RESUMEN
BACKGROUND: Human hexokinase 2 (HK2) plays an important role in regulating Warburg effect, which metabolizes glucose to lactate acid even in the presence of ample oxygen and provides intermediate metabolites to support cancer cell proliferation and tumor growth. HK2 overexpression has been observed in various types of cancers and targeting HK2-driven Warburg effect has been suggested as a potential cancer therapeutic strategy. Given that epigenetic enzymes utilize metabolic intermediates as substrates or co-factors to carry out post-translational modification of histones and nucleic acids modifications in cells, we hypothesized that altering HK2 expression could impact the epigenome and, consequently, chromatin stability in yeast. To test this hypothesis, we established genetic models with different yeast hexokinase 2 (HXK2) expression in Saccharomyces cerevisiae yeast cells and investigated the effect of HXK2-dependent metabolism on parental nucleosome transfer, a key DNA replication-coupled epigenetic inheritance process, and chromatin stability. RESULTS: By comparing the growth of mutant yeast cells carrying single deletion of hxk1Δ, hxk2Δ, or double-loss of hxk1Δ hxk2Δ to wild-type cells, we firstly confirmed that HXK2 is the dominant HXK in yeast cell growth. Surprisingly, manipulating HXK2 expression in yeast, whether through overexpression or deletion, had only a marginal impact on parental nucleosome assembly, but a noticeable trend with decrease chromatin instability. However, targeting yeast cells with 2-deoxy-D-glucose (2-DG), a clinical glycolysis inhibitor that has been proposed as an anti-cancer treatment, significantly increased chromatin instability. CONCLUSION: Our findings suggest that in yeast cells lacking HXK2, alternative HXKs such as HXK1 or glucokinase 1 (GLK1) play a role in supporting glycolysis at a level that adequately maintains epigenomic stability. While our study demonstrated an increase in epigenetic instability with 2-DG treatment, the observed effect seemed to occur dependent on non-glycolytic function of Hxk2. Thus, additional research is needed to identify the molecular mechanism through which 2-DG influences chromatin stability.
Asunto(s)
Cromatina , Epigénesis Genética , Hexoquinasa , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Hexoquinasa/metabolismo , Hexoquinasa/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Cromatina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Nucleosomas/metabolismo , Regulación Fúngica de la Expresión GénicaRESUMEN
The nascent polypeptide-associate complex (NAC) is a heterodimeric chaperone complex that binds near the ribosome exit tunnel and is the first point of chaperone contact for newly synthesized proteins. Deletion of the NAC induces embryonic lethality in many multi-cellular organisms. Previous work has shown that the deletion of the NAC rescues cells from prion-induced cytotoxicity. This counterintuitive result led us to hypothesize that NAC disruption would improve viability in cells expressing human misfolding proteins. Here, we show that NAC disruption improves viability in cells expressing expanded polyglutamine and also leads to delayed and reduced aggregation of expanded polyglutamine and changes in polyglutamine aggregate morphology. Moreover, we show that NAC disruption leads to changes in de novo yeast prion induction. These results indicate that the NAC plays a critical role in aggregate organization as a potential therapeutic target in neurodegenerative disorders.
Asunto(s)
Chaperonas Moleculares , Péptidos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Péptidos/metabolismo , Humanos , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Priones/metabolismo , Agregado de Proteínas , Agregación Patológica de Proteínas/metabolismoRESUMEN
Autophagy is a key lysosomal degradative mechanism allowing a prosurvival response to stresses, especially nutrient starvation. Here we investigate the mechanism of autophagy induction in response to sulfur starvation in Saccharomyces cerevisiae. We found that sulfur deprivation leads to rapid and widespread transcriptional induction of autophagy-related (ATG) genes in ways not seen under nitrogen starvation. This distinctive response depends mainly on the transcription activator of sulfur metabolism Met4. Consistently, Met4 is essential for autophagy under sulfur starvation. Depletion of either cysteine, methionine or SAM induces autophagy flux. However, only SAM depletion can trigger strong transcriptional induction of ATG genes and a fully functional autophagic response. Furthermore, combined inactivation of Met4 and Atg1 causes a dramatic decrease in cell survival under sulfur starvation, highlighting the interplay between sulfur metabolism and autophagy to maintain cell viability. Thus, we describe a pathway of sulfur starvation-induced autophagy depending on Met4 and involving SAM as signaling sulfur metabolite.
Asunto(s)
Autofagia , S-Adenosilmetionina , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Transducción de Señal , Azufre , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Autofagia/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Azufre/metabolismo , S-Adenosilmetionina/metabolismo , Regulación Fúngica de la Expresión Génica , Proteínas Relacionadas con la Autofagia/metabolismo , Proteínas Relacionadas con la Autofagia/genética , Metionina/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Proteínas Quinasas , Factores de Transcripción con Cremalleras de Leucina de Carácter BásicoRESUMEN
BACKGROUND: Alzheimer's disease (AD) is the most common neurogenerative disorder without effective treatments. Defects in mitochondrial complex I are thought to contribute to AD pathogenesis. The aim of this study is to explore whether a novel gene therapy transducing yeast complex I gene NDI1 can be used to treat AD with severely reduced complex I function in cell and animal models. METHODS: The differentiated human neural cells were induced by Aß1-42 to establish the AD cell model, and adeno-associated virus serotype 9 (AAV9) was used to transduce yeast NDI1 into the cell model. Aß1-42 was injected into the hippocampus area of the brain to establish the AD mouse model. AAV9-NDI1 was injected stereotaxically into the hippocampus area to test the therapeutic effect. RESULTS: The expressed yeast complex I had an ameliorating effect on the defective function of human complex I and cellular pathological characteristics in the AD cell model. Furthermore, AAV9-NDI1 gene therapy in the hippocampus had a therapeutic effect on various aspects of mitochondrial function, histopathological characteristics and neurological defects in the AD mouse model. In addition, AAV9-NDI1 injection into the hippocampus of normal mice did not cause any adverse effect. CONCLUSIONS: Compensating mitochondrial complex I function with yeast NDI1 is effective for gene therapy in Aß-induced AD cell and mouse models. The results of this study offer a novel strategy and approach for treating AD types characterized by complex I abnormalities.
Asunto(s)
Enfermedad de Alzheimer , Péptidos beta-Amiloides , Modelos Animales de Enfermedad , Complejo I de Transporte de Electrón , Terapia Genética , Mitocondrias , Animales , Enfermedad de Alzheimer/terapia , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/genética , Complejo I de Transporte de Electrón/metabolismo , Complejo I de Transporte de Electrón/genética , Humanos , Péptidos beta-Amiloides/metabolismo , Mitocondrias/metabolismo , Dependovirus/genética , Hipocampo/patología , Hipocampo/metabolismo , Ratones , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Neuronas/metabolismo , Neuronas/patología , Fragmentos de Péptidos , MasculinoRESUMEN
Loss of Heterozygosity (LOH) due to mitotic recombination is frequently associated with the development of various cancers (e.g. retinoblastoma). LOH is also an important source of genetic diversity, especially in organisms where meiosis is infrequent. Irc20 is a putative helicase, and E3 ubiquitin ligase involved in DNA double-strand break repair pathway. We analyzed genome-wide LOH events, gross chromosomal changes, small insertion-deletions and single nucleotide mutations in eleven S. cerevisiae mutation accumulation lines of irc20∆, which underwent 50 mitotic bottlenecks. LOH enhancement in irc20∆ was small (1.6 fold), but statistically significant as compared to the wild type. Short (≤ 1â¯kb) and long (> 10â¯kb) LOH tracts were significantly enhanced in irc20∆. Both interstitial and terminal LOH events were also significantly enhanced in irc20∆ compared to the wild type. LOH events in irc20∆ were more telomere proximal and away from centromeres compared to the wild type. Gross chromosomal changes, single nucleotide mutations and in-dels were comparable between irc20∆ and wild type. Locus based and genome-wide analysis of meiotic recombination showed that meiotic crossover frequencies are not altered in irc20∆. These results suggest Irc20 primarily regulates mitotic recombination and does not affect meiotic crossovers. Our results suggest that the IRC20 gene is important for regulating LOH frequency and distribution.
Asunto(s)
Pérdida de Heterocigocidad , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , ADN Helicasas/metabolismo , ADN Helicasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Mitosis , Mutación , Reparación del ADN , Meiosis , Roturas del ADN de Doble CadenaRESUMEN
Budding yeast, Saccharomyces cerevisiae, is widely used as a model organism to study the genetics underlying eukaryotic cellular processes and growth critical to cancer development, such as cell division and cell cycle progression. The budding yeast cell cycle is also one of the best-studied dynamical systems owing to its thoroughly resolved genetics. However, the dynamics underlying the crucial cell cycle decision point called the START transition, at which the cell commits to a new round of DNA replication and cell division, are under-studied. The START machinery involves a central cyclin-dependent kinase; cyclins responsible for starting the transition, bud formation, and initiating DNA synthesis; and their transcriptional regulators. However, evidence has shown that the mechanism is more complicated than a simple irreversible transition switch. Activating a key transcription regulator SBF requires the phosphorylation of its inhibitor, Whi5, or an SBF/MBF monomeric component, Swi6, but not necessarily both. Also, the timing and mechanism of the inhibitor Whi5's nuclear export, while important, are not critical for the timing and execution of START. Therefore, there is a need for a consolidated model for the budding yeast START transition, reconciling regulatory and spatial dynamics. We built a detailed mathematical model (START-BYCC) for the START transition in the budding yeast cell cycle based on established molecular interactions and experimental phenotypes. START-BYCC recapitulates the underlying dynamics and correctly emulates key phenotypic traits of ~150 known START mutants, including regulation of size control, localization of inhibitor/transcription factor complexes, and the nutritional effects on size control. Such a detailed mechanistic understanding of the underlying dynamics gets us closer towards deconvoluting the aberrant cellular development in cancer.
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Ciclo Celular , Modelos Biológicos , Saccharomyces cerevisiae , Ciclo Celular/fisiología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Replicación del ADN , Biología Computacional , Saccharomycetales/genética , Saccharomycetales/metabolismo , Saccharomycetales/fisiología , Quinasas Ciclina-Dependientes/metabolismo , Quinasas Ciclina-Dependientes/genética , Fosforilación , Proteínas RepresorasRESUMEN
Homologous recombination (HR) is essential for the maintenance of genome stability. During HR, Replication Protein A (RPA) rapidly coats the 3'-tailed single-strand DNA (ssDNA) generated by end resection. Then, the ssDNA-bound RPA must be timely replaced by Rad51 recombinase to form Rad51 nucleoprotein filaments that drive homology search and HR repair. How cells regulate Rad51 assembly dynamics and coordinate RPA and Rad51 actions to ensure proper HR remains poorly understood. Here, we identified that Rtt105, a Ty1 transposon regulator, acts to stimulate Rad51 assembly and orchestrate RPA and Rad51 actions during HR. We found that Rtt105 interacts with Rad51 in vitro and in vivo and restrains the adenosine 5' triphosphate (ATP) hydrolysis activity of Rad51. We showed that Rtt105 directly stimulates dynamic Rad51-ssDNA assembly, strand exchange, and D-loop formation in vitro. Notably, we found that Rtt105 physically regulates the binding of Rad51 and RPA to ssDNA via different motifs and that both regulations are necessary and epistatic in promoting Rad51 nucleation, strand exchange, and HR repair. Consequently, disrupting either of the interactions impaired HR and conferred DNA damage sensitivity, underscoring the importance of Rtt105 in orchestrating the actions of Rad51 and RPA. Our work reveals additional layers of mechanisms regulating Rad51 filament dynamics and the coordination of HR.
Asunto(s)
ADN de Cadena Simple , Recombinasa Rad51 , Reparación del ADN por Recombinación , Proteína de Replicación A , Proteínas de Saccharomyces cerevisiae , Recombinasa Rad51/metabolismo , Proteína de Replicación A/metabolismo , Proteína de Replicación A/genética , ADN de Cadena Simple/metabolismo , ADN de Cadena Simple/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Unión ProteicaRESUMEN
Loss of function mutations in the checkpoint kinase gene CHEK2 are associated with increased risk of breast and other cancers. Most of the 3,188 unique amino acid changes that can result from non-synonymous single nucleotide variants (SNVs) of CHEK2, however, have not been tested for their impact on the function of the CHEK2-enocded protein (CHK2). One successful approach to testing the function of variants has been to test for their ability to complement mutations in the yeast ortholog of CHEK2, RAD53. This approach has been used to provide functional information on over 100 CHEK2 SNVs and the results align with functional assays in human cells and known pathogenicity. Here we tested all but two of the 4,887 possible SNVs in the CHEK2 open reading frame for their ability to complement RAD53 mutants using a high throughput technique of deep mutational scanning (DMS). Among the non-synonymous changes, 770 were damaging to protein function while 2,417 were tolerated. The results correlate well with previous structure and function data and provide a first or additional functional assay for all the variants of uncertain significance identified in clinical databases. Combined, this approach can be used to help predict the pathogenicity of CHEK2 variants of uncertain significance that are found in susceptibility screening and could be applied to other cancer risk genes.
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Quinasa de Punto de Control 2 , Polimorfismo de Nucleótido Simple , Quinasa de Punto de Control 2/genética , Humanos , Proteínas de Ciclo Celular/genética , Mutación , Mutación con Pérdida de Función , Sistemas de Lectura Abierta/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
BACKGROUND: Global warming causes an increase in the levels of sugars in grapes and hence in ethanol after wine fermentation. Therefore, alcohol reduction is a major target in modern oenology. Deletion of the MKS1 gene, a negative regulator of the Retrograde Response pathway, in Saccharomyces cerevisiae was reported to increase glycerol and reduce ethanol and acetic acid in wine. This study aimed to obtain mutants with a phenotype similar to that of the MKS1 deletion strain by subjecting commercial S. cerevisiae wine strains to an adaptive laboratory evolution (ALE) experiment with the lysine toxic analogue S-(2-aminoethyl)-L-cysteine (AEC). RESULTS: In laboratory-scale wine fermentation, isolated AEC-resistant mutants overproduced glycerol and reduced acetic acid. In some cases, ethanol was also reduced. Whole-genome sequencing revealed point mutations in the Retrograde Response activator Rtg2 and in the homocitrate synthases Lys20 and Lys21. However, only mutations in Rtg2 were responsible for the overactivation of the Retrograde Response pathway and ethanol reduction during vinification. Finally, wine fermentation was scaled up in an experimental cellar for one evolved mutant to confirm laboratory-scale results, and any potential negative sensory impact was ruled out. CONCLUSIONS: Overall, we have shown that hyperactivation of the Retrograde Response pathway by ALE with AEC is a valid approach for generating ready-to-use mutants with a desirable phenotype in winemaking.
Asunto(s)
Cisteína , Etanol , Fermentación , Glicerol , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Vino , Etanol/metabolismo , Vino/análisis , Glicerol/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Cisteína/metabolismo , Evolución Molecular Dirigida , Mutación , Ácido Acético/metabolismoRESUMEN
In various eukaryotic kingdoms, long terminal repeat (LTR) retrotransposons repress transcription by infiltrating heterochromatin generated within their elements. In contrast, the budding yeast LTR retrotransposon Ty1 does not itself undergo transcriptional repression, although it is capable of repressing the transcription of the inserted genes within it. In this study, we identified a DNA region within Ty1 that exerts its silencing effect via sequence orientation. We identified a DNA region within the Ty1 group-specific antigen (GAG) gene that causes gene silencing, termed GAG silencing (GAGsi), in which the silent chromatin in the GAGsi region is created by euchromatin-specific histone modifications. A characteristic inverted repeat (IR) sequence is present at the 5' end of this region, forming a chromatin boundary between promoter-specific chromatin upstream of the IR sequence and silent chromatin downstream of the IR sequence. In addition, Esc2 and Rad57, which are involved in DNA repair, were required for GAGsi silencing. Finally, the chromatin boundary was required for the transcription of Ty1 itself. Thus, the GAGsi sequence contributes to the creation of a chromatin environment that promotes Ty1 transcription.
Asunto(s)
Cromatina , Silenciador del Gen , Retroelementos , Saccharomyces cerevisiae , Retroelementos/genética , Cromatina/genética , Cromatina/metabolismo , Saccharomyces cerevisiae/genética , Elementos Aisladores/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Secuencias Repetidas Terminales/genética , Regulación Fúngica de la Expresión Génica , Transcripción Genética , Productos del Gen gag/genética , Productos del Gen gag/metabolismoRESUMEN
Fluctuations in the initiation rate of transcription, the first step in gene expression, ensue from the stochastic behavior of the molecular process that controls transcription. In steady state, the regulatory process is often assumed to operate reversibly, i.e., in equilibrium. However, reversibility imposes fundamental limits to information processing. For instance, the assumption of equilibrium is difficult to square with the precision with which the regulatory process executes its task in eukaryotes. Here we provide evidence - from microscopic analyses of the transcription dynamics at a single gene copy of yeast - that the regulatory process for transcription is cyclic and irreversible (out of equilibrium). The necessary coupling to reservoirs of free energy occurs via sequence-specific transcriptional activators and the recruitment, in part, of ATP-dependent chromatin remodelers. Our findings may help explain how eukaryotic cells reconcile the dual but opposing requirements for fast regulatory kinetics and high regulatory specificity.
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Regulación Fúngica de la Expresión Génica , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Transcripción Genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Ensamble y Desensamble de Cromatina , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Cinética , Adenosina Trifosfato/metabolismoRESUMEN
Depending on their phosphorylation status, derivatives of phosphatidylinositol play important roles in vesicle identity, recognition and intracellular trafficking processes. In eukaryotic cells, phosphatidylinositol-4 phosphate pools generated by specific kinases are key determinants of the conventional secretion pathways. Earlier work in yeast has classified phosphatidylinositol-4 kinases in two types, Stt4p and Pik1p belonging to type III and Lsb6p to type II, with distinct cellular localizations and functions. Eurotiomycetes appear to lack Pik1p homologues. In Aspergillus nidulans, unlike homologues in other fungi, AnLsb6 is associated to late Golgi membranes and when heterologously overexpressed, it compensates for the thermosensitive phenotype in a Saccharomyces cerevisiae pik1 mutant, whereas its depletion leads to disorganization of Golgi-associated PHOSBP-labelled membranes, that tend to aggregate dependent on functional Rab5 GTPases. Evidence provided herein, indicates that the single type II phosphatidylinositol-4 kinase AnLsb6 is the main contributor for decorating secretory vesicles with relevant phosphatidylinositol-phosphate species, which navigate essential cargoes following the route of apical polarization via endocytic recycling.
Asunto(s)
1-Fosfatidilinositol 4-Quinasa , Endocitosis , Aparato de Golgi , Saccharomyces cerevisiae , 1-Fosfatidilinositol 4-Quinasa/metabolismo , 1-Fosfatidilinositol 4-Quinasa/genética , Aspergillus nidulans/metabolismo , Aspergillus nidulans/genética , Aspergillus nidulans/enzimología , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Aparato de Golgi/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Transporte de Proteínas , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Yeast COMPASS (complex of proteins associated with Set1) and human MLL (mixed-lineage leukemia) complexes are histone H3 lysine 4 methyltransferases with critical roles in gene regulation and embryonic development. Both complexes share a conserved C-terminal SET domain, responsible for catalyzing histone H3 K4 methylation on nucleosomes. Notably, their catalytic activity toward nucleosomes is enhanced and optimized with assembly of auxiliary subunits. In this review, we aim to illustrate the recent X-ray and cryo-EM structures of yeast COMPASS and human MLL1 core complexes bound to either unmodified nucleosome core particle (NCP) or H2B mono-ubiquitinated NCP (H2Bub.NCP). We further delineate how each auxiliary component of the complex contributes to the NCP and ubiquitin recognition to maximize the methyltransferase activity.
Asunto(s)
N-Metiltransferasa de Histona-Lisina , Proteína de la Leucemia Mieloide-Linfoide , Nucleosomas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Humanos , Nucleosomas/metabolismo , N-Metiltransferasa de Histona-Lisina/química , N-Metiltransferasa de Histona-Lisina/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/química , Proteína de la Leucemia Mieloide-Linfoide/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Histonas/metabolismo , Histonas/química , Histonas/genética , Microscopía por Crioelectrón/métodosRESUMEN
The compaction of chromatin into mitotic chromosomes is essential for faithful transmission of the genome during cell division. In eukaryotes, chromosome morphogenesis is regulated by the condensin complex, though the exact mechanism used to target condensin to chromatin and initiate condensation is not understood. Here, we reveal that condensin contains an intrinsically disordered region (IDR) that modulates its association with chromatin in early mitosis and exhibits phase separation. We describe DNA-binding motifs within the IDR that, upon deletion, inflict striking defects in chromosome condensation and segregation, ill-timed condensin turnover on chromatin, and cell death. Importantly, we demonstrate that the condensin IDR can impart cell cycle regulatory functions when transferred to other subunits within the complex, indicating its autonomous nature. Collectively, our study unveils the molecular basis for the initiation of chromosome condensation in early mitosis and how this process ultimately promotes genomic stability and faultless cell division.
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Adenosina Trifosfatasas , Proteínas de Unión al ADN , Mitosis , Complejos Multiproteicos , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Complejos Multiproteicos/metabolismo , Adenosina Trifosfatasas/metabolismo , Cromatina/metabolismo , ADN/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Cromosomas/metabolismo , Unión Proteica , Segregación Cromosómica , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Saccharomyces cerevisiae has long been used as a model organism to study genome instability. The SAM1 and SAM2 genes encode AdoMet synthetases, which generate S-AdenosylMethionine (AdoMet) from Methionine (Met) and ATP. Previous work from our group has shown that deletions of the SAM1 and SAM2 genes cause changes to AdoMet levels and impact genome instability in opposite manners. AdoMet is a key product of methionine metabolism and the major methyl donor for methylation events of proteins, RNAs, small molecules, and lipids. The methyl cycle is interrelated to the folate cycle which is involved in de novo synthesis of purine and pyrimidine deoxyribonucleotides (dATP, dTTP, dCTP, and dGTP). AdoMet also plays a role in polyamine production, essential for cell growth and used in detoxification of reactive oxygen species (ROS) and maintenance of the redox status in cells. This is also impacted by the methyl cycle's role in production of glutathione, another ROS scavenger and cellular protectant. We show here that sam2∆/sam2∆ cells, previously characterized with lower levels of AdoMet and higher genome instability, have a higher level of each dNTP (except dTTP), contributing to a higher overall dNTP pool level when compared to wildtype. Unchecked, these increased levels can lead to multiple types of DNA damage which could account for the genome instability increases in these cells.
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S-Adenosilmetionina , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , S-Adenosilmetionina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Inestabilidad Genómica , Desoxirribonucleótidos/metabolismo , Nucleótidos/metabolismo , Metionina/metabolismoRESUMEN
We have investigated the interplay between glycolytic oscillations and intracellular K + ${{\rm{K}}}^{+}$ concentration in the yeast Saccharomyces cerevisiae. Intracellular K + ${{\rm{K}}}^{+}$ concentration was measured using the fluorophore potassium-binding benzofuranisophthalate (PBFI). We found that K + ${{\rm{K}}}^{+}$ is an essential ion for the occurrence of glycolytic oscillations and that intracellular K + ${{\rm{K}}}^{+}$ concentration oscillates synchronously with other variables such as nicotinamide adenine dinucleotide hydride (NADH), intracellular adenosine triphosphate (ATP), and mitochondrial membrane potential. We also investigated if glycolysis and intracellular K + ${{\rm{K}}}^{+}$ concentration oscillate in a number of yeast strains with mutations in K + ${{\rm{K}}}^{+}$ transporters in the plasma membrane, mitochondrial membrane and in the vacuolar membrane. Most of these strains are still capable of showing glycolytic oscillations, but two strains are not: (i) a strain with a deletion in the mitochondrial Mdm38p K + ∕ H + ${{\rm{K}}}^{+}\unicode{x02215}{{\rm{H}}}^{+}$ transporter and (ii) a strain with deletion of the late endosomal Nhx1p K + ∕ H + ${{\rm{K}}}^{+}\unicode{x02215}{{\rm{H}}}^{+}$ ( Na + ∕ H + ${\text{Na}}^{+}\unicode{x02215}{{\rm{H}}}^{+}$ ) transporter. In these two mutant strains intracellular K + ${{\rm{K}}}^{+}$ concentration seems to be low, indicating that the two transporters may be involved in transport of K + ${{\rm{K}}}^{+}$ into the cytosol. In the strain, Mdm38p Δ ${\rm{\Delta }}$ oscillations in glycolysis could be restored by addition of the K + ∕ H + ${{\rm{K}}}^{+}\unicode{x02215}{{\rm{H}}}^{+}$ exchange ionophore nigericin. Furthermore, in two nonoscillating mutant strains with a defective V-ATPase and deletion of the Arp1p protein the intracellular K + ${{\rm{K}}}^{+}$ is relatively high, suggesting that the V-ATPase is essential for transport of K + ${{\rm{K}}}^{+}$ out of the cytosol and that the cytoskeleton may be involved in binding K + ${{\rm{K}}}^{+}$ to reduce the concentration of free ion in the cytosol. Analyses of the time series of oscillations of NADH, ATP, mitochondrial membrane potential, and potassium concentration using data-driven modeling corroborate the conjecture that K + ${{\rm{K}}}^{+}$ ion is essential for the emergence of oscillations and support the experimental findings using mutant strains.
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Glucólisis , Potasio , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Potasio/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , NAD/metabolismo , Potencial de la Membrana MitocondrialRESUMEN
Over the past 30 years, much has been learned regarding iron homeostatic regulation in budding yeast, S. cerevisiae, including the identity of many of the proteins and molecular-level regulatory mechanisms involved. Most advances have involved inferring such mechanisms based on the analysis of iron-dysregulation phenotypes arising in various genetic mutant strains. Still lacking is a cellular- or system-level understanding of iron homeostasis. These experimental advances are summarized in this review, and a method for developing cellular-level regulatory mechanisms in yeast is presented. The method employs the results of Mössbauer spectroscopy of whole cells and organelles, iron quantification of the same, and ordinary differential equation-based mathematical models. Current models are simplistic when compared to the complexity of iron homeostasis in real cells, yet they hold promise as a useful, perhaps even required, complement to the popular genetics-based approach. The fundamental problem in comprehending cellular regulatory mechanisms is that, given the complexities involved, different molecular-level mechanisms can often give rise to virtually indistinguishable cellular phenotypes. Mathematical models cannot eliminate this problem, but they can minimize it.
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Homeostasis , Hierro , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Hierro/metabolismo , Simulación por Computador , Modelos Biológicos , Espectroscopía de Mossbauer/métodos , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The URH1p enzyme from the yeast Saccharomyces cerevisiae has gained significant interest due to its role in nitrogenous base metabolism, particularly involving uracil and nicotinamide salvage. Indeed, URH1p was initially classified as a nucleoside hydrolase (NH) with a pronounced preference for uridine substrate but was later shown to also participate in a Preiss-Handler-dependent pathway for recycling of both endogenous and exogenous nicotinamide riboside (NR) towards NAD+ synthesis. Here, we present the detailed enzymatic and structural characterisation of the yeast URH1p enzyme, a member of the group I NH family of enzymes. We show that the URH1p has similar catalytic efficiencies for hydrolysis of NR and uridine, advocating a dual role of the enzyme in both NAD+ synthesis and nucleobase salvage. We demonstrate that URH1p has a monomeric structure that is unprecedented for members of the NH homology group I, showing that oligomerisation is not strictly required for the N-ribosidic activity in this family of enzymes. The size, thermal stability and activity of URH1p towards the synthetic substrate 5-fluoruridine, a riboside precursor of the antitumoral drug 5-fluorouracil, make the enzyme an attractive tool to be employed in gene-directed enzyme-prodrug activation therapy against solid tumours.
Asunto(s)
Niacinamida , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Niacinamida/análogos & derivados , Niacinamida/metabolismo , Niacinamida/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Relación Estructura-Actividad , Compuestos de Piridinio/metabolismo , Compuestos de Piridinio/química , N-Glicosil Hidrolasas/metabolismo , N-Glicosil Hidrolasas/genética , N-Glicosil Hidrolasas/química , Uridina/metabolismo , Uridina/análogos & derivados , Uridina/química , Especificidad por Sustrato , Humanos , Modelos MolecularesRESUMEN
A large number of recombinant plasmids for the yeast Saccharomyces cerevisiae have been constructed and accumulated over the past four decades. It is desirable to apply the recombinant plasmid resources to Saccharomyces sensu stricto species group, which contains an increasing number of natural isolate and industrial strains. The application to the group encounters a difficulty. Natural isolates and industrial strains are exclusively prototrophic and polyploid, whereas direct application of most conventional plasmid resources imposes a prerequisite in host yeast strains of an auxotrophic mutation (i.e., leu2) that is rescued by a selection gene (e.g., LEU2) on the recombinant plasmids. To solve the difficulty, we aimed to generate leu2 mutants from yeast strains belonging to the yeast Saccharomyces sensu stricto species group by DNA editing. First, we modified an all-in-one type CRISPR-Cas9 plasmid pML104 by adding an antibiotic-resistance gene and designing guide sequences to target the LEU2 gene and to enable wide application in this yeast group. Then, the resulting CRISPR-Cas9 plasmids were exploited to seven strains belonging to five species of the group, including natural isolate, industrial, and allopolyploid strains. Colonies having the designed mutations in the gene appeared successfully by introducing the plasmids and assisting oligonucleotides to the strains. Most of the plasmids and resultant leu2- mutants produced in this study will be deposited in several repository organizations. KEY POINTS: ⢠All-in-one type CRISPR-Cas9 plasmids targeting LEU2 gene were designed for broad application to Saccharomyces sensu stricto group species strains ⢠Application of the plasmids generated leu2 mutants from strains including natural isolates, industrial, and allopolyploid strains ⢠The easy conversion to leu2 mutants permits free access to recombinant plasmids having a LEU2 gene.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Mutación , Plásmidos , Poliploidía , Plásmidos/genética , Edición Génica/métodos , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces/genética , Saccharomyces cerevisiae/genética , 3-Isopropilmalato Deshidrogenasa/genética , 3-Isopropilmalato Deshidrogenasa/metabolismo , Genoma Fúngico/genéticaRESUMEN
While that ROS causes DNA damage is well documented, there has been limited investigation into whether DNA damages and their repair processes can conversely induce oxidative stress. By generating a site-specific DNA double strand break (DSB) via I-SceI endonuclease expression in S. cerevisiae without damaging other cellular components, this study demonstrated that DNA repair does trigger oxidative stress. Deleting genes participating in the initiation of the resection step of homologous recombination (HR), like the MRX complex, resulted in stimulation of ROS. In contrast, deleting genes acting downstream of HR resection suppressed ROS levels. Additionally, blocking non-homologous end joining (NHEJ) also suppressed ROS. Further analysis identified Rad53 as a key player that relays DNA damage signals to alter redox metabolism in an HR-specific manner. These results suggest both HR and NHEJ can drive metabolism changes and oxidative stress, with NHEJ playing a more prominent role in ROS stimulation. Further analysis revealed a correlation between DSB-induced ROS increase and enhanced activity of NADPH oxidase Yno1 and various antioxidant enzymes. Deleting the antioxidant gene SOD1 induced synthetic lethality in HR-deficient mutants like mre11Δ and rad51Δ upon DSB induction. These findings uncover a significant interplay between DNA repair mechanisms and cellular metabolism, providing insights into understanding the side effects of genotoxic therapies and potentially aiding development of more effective cancer treatment strategies.