RESUMO
The yeast Rif2 protein is known to inhibit Mre11 nuclease and the activation of Tel1 kinase through a short motif termed MIN, which binds the Rad50 subunit and simulates its ATPase activity in vitro. The mechanism by which Rif2 restrains Tel1 activation and the consequences of this inhibition at DNA double-strand breaks (DSBs) are poorly understood. In this study, we employed AlphaFold-Multimer modelling to pinpoint and validate the interaction surface between Rif2 MIN and Rad50. We also engineered the rif2-S6E mutation that enhances the inhibitory effect of Rif2 by increasing Rif2-Rad50 interaction. Unlike rif2Δ, the rif2-S6E mutation impairs hairpin cleavage. Furthermore, it diminishes Tel1 activation by inhibiting Tel1 binding to DSBs while leaving MRX association unchanged, indicating that Rif2 can directly inhibit Tel1 recruitment to DSBs. Additionally, Rif2S6E reduces Tel1-MRX interaction and increases stimulation of ATPase by Rad50, indicating that Rif2 binding to Rad50 induces an ADP-bound MRX conformation that is not suitable for Tel1 binding. The decreased Tel1 recruitment to DSBs in rif2-S6E cells impairs DSB end-tethering and this bridging defect is suppressed by expressing a Tel1 mutant variant that increases Tel1 persistence at DSBs, suggesting a direct role for Tel1 in the bridging of DSB ends.
Assuntos
Proteínas de Ligação a DNA , Proteínas Serina-Treonina Quinases , Proteínas de Saccharomyces cerevisiae , Proteínas de Ligação a Telômeros , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , DNA/genética , DNA/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ligação a Telômeros/metabolismoRESUMO
Repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) requires that the 5'-terminated DNA strands are resected to generate single-stranded DNA overhangs. This process is initiated by a short-range resection catalyzed by the MRX (Mre11-Rad50-Xrs2) complex, which is followed by a long-range step involving the nucleases Exo1 and Dna2. Here we show that the Saccharomyces cerevisiae ATP-dependent chromatin-remodeling protein Chd1 participates in both short- and long-range resection by promoting MRX and Exo1 association with the DSB ends. Furthermore, Chd1 reduces histone occupancy near the DSB ends and promotes DSB repair by HR. All these functions require Chd1 ATPase activity, supporting a role for Chd1 in the opening of chromatin at the DSB site to facilitate MRX and Exo1 processing activities.
Assuntos
Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/fisiologia , Exodesoxirribonucleases/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/genética , Genes Fúngicos , Histonas/isolamento & purificaçãoRESUMO
DNA is exposed to both endogenous and exogenous DNA damaging agents that chemically modify it. To counteract the deleterious effects exerted by DNA lesions, eukaryotic cells have evolved a network of cellular pathways, termed DNA damage response (DDR). The DDR comprises both mechanisms devoted to repair DNA lesions and signal transduction pathways that sense DNA damage and transduce this information to specific cellular targets. These targets, in turn, impact a wide range of cellular processes including DNA replication, DNA repair and cell cycle transitions. The importance of the DDR is highlighted by the fact that DDR inactivation is commonly found in cancer and causes many different human diseases. The protein kinases ATM and ATR, as well as their budding yeast orthologs Tel1 and Mec1, act as master regulators of the DDR. The initiating events in the DDR entail both DNA lesion recognition and assembly of protein complexes at the damaged DNA sites. Here, we review what is known about the early steps of the DDR.
Assuntos
Dano ao DNA , DNA/análise , Saccharomyces cerevisiae/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Ciclo Celular , Reparo do DNA , Replicação do DNA , DNA de Cadeia Simples/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-ets/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces , Transdução de Sinais , Xenopus laevis , Variante 6 da Proteína do Fator de Translocação ETSRESUMO
Effective stratification of cancer patients on the basis of their molecular make-up is a key open challenge. Given the altered and heterogenous nature of cancer metabolism, we here propose to use the overall expression of central carbon metabolism as biomarker to characterize groups of patients with important characteristics, such as response to ad-hoc therapeutic strategies and survival expectancy. To this end, we here introduce the data integration framework named Metabolic Reaction Enrichment Analysis (MaREA), which strives to characterize the metabolic deregulations that distinguish cancer phenotypes, by projecting RNA-seq data onto metabolic networks, without requiring metabolic measurements. MaREA computes a score for each network reaction, based on the expression of the set of genes encoding for the associated enzyme(s). The scores are first used as features for cluster analysis and then to rank and visualize in an organized fashion the metabolic deregulations that distinguish cancer sub-types. We applied our method to recent lung and breast cancer RNA-seq datasets from The Cancer Genome Atlas and we were able to identify subgroups of patients with significant differences in survival expectancy. We show how the prognostic power of MaREA improves when an extracted and further curated core model focusing on central carbon metabolism is used rather than the genome-wide reference network. The visualization of the metabolic differences between the groups with best and worst prognosis allowed to identify and analyze key metabolic properties related to cancer aggressiveness. Some of these properties are shared across different cancer (sub) types, e.g., the up-regulation of nucleic acid and amino acid synthesis, whereas some other appear to be tumor-specific, such as the up- or down-regulation of the phosphoenolpyruvate carboxykinase reaction, which display different patterns in distinct tumor (sub)types. These results might be soon employed to deliver highly automated diagnostic and prognostic strategies for cancer patients.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Análise de Sequência de RNA/métodos , Transcriptoma , Adenocarcinoma/diagnóstico , Adenocarcinoma/metabolismo , Algoritmos , Biópsia , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/metabolismo , Carbono/metabolismo , Análise por Conglomerados , Perfilação da Expressão Gênica , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/metabolismo , Redes e Vias Metabólicas , Reconhecimento Automatizado de Padrão , PrognósticoRESUMO
The MRX complex together with Sae2 initiates resection of DNA double-strand breaks (DSBs) to generate single-stranded DNA (ssDNA) that triggers homologous recombination. The absence of Sae2 not only impairs DSB resection, but also causes prolonged MRX binding at the DSBs that leads to persistent Tel1- and Rad53-dependent DNA damage checkpoint activation and cell cycle arrest. Whether this enhanced checkpoint signaling contributes to the DNA damage sensitivity and/or the resection defect of sae2Δ cells is not known. By performing a genetic screen, we identify rad53 and tel1 mutant alleles that suppress both the DNA damage hypersensitivity and the resection defect of sae2Δ cells through an Sgs1-Dna2-dependent mechanism. These suppression events do not involve escaping the checkpoint-mediated cell cycle arrest. Rather, defective Rad53 or Tel1 signaling bypasses Sae2 function at DSBs by decreasing the amount of Rad9 bound at DSBs. As a consequence, reduced Rad9 association to DNA ends relieves inhibition of Sgs1-Dna2 activity, which can then compensate for the lack of Sae2 in DSB resection and DNA damage resistance. We propose that persistent Tel1 and Rad53 checkpoint signaling in cells lacking Sae2 increases the association of Rad9 at DSBs, which in turn inhibits DSB resection by limiting the activity of the Sgs1-Dna2 resection machinery.
Assuntos
Proteínas de Ciclo Celular/genética , Quinase do Ponto de Checagem 2/genética , Quebras de DNA de Cadeia Dupla , Endonucleases/genética , Instabilidade Genômica/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas de Saccharomyces cerevisiae/genética , Ciclo Celular/genética , Dano ao DNA/genética , DNA Helicases/genética , Recombinação Homóloga/genética , Hipersensibilidade/genética , Fosforilação , RecQ Helicases/genética , Saccharomyces cerevisiae/genéticaRESUMO
Homologous recombination is initiated by the nucleolytic degradation (resection) of DNA double-strand breaks (DSBs). DSB resection is a two-step process. In the short-range step, the MRX (Mre11-Rad50-Xrs2) complex, together with Sae2, incises the 5'-terminated strand at the DSB end and resects back toward the DNA end. Then, the long-range resection nucleases Exo1 and Dna2 further elongate the resected DNA tracts. We found that mutations lowering proteasome functionality bypass the need for Sae2 in DSB resection. In particular, the dysfunction of the proteasome subunit Rpn11 leads to hyper-resection and increases the levels of both Exo1 and Dna2 to such an extent that it allows the bypass of the requirement for either Exo1 or Dna2, but not for both. These observations, along with the finding that Exo1 and Dna2 are ubiquitylated, indicate a role of the proteasome in restraining DSB resection by negatively controlling the abundance of the long-range resection nucleases.
RESUMO
DNA damage elicits a checkpoint response depending on the Mec1/ATR kinase, which detects the presence of single-stranded DNA and activates the effector kinase Rad53/CHK2. In Saccharomyces cerevisiae, one of the signaling circuits leading to Rad53 activation involves the evolutionarily conserved 9-1-1 complex, which acts as a platform for the binding of Dpb11 and Rad9 (referred to as the 9-1-1 axis) to generate a protein complex that allows Mec1 activation. By examining the effects of both loss-of-function and hypermorphic mutations, here, we show that the Cdc55 and Tpd3 subunits of the PP2A phosphatase counteract activation of the 9-1-1 axis. The lack of this inhibitory function results in DNA-damage sensitivity, sustained checkpoint-mediated cell-cycle arrest, and impaired resection of DNA double-strand breaks. This PP2A anti-checkpoint role depends on the capacity of Cdc55 to interact with Ddc1 and to counteract Ddc1-Dpb11 complex formation by preventing Dpb11 recognition of Ddc1 phosphorylated on Thr602.
Assuntos
Proteínas Serina-Treonina Quinases , Proteínas de Saccharomyces cerevisiae , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Dano ao DNA , Fosforilação , DNA/metabolismo , Quinase do Ponto de Checagem 2/genéticaRESUMO
In Saccharomyces cerevisiae, the protein kinase A (PKA) plays a central role in the control of metabolism, stress resistance and cell cycle progression. In a previous work, we used a FRET-based A-kinase activity reporter (AKAR3 probe) to monitor changes in PKA activity in vivo in single S. cerevisiae cells. Since this procedure is quite complex and time-consuming, in this work we used the AKAR3 probe (evenly distributed within the cells) and the plate reader Victor-X3™ (Perkin Elmer®) to measure PKA activity in vivo in a whole cell population. We show that in wild type strains, the FRET increases after addition of glucose to glucose-starved cells, while no changes are observed when this sugar is added to strains with either absent or attenuated PKA activity. Moreover, using the pm-AKAR3 probe, mainly expressed at the plasma membrane and partially at the vacuolar membrane, we could monitor PKA activity from the starting site of the signal to internal regions, where the signal is propagated. Finally, we also show evidence for direct activation of PKA by glucose, independent of cAMP. In conclusion, our data show that AKAR3 and pm-AKAR3 probes are useful biosensors to monitor PKA activity in a S. cerevisiae cell population using a plate reader.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Transferência Ressonante de Energia de Fluorescência , Glucose/metabolismo , Fosforilação , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Studies performed in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe have led the way in defining the DNA damage checkpoint and in identifying most of the proteins involved in this regulatory network, which turned out to have structural and functional equivalents in humans. Subsequent experiments revealed that the checkpoint is an elaborate signal transduction pathway that has the ability to sense and signal the presence of damaged DNA and transduce this information to influence a multifaceted cellular response that is essential for cancer avoidance. This review focuses on the work that was done in Saccharomyces cerevisiae to articulate the checkpoint concept, to identify its players and the mechanisms of activation and deactivation.
RESUMO
Early work by Muller and McClintock discovered that the physical ends of linear chromosomes, named telomeres, possess an inherent ability to escape unwarranted fusions. Since then, extensive research has shown that this special feature relies on specialized proteins and structural properties that confer identity to the chromosome ends, thus allowing cells to distinguish them from intrachromosomal DNA double-strand breaks. Due to the inability of conventional DNA replication to fully replicate the chromosome ends and the downregulation of telomerase in most somatic human tissues, telomeres shorten as cells divide and lose this protective capacity. Telomere attrition causes the activation of the DNA damage checkpoint that leads to a cell-cycle arrest and the entering of cells into a nondividing state, called replicative senescence, that acts as a barrier against tumorigenesis. However, downregulation of the checkpoint overcomes this barrier and leads to further genomic instability that, if coupled with re-stabilization of telomeres, can drive tumorigenesis. This review focuses on the key experiments that have been performed in the model organism Saccharomyces cerevisiae to uncover the mechanisms that protect the chromosome ends from eliciting a DNA damage response, the conservation of these pathways in mammals, as well as the consequences of their loss in human cancer.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Telomerase , Encurtamento do Telômero , Animais , Humanos , Carcinogênese , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Telomerase/genética , Telomerase/metabolismo , Telômero/genética , Telômero/metabolismoRESUMO
Budding yeast Dpb4 (POLE3/CHRAC17 in mammals) is a highly conserved histone fold protein that is shared by two protein complexes: the chromatin remodeler ISW2/hCHRAC and the DNA polymerase ε (Pol ε) holoenzyme. In Saccharomyces cerevisiae, Dpb4 forms histone-like dimers with Dls1 in the ISW2 complex and with Dpb3 in the Pol ε complex. Here, we show that Dpb4 plays two functions in sensing and processing DNA double-strand breaks (DSBs). Dpb4 promotes histone removal and DSB resection by interacting with Dls1 to facilitate the association of the Isw2 ATPase to DSBs. Furthermore, it promotes checkpoint activation by interacting with Dpb3 to facilitate the association of the checkpoint protein Rad9 to DSBs. Persistence of both Isw2 and Rad9 at DSBs is enhanced by the A62S mutation that is located in the Dpb4 histone fold domain and increases Dpb4 association at DSBs. Thus, Dpb4 exerts two distinct functions at DSBs depending on its interactors.
Assuntos
Quebras de DNA de Cadeia Dupla , DNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/metabolismo , Montagem e Desmontagem da Cromatina , DNA/metabolismo , Dano ao DNA , Reparo do DNA , Histonas/metabolismo , Mutação , Fatores de TranscriçãoRESUMO
DNA double-strand breaks (DSBs) are highly cytotoxic lesions that must be repaired to ensure genomic stability and avoid cell death. The cellular response to DSBs is initiated by the evolutionarily conserved Mre11-Rad50-Xrs2/NBS1 (MRX/MRN) complex that has structural and catalytic functions. Furthermore, it is responsible for DSB signaling through the activation of the checkpoint kinase Tel1/ATM. Here, we review functions and regulation of the MRX/MRN complex in DSB processing in a chromatin context, as well as its interplay with Tel1/ATM.