolonged incubation times, the stability of the cell wall is compromised, resulting in cell lysis. This separation defect is due to a reduction in expression of CTS1 (the gene encoding chitinase) and a group of genes involved in cell separation (such as ENG1,SCW11, DSE1 and DSE2). Interestingly, these genes are specifically regulated by the transcription factor Ace2p, suggesting that Swm1p is required for normal expression of Ace2p-dependent genes during growth at high temperatures. Although no defect in Ace2p localization can be observed at 28 degrees C, this transcription factor is unable to enter the nucleus of the daughter cell during growth at 38 degrees C. Under these growth conditions, swm1 cells undergo a delay in exit from mitosis, as determined by analysis of Clb2p degradation and Cdc28p-Clb2p kinase assays, and this could be the reason for the cytoplasmic localization of Ace2p.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Regulação Fúngica da Expressão Gênica , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Fatores de Transcrição/fisiologia , Complexos Ubiquitina-Proteína Ligase/fisiologia , Ciclossomo-Complexo Promotor de Anáfase , Quitinases/metabolismo , Mutação , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/metabolismo , Temperatura , Fatores de Tempo , Complexos Ubiquitina-Proteína Ligase/genética , Ubiquitina-Proteína LigasesRESUMO
Protein kinase GCN2 regulates translation initiation by phosphorylating eukaryotic initiation factor 2alpha (eIF2alpha), impeding general protein synthesis but specifically inducing translation of GCN4, a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cerevisiae. GCN2 activity is stimulated in amino acid-deprived cells through binding of uncharged tRNA to a domain related to histidyl tRNA synthetase. We show that GCN2 is phosphorylated by another kinase on serine 577, located N-terminal to the kinase domain. Mutation of Ser-577 to alanine produced partial activation of GCN2 in nonstarved cells, increasing the level of phosphorylated eIF2alpha, derepressing GCN4 expression, and elevating the cellular levels of tryptophan and histidine. The Ala-577 mutation also increased the tRNA binding affinity of purified GCN2, which can account for the elevated kinase activity of GCN2-S577A in nonstarved cells where uncharged tRNA levels are low. Whereas Ser-577 remains phosphorylated in amino acid-starved cells, its dephosphorylation could mediate GCN2 activation in other stress or starvation conditions by lowering the threshold of uncharged tRNA required to activate the protein.