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
Mec1ATR mediates the DNA damage response (DDR), integrating chromosomal signals and mechanical stimuli. We show that the PP2A phosphatases, ceramide-activated enzymes, couple cell metabolism with the DDR. Using genomic screens, metabolic analysis, and genetic and pharmacological studies, we found that PP2A attenuates the DDR and that three metabolic circuits influence the DDR by modulating PP2A activity. Irc21, a putative cytochrome b5 reductase that promotes the condensation reaction generating dihydroceramides (DHCs), and Ppm1, a PP2A methyltransferase, counteract the DDR by activating PP2A; conversely, the nutrient-sensing TORC1-Tap42 axis sustains DDR activation by inhibiting PP2A. Loss-of-function mutations in IRC21, PPM1, and PP2A and hyperactive tap42 alleles rescue mec1 mutants. Ceramides synergize with rapamycin, a TORC1 inhibitor, in counteracting the DDR. Hence, PP2A integrates nutrient-sensing and metabolic pathways to attenuate the Mec1ATR response. Our observations imply that metabolic changes affect genome integrity and may help with exploiting therapeutic options and repositioning known drugs.
Assuntos
Dano ao DNA , Reparo do DNA , DNA Fúngico/metabolismo , Metabolismo Energético , Genoma Fúngico , Instabilidade Genômica , Proteína Fosfatase 2/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Ceramidas/metabolismo , Ceramidas/farmacologia , Citocromo-B(5) Redutase/genética , Citocromo-B(5) Redutase/metabolismo , Reparo do DNA/efeitos dos fármacos , DNA Fúngico/genética , Ativação Enzimática , Regulação Fúngica da Expressão Gênica , Genoma Fúngico/efeitos dos fármacos , Instabilidade Genômica/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Metabolômica , Mutação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Metiltransferases/genética , Proteínas Metiltransferases/metabolismo , Proteína Fosfatase 2/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/genética , Sirolimo/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
DNA double-strand breaks (DSBs) can be repaired by either homologous recombination (HR) or non-homologous end-joining (NHEJ). NHEJ is induced by the binding to DSBs of the Ku70-Ku80 heterodimer, which acts as a hub for the recruitment of downstream NHEJ components. An important issue in DSB repair is the maintenance of the DSB ends in close proximity, a function that in yeast involves the MRX complex and Sae2. Here, we provide evidence that Ku contributes to keep the DNA ends tethered to each other. The ku70-C85Y mutation, which increases Ku affinity for DNA and its persistence very close to the DSB ends, enhances DSB end-tethering and suppresses the end-tethering defect of sae2Δ cells. Impairing histone removal around DSBs either by eliminating Tel1 kinase activity or nucleosome remodelers enhances Ku persistence at DSBs and DSB bridging, suggesting that Tel1 antagonizes the Ku function in supporting end-tethering by promoting nucleosome removal and possibly Ku sliding inwards. As Ku provides a block to DSB resection, this Tel1 function can be important to regulate the mode by which DSBs are repaired.
Assuntos
Proteínas de Ligação a DNA , Proteínas Serina-Treonina Quinases , Proteínas de Saccharomyces cerevisiae , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Reparo do DNA , Proteínas de Ligação a DNA/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Autoantígeno Ku/metabolismo , Nucleossomos , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
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
Homologous recombination is triggered by nucleolytic degradation (resection) of DNA double-strand breaks (DSBs). DSB resection requires the Mre11-Rad50-Xrs2 (MRX) complex, which promotes the activity of Exo1 nuclease through a poorly understood mechanism. Here, we describe the Mre11-R10T mutant variant that accelerates DSB resection compared to wild-type Mre11 by potentiating Exo1-mediated processing. This increased Exo1 resection activity leads to a decreased association of the Ku complex to DSBs and an enhanced DSB resection in G1, indicating that Exo1 has a direct function in preventing Ku association with DSBs. Molecular dynamics simulations show that rotation of the Mre11 capping domains is able to induce unwinding of double-strand DNA (dsDNA). The R10T substitution causes altered orientation of the Mre11 capping domain that leads to persistent melting of the dsDNA end. We propose that MRX creates a specific DNA end structure that promotes Exo1 resection activity by facilitating the persistence of this nuclease on the DSB ends, uncovering a novel MRX function in DSB resection.
Assuntos
Quebras de DNA de Cadeia Dupla , DNA Fúngico/metabolismo , Exodesoxirribonucleases/metabolismo , Complexos Multiproteicos/metabolismo , Saccharomyces cerevisiae/metabolismo , DNA Fúngico/genética , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/genética , Complexos Multiproteicos/genética , Domínios Proteicos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The cellular response to DNA double-strand breaks (DSBs) is initiated by the Mre11-Rad50-Xrs2 (MRX) complex that has structural and catalytic functions. MRX association at DSBs is counteracted by Rif2, which is known to interact with Rap1 that binds telomeric DNA through two tandem Myb-like domains. Whether and how Rap1 acts at DSBs is unknown. Here we show that Rif2 inhibits MRX association to DSBs in a manner dependent on Rap1, which binds to DSBs and promotes Rif2 association to them. Rap1 in turn can negatively regulate MRX function at DNA ends also independently of Rif2. In fact, a characterization of Rap1 mutant variants shows that Rap1 binding to DNA through both Myb-like domains results in formation of Rap1-DNA complexes that control MRX functions at both DSBs and telomeres primarily through Rif2. By contrast, Rap1 binding to DNA through a single Myb-like domain results in formation of high stoichiometry complexes that act at DNA ends mostly in a Rif2-independent manner. Altogether these findings indicate that the DNA binding modes of Rap1 influence its functional properties, thus highlighting the structural plasticity of this protein.
Assuntos
DNA Fúngico/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Homeostase do Telômero , Proteínas de Ligação a Telômeros/metabolismo , Telômero/metabolismo , Fatores de Transcrição/metabolismo , Alelos , Quebras de DNA de Cadeia Dupla , Dano ao DNA , Modelos Biológicos , Mutação/genética , Ligação Proteica , Saccharomyces cerevisiae/citologia , Complexo Shelterina , Transcrição GênicaRESUMO
The natural ends of linear chromosomes resemble those of accidental double-strand breaks (DSBs). DSBs induce a multifaceted cellular response that promotes the repair of lesions and slows down cell cycle progression. This response is not elicited at chromosome ends, which are organized in nucleoprotein structures called telomeres. Besides counteracting DSB response through specialized telomere-binding proteins, telomeres also prevent chromosome shortening. Despite of the different fate of telomeres and DSBs, many proteins involved in the DSB response also localize at telomeres and participate in telomere homeostasis. In particular, the DSB master regulators Tel1/ATM and Mec1/ATR contribute to telomere length maintenance and arrest cell cycle progression when chromosome ends shorten, thus promoting a tumor-suppressive process known as replicative senescence. During senescence, the actions of both these apical kinases and telomere-binding proteins allow checkpoint activation while bulk DNA repair activities at telomeres are still inhibited. Checkpoint-mediated cell cycle arrest also prevents further telomere erosion and deprotection that would favor chromosome rearrangements, which are known to increase cancer-associated genome instability. This review summarizes recent insights into functions and regulation of Tel1/ATM and Mec1/ATR at telomeres both in the presence and in the absence of telomerase, focusing mainly on discoveries in budding yeast.
Assuntos
Dano ao DNA , Reparo do DNA/genética , Proteínas Quinases/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Telômero/genética , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , DNA/genética , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Humanos , Modelos Genéticos , Proteínas Proto-Oncogênicas c-ets/metabolismo , Proteínas Repressoras/metabolismo , Telomerase/metabolismo , Telômero/metabolismo , Variante 6 da Proteína do Fator de Translocação ETSRESUMO
Activation of the checkpoint protein Tel1 requires the Mre11-Rad50-Xrs2 (MRX) complex, which recruits Tel1 at DNA double-strand breaks (DSBs) through direct interaction between Tel1 and Xrs2. However, in vitro Tel1 activation by MRX requires ATP binding to Rad50, suggesting a role also for the MR subcomplex in Tel1 activation. Here we describe two separation-of-functions alleles, mre11-S499P and rad50-A78T, which we show to specifically affect Tel1 activation without impairing MRX functions in DSB repair. Both Mre11-S499P and Rad50-A78T reduce Tel1-MRX interaction leading to poor Tel1 association at DSBs and consequent loss of Tel1 activation. The Mre11-S499P variant reduces Mre11-Rad50 interaction, suggesting an important role for MR complex formation in Tel1 activation. Molecular dynamics simulations show that the wild type MR subcomplex bound to ATP lingers in a tightly 'closed' conformation, while ADP presence leads to the destabilization of Rad50 dimer and of Mre11-Rad50 association, both events being required for MR conformational transition to an open state. By contrast, MRA78T undertakes complex opening even if Rad50 is bound to ATP, indicating that defective Tel1 activation caused by MRA78T results from destabilization of the ATP-bound conformational state.
Assuntos
Proteínas de Ligação a DNA/genética , Endodesoxirribonucleases/genética , Exodesoxirribonucleases/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas de Saccharomyces cerevisiae/genética , Ativação Transcricional/genética , Trifosfato de Adenosina/genética , Proteínas Mutadas de Ataxia Telangiectasia/genética , Dano ao DNA/genética , Reparo do DNA/genética , DNA Fúngico/genética , Conformação Molecular , Complexos Multiproteicos/genética , Ligação Proteica/genética , Multimerização Proteica/genética , Saccharomyces cerevisiae/genética , Transdução de Sinais/genéticaRESUMO
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
Nucleolytic processing by nucleases can be a relevant mechanism to allow repair/restart of stalled replication forks. However, nuclease action needs to be controlled to prevent overprocessing of damaged replication forks that can be detrimental to genome stability. The checkpoint protein Rad9/53BP1 is known to limit nucleolytic degradation (resection) of DNA double-strand breaks (DSBs) in both yeast and mammals. Here, we show that loss of the inhibition that Rad9 exerts on resection exacerbates the sensitivity to replication stress of Mec1/ATR-defective yeast cells by exposing stalled replication forks to Dna2-dependent degradation. This Rad9 protective function is independent of checkpoint activation and relies mainly on Rad9-Dpb11 interaction. We propose that Rad9/53BP1 supports cell viability by protecting stalled replication forks from extensive resection when the intra-S checkpoint is not fully functional.
Assuntos
Replicação do DNA , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Proteínas Serina-Treonina Quinases/deficiência , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Proteínas de Ciclo Celular/metabolismo , Viabilidade Microbiana , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Estresse FisiológicoRESUMO
In both yeast and mammals, the topoisomerase poison camptothecin (CPT) induces fork reversal, which has been proposed to stabilize replication forks, thus providing time for the repair of CPT-induced lesions and supporting replication restart. We show that Tel1, the Saccharomyces cerevisiae orthologue of human ATM kinase, stabilizes CPT-induced reversed forks by counteracting their nucleolytic degradation by the MRX complex. Tel1-lacking cells are hypersensitive to CPT specifically and show less reversed forks in the presence of CPT The lack of Mre11 nuclease activity restores wild-type levels of reversed forks in CPT-treated tel1Δ cells without affecting fork reversal in wild-type cells. Moreover, Mrc1 inactivation prevents fork reversal in wild-type, tel1Δ, and mre11 nuclease-deficient cells and relieves the hypersensitivity of tel1Δ cells to CPT Altogether, our data indicate that Tel1 counteracts Mre11 nucleolytic activity at replication forks that undergo Mrc1-mediated reversal in the presence of CPT.
Assuntos
Proteínas de Ciclo Celular/genética , Replicação do DNA/genética , Endodesoxirribonucleases/genética , Exodesoxirribonucleases/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Mutadas de Ataxia Telangiectasia/genética , Camptotecina/farmacologia , Reparo do DNA/genética , Replicação do DNA/efeitos dos fármacos , DNA Topoisomerases/genética , Humanos , Mutação , Saccharomyces cerevisiae/genéticaRESUMO
Sae2 cooperates with the Mre11-Rad50-Xrs2 (MRX) complex to initiate resection of DNA double-strand breaks (DSBs) and to maintain the DSB ends in close proximity to allow their repair. How these diverse MRX-Sae2 functions contribute to DNA damage resistance is not known. Here, we describe mre11 alleles that suppress the hypersensitivity of sae2Δ cells to genotoxic agents. By assessing the impact of these mutations at the cellular and structural levels, we found that all the mre11 alleles that restore sae2Δ resistance to both camptothecin and phleomycin affect the Mre11 N-terminus and suppress the resection defect of sae2Δ cells by lowering MRX and Tel1 association to DSBs. As a consequence, the diminished Tel1 persistence potentiates Sgs1-Dna2 resection activity by decreasing Rad9 association to DSBs. By contrast, the mre11 mutations restoring sae2Δ resistance only to phleomycin are located in Mre11 C-terminus and bypass Sae2 function in end-tethering but not in DSB resection, possibly by destabilizing the Mre11-Rad50 open conformation. These findings unmask the existence of structurally distinct Mre11 domains that support resistance to genotoxic agents by mediating different processes.
Assuntos
Quebras de DNA de Cadeia Dupla , DNA Helicases/metabolismo , Reparo do DNA , Endodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Antineoplásicos/farmacologia , Camptotecina/farmacologia , DNA Helicases/química , DNA Helicases/genética , Farmacorresistência Fúngica/efeitos dos fármacos , Farmacorresistência Fúngica/genética , Endodesoxirribonucleases/química , Endodesoxirribonucleases/genética , Endonucleases/química , Endonucleases/genética , Endonucleases/metabolismo , Exodesoxirribonucleases/química , Exodesoxirribonucleases/genética , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Mutação , Fleomicinas/farmacologia , Domínios Proteicos , Multimerização Proteica/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The evolutionarily conserved Mre11-Rad50-Xrs2 (MRX) complex cooperates with the Sae2 protein in initiating resection of DNA double-strand breaks (DSBs) and in maintaining the DSB ends tethered to each other for their accurate repair. How these MRX-Sae2 functions contribute to DNA damage resistance is not understood. By taking advantage of mre11 alleles that suppress the hypersensitivity of sae2∆ cells to genotoxic agents, we have recently found that Mre11 can be divided in two structurally distinct domains that support resistance to genotoxic agents by mediating different processes. While the Mre11 N-terminal domain impacts on the resection activity of long-range resection nucleases by mediating MRX and Tel1/ATM association to DNA DSBs, the C-terminus influences the MRX-tethering activity by its virtue to interact with Rad50. Given the evolutionary conservation of the MRX complex, our results have implications for understanding the consequences of its dysfunctions in human diseases.
Assuntos
Dano ao DNA , DNA Fúngico/metabolismo , Endodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , DNA Fúngico/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/genética , Endonucleases/genética , Endonucleases/metabolismo , Exodesoxirribonucleases/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The cellular response to DNA double-strand breaks (DSBs) is initiated by the MRX/MRN complex (Mre11-Rad50-Xrs2 in yeast; Mre11-Rad50-Nbs1 in mammals), which recruits the checkpoint kinase Tel1/ATM to DSBs. In Saccharomyces cerevisiae, the role of Tel1 at DSBs remains enigmatic, as tel1Δ cells do not show obvious hypersensitivity to DSB-inducing agents. By performing a synthetic phenotype screen, we isolated a rad50-V1269M allele that sensitizes tel1Δ cells to genotoxic agents. The MRV1269MX complex associates poorly to DNA ends, and its retention at DSBs is further reduced by the lack of Tel1. As a consequence, tel1Δ rad50-V1269M cells are severely defective both in keeping the DSB ends tethered to each other and in repairing a DSB by either homologous recombination (HR) or nonhomologous end joining (NHEJ). These data indicate that Tel1 promotes MRX retention to DSBs and this function is important to allow proper MRX-DNA binding that is needed for end-tethering and DSB repair. The role of Tel1 in promoting MRX accumulation to DSBs is counteracted by Rif2, which is recruited to DSBs. We also found that Rif2 enhances ATP hydrolysis by MRX and attenuates MRX function in end-tethering, suggesting that Rif2 can regulate MRX activity at DSBs by modulating ATP-dependent conformational changes of Rad50.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Proteínas de Ligação a Telômeros/fisiologia , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/metabolismo , Recombinação Homóloga , Hidrólise , Dados de Sequência Molecular , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Eukaryotic cells preserve genome integrity upon DNA damage by activating a signaling network that promotes DNA repair and controls cell cycle progression. One of the most severe DNA damage is the DNA double-strand break (DSB), whose 5Î ends can be nucleolitically resected by multiple nucleases to create 3Î-ended single-stranded DNA tails that trigger DSB repair by homologous recombination. Here, we identify the Saccharomyces cerevisiae RNA binding protein Npl3 as a new player in DSB resection. Npl3 is related to both the metazoan serine-arginine-rich and the heterogeneous nuclear ribonucleo-proteins. NPL3 deletion impairs the generation of long ssDNA tails at the DSB ends, whereas it does not exacerbate the resection defect of exo1Δ cells. Furthermore, either the lack of Npl3 or the inactivation of its RNA-binding domains causes decrease of the exonuclease Exo1 protein levels as well as generation of unusual and extended EXO1 RNA species. These findings, together with the observation that EXO1 overexpression partially suppresses the resection defect of npl3Δ cells, indicate that Npl3 participates in DSB resection by promoting the proper biogenesis of EXO1 mRNA.
Assuntos
Reparo do DNA , DNA Fúngico/genética , Exodesoxirribonucleases/genética , Proteínas Nucleares/fisiologia , Proteínas de Ligação a RNA/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/genética , Pontos de Checagem do Ciclo Celular , Quebras de DNA de Cadeia Dupla , DNA Fúngico/metabolismo , DNA de Cadeia Simples/metabolismo , Indução Enzimática , Epistasia Genética , Exodesoxirribonucleases/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Regulação Fúngica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Telomeric DNA consists of repetitive G-rich sequences that terminate with a 3Î-ended single stranded overhang (G-tail), which is important for telomere extension by telomerase. Several proteins, including the CST complex, are necessary to maintain telomere structure and length in both yeast and mammals. Emerging evidence indicates that RNA processing factors play critical, yet poorly understood, roles in telomere metabolism. Here, we show that the lack of the RNA processing proteins Xrn1 or Rrp6 partially bypasses the requirement for the CST component Cdc13 in telomere protection by attenuating the activation of the DNA damage checkpoint. Xrn1 is necessary for checkpoint activation upon telomere uncapping because it promotes the generation of single-stranded DNA. Moreover, Xrn1 maintains telomere length by promoting the association of Cdc13 to telomeres independently of ssDNA generation and exerts this function by downregulating the transcript encoding the telomerase inhibitor Rif1. These findings reveal novel roles for RNA processing proteins in the regulation of telomere metabolism with implications for genome stability in eukaryotes.
Assuntos
Exorribonucleases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Homeostase do Telômero , Telômero/metabolismo , DNA de Cadeia Simples/metabolismo , Exorribonucleases/genética , Exorribonucleases/fisiologia , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Complexo Multienzimático de Ribonucleases do Exossomo/fisiologia , Mutação , Processamento Pós-Transcricional do RNA , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologia , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo , TemperaturaRESUMO
Tel1/ATM and Mec1/ATR checkpoint kinases are activated by DNA double-strand breaks (DSBs). Mec1/ATR recruitment to DSBs requires the formation of RPA-coated single-stranded DNA (ssDNA), which arises from 5'-3' nucleolytic degradation (resection) of DNA ends. Here, we show that Saccharomyces cerevisiae Mec1 regulates resection of the DSB ends. The lack of Mec1 accelerates resection and reduces the loading to DSBs of the checkpoint protein Rad9, which is known to inhibit ssDNA generation. Extensive resection is instead inhibited by the Mec1-ad mutant variant that increases the recruitment near the DSB of Rad9, which in turn blocks DSB resection by both Rad53-dependent and Rad53-independent mechanisms. The mec1-ad resection defect leads to prolonged persistence at DSBs of the MRX complex that causes unscheduled Tel1 activation, which in turn impairs checkpoint switch off. Thus, Mec1 regulates the generation of ssDNA at DSBs, and this control is important to coordinate Mec1 and Tel1 signaling activities at these breaks.
Assuntos
Quebras de DNA de Cadeia Dupla , DNA de Cadeia Simples , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Transdução de Sinais , Adaptação Biológica , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Imunoprecipitação da Cromatina , DNA Fúngico/genética , Proteínas de Ligação a DNA , Ativação Enzimática , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Genes cdc , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mutação , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
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
The repair of DNA double-strand breaks (DSBs) is crucial for maintaining genome stability. The Saccharomyces cerevisiae protein Tbf1, which is characterized by a Myb domain and is related to mammalian TRF1 and TRF2, has been proposed to act as a transcriptional activator. Here, we show that Tbf1 and its interacting protein Vid22 are new players in the response to DSBs. Inactivation of either TBF1 or VID22 causes hypersensitivity to DSB-inducing agents and shows strong negative interactions with mutations affecting homologous recombination. Furthermore, Tbf1 and Vid22 are recruited to an HO-induced DSB, where they promote both resection of DNA ends and repair by non-homologous end joining. Finally, inactivation of either Tbf1 or Vid22 impairs nucleosome eviction around the DSB, suggesting that these proteins promote efficient repair of the break by influencing chromatin identity in its surroundings.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades/genética , Proteínas de Ligação a DNA/fisiologia , Proteínas de Membrana/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/genética , Fatores de Transcrição/fisiologia , Montagem e Desmontagem da Cromatina/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Epistasia Genética/genética , Histonas/metabolismo , Recombinação Homóloga/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Biológicos , Organismos Geneticamente Modificados , Ligação Proteica/genética , Processamento de Proteína Pós-Traducional/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Homologous recombination requires nucleolytic degradation (resection) of DNA double-strand break (DSB) ends. In Saccharomyces cerevisiae, the MRX complex and Sae2 are involved in the onset of DSB resection, whereas extensive resection requires Exo1 and the concerted action of Dna2 and Sgs1. Here, we show that the checkpoint protein Rad9 limits the action of Sgs1/Dna2 in DSB resection by inhibiting Sgs1 binding/persistence at the DSB ends. When inhibition by Rad9 is abolished by the Sgs1-ss mutant variant or by deletion of RAD9, the requirement for Sae2 and functional MRX in DSB resection is reduced. These results provide new insights into how early and long-range resection is coordinated.