RESUMO
The Mec1/ATR kinase is crucial for genome stability, yet the mechanism by which it prevents gross chromosomal rearrangements (GCRs) remains unknown. Here we find that in cells with deficient Mec1 signaling, GCRs accumulate due to the deregulation of multiple steps in homologous recombination (HR). Mec1 primarily suppresses GCRs through its role in activating the canonical checkpoint kinase Rad53, which ensures the proper control of DNA end resection. Upon loss of Rad53 signaling and resection control, Mec1 becomes hyperactivated and triggers a salvage pathway in which the Sgs1 helicase is recruited to sites of DNA lesions via the 911-Dpb11 scaffolds and phosphorylated by Mec1 to favor heteroduplex rejection and limit HR-driven GCR accumulation. Fusing an ssDNA recognition domain to Sgs1 bypasses the requirement of Mec1 signaling for GCR suppression and nearly eliminates D-loop formation, thus preventing non-allelic recombination events. We propose that Mec1 regulates multiple steps of HR to prevent GCRs while ensuring balanced HR usage when needed for promoting tolerance to replication stress.
Assuntos
Recombinação Homóloga , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Serina-Treonina Quinases , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Quinase do Ponto de Checagem 2/metabolismo , Quinase do Ponto de Checagem 2/genética , RecQ Helicases/metabolismo , RecQ Helicases/genética , Transdução de Sinais , Fosforilação , Aberrações Cromossômicas , Rearranjo GênicoRESUMO
The Mec1 and Rad53 kinases play a central role during acute replication stress in budding yeast. They are also essential for viability in normal growth conditions, but the signal that activates the Mec1-Rad53 pathway in the absence of exogenous insults is currently unknown. Here, we show that this pathway is active at the onset of normal S phase because deoxyribonucleotide triphosphate (dNTP) levels present in G1 phase may not be sufficient to support processive DNA synthesis and impede DNA replication. This activation can be suppressed experimentally by increasing dNTP levels in G1 phase. Moreover, we show that unchallenged cells entering S phase in the absence of Rad53 undergo irreversible fork collapse and mitotic catastrophe. Together, these data indicate that cells use suboptimal dNTP pools to detect the onset of DNA replication and activate the Mec1-Rad53 pathway, which in turn maintains functional forks and triggers dNTP synthesis, allowing the completion of DNA replication.
Assuntos
Replicação do DNA/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fase S/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2/genética , Quinase do Ponto de Checagem 2/metabolismo , Desoxirribonucleotídeos/genética , Desoxirribonucleotídeos/metabolismo , Regulação Fúngica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Mitose , Proteínas Serina-Treonina Quinases/genética , Origem de Replicação , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Mec1 is a DNA damage sensor, which performs an essential role in the DNA damage response pathway and glucose starvation-induced autophagy. However, the functions of Mec1 in autophagy remain unclear. In response to glucose starvation, Mec1 forms puncta, which are recruited to mitochondria through the adaptor protein Ggc1. Here, we show that Mec1 puncta also contact the phagophore assembly site (PAS) via direct binding with Atg13. Functional analysis of the Atg13-Mec1 interaction revealed two previously unrecognized protein regions, the Mec1-Binding Region (MBR) on Atg13 and the Atg13-Binding Region (ABR) on Mec1, which mediate their mutual association under glucose starvation conditions. Disruption of the MBR or ABR impairs the recruitment of Mec1 puncta and Atg13 to the PAS, consequently blocking glucose starvation-induced autophagy. Additionally, the MBR and ABR regions are also crucial for DNA damage-induced autophagy. We thus propose that Mec1 regulates glucose starvation-induced autophagy by controlling Atg13 recruitment to the PAS.
Assuntos
Proteínas de Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Quinases/metabolismo , Glucose/metabolismo , Autofagia/fisiologia , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismoRESUMO
The Mec1/ATR kinase coordinates multiple cellular responses to replication stress. In addition to its canonical role in activating the checkpoint kinase Rad53, Mec1 also plays checkpoint-independent roles in genome maintenance that are not well understood. Here we used a combined genetic-phosphoproteomic approach to manipulate Mec1 activation and globally monitor Mec1 signaling, allowing us to delineate distinct checkpoint-independent modes of Mec1 action. Using cells in which endogenous Mec1 activators were genetically ablated, we found that expression of "free" Mec1 activation domains (MADs) can robustly activate Mec1 and rescue the severe DNA replication and growth defects of these cells back to wild-type levels. However, unlike the activation mediated by endogenous activator proteins, "free" MADs are unable to stimulate Mec1-mediated suppression of gross chromosomal rearrangements (GCRs), revealing that Mec1's role in genome maintenance is separable from a previously unappreciated proreplicative function. Both Mec1's functions in promoting replication and suppressing GCRs are independent of the downstream checkpoint kinases. Additionally, Mec1-dependent GCR suppression seems to require localized Mec1 action at DNA lesions, which correlates with the phosphorylation of activator-proximal substrates involved in homologous recombination-mediated DNA repair. These findings establish that Mec1 initiates checkpoint signaling, promotes DNA replication, and maintains genetic stability through distinct modes of action.
Assuntos
Pontos de Checagem do Ciclo Celular/genética , Replicação do DNA/genética , Genoma Fúngico/genética , Saccharomyces cerevisiae/genética , Transdução de Sinais/genética , Ativação Enzimática/genética , Instabilidade Genômica/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mutação , Fosforilação , Domínios Proteicos/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteômica , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The Mec1/ATR kinase is crucial for genome maintenance in response to a range of genotoxic insults, but it remains unclear how it promotes context-dependent signaling and DNA repair. Using phosphoproteomic analyses, we uncovered a distinctive Mec1/ATR signaling response triggered by extensive nucleolytic processing (resection) of DNA ends. Budding yeast cells lacking Rad9, a checkpoint adaptor and an inhibitor of resection, exhibit a selective increase in Mec1-dependent phosphorylation of proteins associated with single-strand DNA (ssDNA) transactions, including the ssDNA-binding protein Rfa2, the translocase/ubiquitin ligase Uls1, and the Sgs1-Top3-Rmi1 (STR) complex that regulates homologous recombination (HR). Extensive Mec1-dependent phosphorylation of the STR complex, mostly on the Sgs1 helicase subunit, promotes an interaction between STR and the DNA repair scaffolding protein Dpb11. Fusion of Sgs1 to phosphopeptide-binding domains of Dpb11 strongly impairs HR-mediated repair, supporting a model whereby Mec1 signaling regulates STR upon hyper-resection to influence recombination outcomes. Overall, the identification of a distinct Mec1 signaling response triggered by hyper-resection highlights the multi-faceted action of this kinase in the coordination of checkpoint signaling and HR-mediated DNA repair.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Reparo do DNA/genética , Reparo do DNA/fisiologia , Recombinação Homóloga/genética , Recombinação Homóloga/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Serina-Treonina Quinases/genética , RecQ Helicases/genética , RecQ Helicases/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
Upon replication stress, budding yeast checkpoint kinase Mec1ATR triggers the downregulation of transcription, thereby reducing the level of RNA polymerase (RNAP) on chromatin to facilitate replication fork progression. Here, we identify a hydroxyurea-induced phosphorylation site on Mec1, Mec1-S1991, that contributes to the eviction of RNAPII and RNAPIII during replication stress. The expression of the non-phosphorylatable mec1-S1991A mutant reduces replication fork progression genome-wide and compromises survival on hydroxyurea. This defect can be suppressed by destabilizing chromatin-bound RNAPII through a TAP fusion to its Rpb3 subunit, suggesting that lethality in mec1-S1991A mutants arises from replication-transcription conflicts. Coincident with a failure to repress gene expression on hydroxyurea in mec1-S1991A cells, highly transcribed genes such as GAL1 remain bound at nuclear pores. Consistently, we find that nuclear pore proteins and factors controlling RNAPII and RNAPIII are phosphorylated in a Mec1-dependent manner on hydroxyurea. Moreover, we show that Mec1 kinase also contributes to reduced RNAPII occupancy on chromatin during an unperturbed S phase by promoting degradation of the Rpb1 subunit.
Assuntos
Replicação do DNA , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , RNA Polimerase III/genética , RNA Polimerase II/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Cromatina/química , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Galactoquinase/genética , Galactoquinase/metabolismo , Regulação Fúngica da Expressão Gênica , Hidroxiureia/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fosfoproteínas , Fosforilação , Proteínas Serina-Treonina Quinases/genética , RNA Polimerase II/metabolismo , RNA Polimerase III/metabolismo , Fase S/efeitos dos fármacos , Fase S/genética , Saccharomyces cerevisiae/genética , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genética , Transcrição GênicaRESUMO
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 replication stress threatens ordinary DNA synthesis. The evolutionarily conserved DNA replication stress response pathway involves sensor kinase Mec1/ATR, adaptor protein Mrc1/Claspin, and effector kinase Rad53/Chk1, which spurs a host of changes to stabilize replication forks and maintain genome integrity. DNA replication forks consist of largely distinct sets of proteins at leading and lagging strands that function autonomously in DNA synthesis in vitro. In this article, we discuss eSPAN and BrdU-IP-ssSeq, strand-specific sequencing technologies that permit analysis of protein localization and DNA synthesis at individual strands in budding yeast. Using these approaches, we show that under replication stress Rad53 stalls DNA synthesis on both leading and lagging strands. On lagging strands, it stimulates PCNA unloading, and on leading strands, it attenuates the replication function of Mrc1-Tof1. We propose that in doing so, Rad53 couples leading and lagging strand DNA synthesis during replication stress, thereby preventing the emergence of harmful ssDNA.
Assuntos
Proteínas de Saccharomyces cerevisiae , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2/genética , Quinase do Ponto de Checagem 2/metabolismo , DNA/metabolismo , Replicação do DNA/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
DNA replication is a highly precise process which usually functions in a perfect rhythm with cell cycle progression. However, cells are constantly faced with various kinds of obstacles such as blocks in DNA replication, lack of availability of precursors and improper chromosome alignment. When these problems are not addressed, they may lead to chromosome instability and the accumulation of mutations, and even cell death. Therefore, the cell has developed response mechanisms to keep most of these situations under control. Of the many factors that participate in this DNA damage response, members of the family of phosphatidylinositol 3-kinase-related protein kinases (PIKKs) orchestrate the response landscape. Our understanding of two members of the PIKK family, human ATR (yeast Mec1) and ATM (yeast Tel1), and their associated partner proteins, has shown substantial progress through recent biochemical and structural studies. Emerging structural information of these unique kinases show common features that reveal the mechanism of kinase activity.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Serina-Treonina Quinases , Proteínas de Saccharomyces cerevisiae , Proteínas Mutadas de Ataxia Telangiectasia/genética , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Dano ao DNA , Humanos , Proteínas Serina-Treonina Quinases/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
ATP-dependent chromatin remodeling complexes alter chromatin structure through interactions with chromatin substrates such as DNA, histones, and nucleosomes. However, whether chromatin remodeling complexes have the ability to regulate nonchromatin substrates remains unclear. Saccharomyces cerevisiae checkpoint kinase Mec1 (ATR in mammals) is an essential master regulator of genomic integrity. Here we found that the SWI/SNF chromatin remodeling complex is capable of regulating Mec1 kinase activity. In vivo, Mec1 activity is reduced by the deletion of Snf2, the core ATPase subunit of the SWI/SNF complex. SWI/SNF interacts with Mec1, and cross-linking studies revealed that the Snf2 ATPase is the main interaction partner for Mec1. In vitro, SWI/SNF can activate Mec1 kinase activity in the absence of chromatin or known activators such as Dpb11. The subunit requirement of SWI/SNF-mediated Mec1 regulation differs from that of SWI/SNF-mediated chromatin remodeling. Functionally, SWI/SNF-mediated Mec1 regulation specifically occurs in S phase of the cell cycle. Together, these findings identify a novel regulator of Mec1 kinase activity and suggest that ATP-dependent chromatin remodeling complexes can regulate nonchromatin substrates such as a checkpoint kinase.
Assuntos
Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Fatores de Transcrição/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Montagem e Desmontagem da Cromatina , Dano ao DNA/fisiologia , Ativação Enzimática , Ativadores de Enzimas/metabolismo , Fase S , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genéticaRESUMO
The RecA-family recombinase Rad51 is the central player in homologous recombination (HR), the faithful pathway for repairing DNA double-strand breaks (DSBs) during both mitosis and meiosis. The behavior of Rad51 protein in vivo is fine-tuned via posttranslational modifications conducted by multiple protein kinases in response to cell cycle cues and DNA lesions. Unrepaired DSBs and ssDNA also activate Mec1ATR and Tel1ATM family kinases to initiate the DNA damage response (DDR) that safeguards genomic integrity. Defects in HR and DDR trigger genome instability and result in cancer predisposition, infertility, developmental defects, neurological diseases or premature aging. Intriguingly, yeast Mec1ATR- and Tel1ATM-dependent phosphorylation promotes Rad51 protein stability during DDR, revealing how Mec1ATR can alleviate proteotoxic stress. Moreover, Mec1ATR- and Tel1ATM-dependent phosphorylation also occurs on DDR-unrelated proteins, suggesting that Mec1ATR and Tel1ATM have a DDR-independent function in protein homeostasis. In this minireview, we first describe how human and budding yeast Rad51 are phosphorylated by multiple protein kinases at different positions to promote homology-directed DNA repair and recombination (HDRR). Then, we discuss recent findings showing that intrinsic structural disorder and Mec1ATR/Tel1ATM-dependent phosphorylation are coordinated in yeast Rad51 to regulate both HR and protein homeostasis.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Serina-Treonina Quinases/genética , Rad51 Recombinase/genética , Proteínas de Saccharomyces cerevisiae/genética , Dano ao DNA/genética , Homeostase/genética , Recombinação Homóloga/genética , Humanos , Meiose/genética , Mitose/genética , Saccharomyces cerevisiae/genéticaRESUMO
DNA replication checkpoint is a cell signaling pathway that is activated in response to perturbed replication. Although it is crucial for maintaining genomic integrity and cell survival, the exact mechanism of the checkpoint signaling remains to be understood. Emerging evidence has shown that RecQ helicases, a large family of helicases that are conserved from bacteria to yeasts and humans, contribute to the replication checkpoint as sensors, adaptors, or regulation targets. Here, we highlight the multiple functions of RecQ helicases in the replication checkpoint in four model organisms and present additional evidence that fission yeast RecQ helicase Rqh1 may participate in the replication checkpoint as a sensor.
Assuntos
Pontos de Checagem do Ciclo Celular/genética , DNA Helicases/genética , Replicação do DNA/genética , RecQ Helicases/genética , Proteínas de Schizosaccharomyces pombe/genética , Humanos , Schizosaccharomyces/genética , Transdução de Sinais/genéticaRESUMO
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
Double-strand break repair by recombination requires a homology search. In yeast, induced breaks move significantly more than undamaged loci. To examine whether DNA damage provokes an increase in chromatin mobility generally, we tracked undamaged loci under DNA-damaging conditions. We found that the yeast checkpoint factors Mec1, Rad9, and Rad53 are required for genome-wide increases in chromatin mobility, but not the repair protein Rad51. Mec1 activation by targeted Ddc1/Ddc2 enhances chromatin mobility even in the absence of damage. Finally, the INO80 chromatin remodeler is shown to act downstream from Mec1 to increase chromatin mobility, highlighting an additional damage-related role of this nucleosome remodeling complex.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Dano ao DNA , Fosfotransferases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Pontos de Checagem do Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/genética , Genoma Fúngico/genética , Modelos Biológicos , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Chromosomes are constantly damaged by exogenous and endogenous factors. To cope with DNA damage, eukaryotic cells are equipped with three phosphatidylinositol 3-kinase-related kinases (PIKKs), such as ATM, ATR, and DNA-PK. PIKKs are structurally related to phosphatidylinositol 3-kinase (lipid kinase), however possess protein kinase activities. The Mre11-Rad50-Nbs1 and the Ku complex interact with and activate ATM and DNA-PKcs at double-stranded DNA breaks (DSBs), respectively. In contrast, ATR responds to various types of DNA lesions by interacting with replication protein A (RPA)-covered single-stranded DNA (ssDNA). Several lines of evidence have established a model in which ATR is activated by interacting with ATR activating proteins including TopBP1 and ETAA1 at DNA lesions in humans, yet the interaction of ATR with RPA-covered ssDNA does not result in ATR activation. In budding yeast, the Mec1-Ddc2 complex (Mec1-Ddc2) corresponds to ATR-ATRIP. Similar to ATR, Mec1 activation is accomplished by interactions with Mec1 activating proteins, which are Ddc1, Dpb11 (TopBP1 homolog) and Dna2. However, recent studies provide results supporting the idea that Mec1ATR is also activated by interacting with RPA-covered ssDNA tracts. These observations suggest that all the ATM, ATR, DNA-PK family proteins can be activated immediately upon DNA damage recognition.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Dano ao DNA , Reparo do DNA , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Eucariotos/enzimologia , Eucariotos/genética , Eucariotos/metabolismo , Humanos , Proteína de Replicação A/metabolismo , Saccharomycetales/enzimologia , Saccharomycetales/genética , Saccharomycetales/metabolismo , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismoRESUMO
During cell proliferation, the genome is constantly threatened by cellular and external factors. When the DNA is damaged, or when its faithful duplication is delayed by DNA polymerase stalling, the cells induce a coordinated response termed the DNA damage response (DDR) or checkpoint. Elg1 forms an RFC-like complex in charge of unloading the DNA polymerase processively factor PCNA during DNA replication and DNA repair. Using checkpoint-inducible strains, a recently published paper (Sau et al. in mBio 10(3):e01159-19. https://doi.org/10.1128/mbio.01159-19, 2019) uncovered a role for Elg1 in eliciting the DNA damage checkpoint (DC), one of the branches of the DDR. The apical kinase Mec1/ATR phosphorylates Elg1, as well as the adaptor proteins Rad9/53BP1 and Dpb11/TopBP1, which are recruited to the site of DNA damage to amplify the checkpoint signal. In the absence of Elg1, Rad9 and Dpb11 are recruited but fail to be phosphorylated and the signal is therefore not amplified. Thus, Elg1 appears to coordinate DNA repair and the induction of the DNA damage checkpoint.
Assuntos
Proteínas de Transporte/metabolismo , Dano ao DNA , Proteínas Fúngicas/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Leveduras/genética , Leveduras/metabolismo , Reparo do DNA , Replicação do DNARESUMO
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
The Saccharomyces cerevisiae Mec1 kinase, the mammalian ATR ortholog, is essential for sensing a variety of DNA lesions and initiating DNA damage response. The Dpb11, a homolog of human TopBP1, functions in activating the Mec1 upon DNA replication stress and DNA damages. Here, we report an affinity purification and ion exchange chromatography method to efficiently purify endogenous Dpb11 under normal expression level directly from yeast whole cell extraction. The final concentration of 5⯵M of high purity and homogeneity biochemical preparation enables functional and structural characterization of the physical interaction between Dpb11 and Mec1-Ddc2 complex. The Dpb11 obtained by endogenous purification strongly stimulates the Mec1 kinase activity and promotes the changes in conformational distribution. This observation suggests the Dpb11 activates Mec1 kinase probably through modulation in the kinase conformations.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática , Humanos , Proteínas Nucleares/metabolismo , Mapas de Interação de ProteínasRESUMO
The 9-1-1 complex is a circular heterotrimeric complex composed of Rad9-Hus1-Rad1. In response to DNA damage, the 9-1-1 complex will be loaded onto the DNA damage site by clamp loader Rad24-RFC to activate the cell cycle checkpoint. The C-terminal of Ddc1/Rad9 is critical for checkpoint activation. However, there is little structural information about the intact 9-1-1 complex and the interaction with Rad24-RFC. Here, we determined the structure of the intact 9-1-1 complex in S. cerevisiae by cryo-Electron Microscopy (cryo-EM) and identified the Ddc1 C-tail module for the first time. We found that the C-terminal of Ddc1 has structural flexibility and it plays a critical role for Mec1/Ddc2 activation in G1/G2 phase. At the same time, we got a glimpse of the structure of Rad24-RFC and captured the interaction between the 9-1-1 complex and Rad24-RFC. The structural information greatly helped us to understand the process of clamp-loading.
Assuntos
Pontos de Checagem do Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ciclo Celular , Quinase do Ponto de Checagem 2/metabolismo , Microscopia Crioeletrônica , Dano ao DNA , Proteínas Nucleares/metabolismo , Fosforilação , Domínios ProteicosRESUMO
The ataxia-telangiectasia mutated/ATM and Rad3-related (ATM/ATR) family proteins are evolutionarily conserved serine/threonine kinases best known for their roles in mediating the DNA damage response. Upon activation, ATM/ATR phosphorylate numerous targets to stabilize stalled replication forks, repair damaged DNA, and inhibit cell cycle progression to ensure survival of the cell and safeguard integrity of the genome. Intriguingly, separation of function alleles of the human ATM and MEC1, the budding yeast ATM/ATR, were shown to confer widespread protein aggregation and acute sensitivity to different types of proteotoxic agents including heavy metal, amino acid analogue, and an aggregation-prone peptide derived from the Huntington's disease protein. Further analyses unveiled that ATM and Mec1 promote resistance to perturbation in protein homeostasis via a mechanism distinct from the DNA damage response. In this minireview, we summarize the key findings and discuss ATM/ATR as a multifaceted signalling protein capable of mediating cellular response to both DNA and protein damage.