RESUMEN
DNA replication is essential for the propagation of life and the development of complex organisms. However, replication is a risky process as it can lead to mutations and chromosomal alterations. Conditions challenging DNA synthesis by replicative polymerases or DNA helix unwinding, generally termed as replication stress, can halt replication fork progression. Stalled replication forks are unstable, and mechanisms exist to protect their integrity, which promote an efficient restart of DNA synthesis and counteract fork collapse characterized by the accumulation of DNA lesions and mutagenic events. DNA replication is a highly regulated process, and several mechanisms control replication timing and integrity both during unperturbed cell cycles and in response to replication stress. Work over the last two decades has revealed that key steps of DNA replication are controlled by conjugation of the small peptide ubiquitin. While ubiquitylation was traditionally linked to protein degradation, the complexity and flexibility of the ubiquitin system in regulating protein function have recently emerged. Here we review the multiple roles exerted by ubiquitin-conjugating enzymes and ubiquitin-specific proteases, as well as readers of ubiquitin chains, in the control of eukaryotic DNA replication and replication-coupled DNA damage tolerance and repair.
Asunto(s)
Replicación del ADN/fisiología , Ubiquitinación/fisiología , Animales , Daño del ADN , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Humanos , Ubiquitina/metabolismo , Ubiquitina/fisiología , Ubiquitina-Proteína Ligasas/fisiología , Proteasas Ubiquitina-Específicas/fisiologíaRESUMEN
Regulation of PCNA ubiquitylation plays a key role in the tolerance to DNA damage in eukaryotes. Although the evolutionary conserved mechanism of PCNA ubiquitylation is well understood, the deubiquitylation of ubPCNA remains poorly characterized. Here, we show that the histone H2B(K123) ubiquitin protease Ubp10 also deubiquitylates ubPCNA in Saccharomyces cerevisiae. Our results sustain that Ubp10-dependent deubiquitylation of the sliding clamp PCNA normally takes place during S phase, likely in response to the simple presence of ubPCNA. In agreement with this, we show that Ubp10 forms a complex with PCNA in vivo. Interestingly, we also show that deletion of UBP10 alters in different ways the interaction of PCNA with DNA polymerase ζ-associated protein Rev1 and with accessory subunit Rev7. While deletion of UBP10 enhances PCNA-Rev1 interaction, it decreases significantly Rev7 binding to the sliding clamp. Finally, we report that Ubp10 counteracts Rad18 E3-ubiquitin ligase activity on PCNA at lysine 164 in such a manner that deregulation of Ubp10 expression causes tolerance impairment and MMS hypersensitivity.
Asunto(s)
Proteínas Nucleares , Antígeno Nuclear de Célula en Proliferación , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Ubiquitina Tiolesterasa , Ubiquitinación , Daño del ADN/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Metilmetanosulfonato/farmacología , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Unión Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina Tiolesterasa/genética , Ubiquitina Tiolesterasa/metabolismo , Ubiquitinación/efectos de los fármacos , Ubiquitinación/genéticaRESUMEN
Reactive oxygen species (ROS) are an important source of cellular damage. When ROS intracellular levels increase, oxidative stress takes place affecting DNA stability and metabolic functions. To prevent these effects, stress-activated protein kinases (SAPKs) delay cell cycle progression and induce a transcriptional response that activates antioxidant mechanisms ensuring cell adaptation and survival. Fission yeast Cdc14-like phosphatase Flp1 (also known as Clp1) has a well-established role in cell cycle regulation. Moreover, Flp1 contributes to checkpoint activation during replication stress. Here, we show that Flp1 has a role in fine-tuning the cellular oxidative stress response. Phosphorylation-dependent nucleolar release of Flp1 in response to oxidative stress conditions plays a role in the cellular transcriptional response. Thus, Flp1 ablation increases the transcriptional response to oxidative stress, in both intensity and duration, upregulating both Atf1/Pcr1- and Pap1-dependent stress induced genes. Remarkably, we found that Flp1 interacts with the Atf1/Pcr1 complex with Pcr1 acting as a direct substrate. Our results provide evidence that Flp1 modulates the oxidative stress response by limiting the Atf1/Pcr1-mediated transcription.
Asunto(s)
Schizosaccharomyces , Schizosaccharomyces/genética , Especies Reactivas de Oxígeno , Estrés Oxidativo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico , Monoéster Fosfórico HidrolasasRESUMEN
The Cdc14 family of serine-threonine phosphatases antagonizes CDK activity by reversing CDK-dependent phosphorylation events. It is well established that the yeast members of this family bring about the M/G1 transition. Budding yeast Cdc14 is essential for CDK inactivation at the end of mitosis and fission yeast Cdc14 homologue Flp1/Clp1 down-regulates Cdc25 to ensure the inactivation of mitotic CDK complexes to trigger cell division. However, the functions of human Cdc14 homologues remain poorly understood. Here we have tested the hypothesis that Cdc14A might regulate Cdc25 mitotic inducers in human cells. We found that increasing levels of Cdc14A delay entry into mitosis by inhibiting Cdk1-cyclin B1 activity. By contrast, lowering the levels of Cdc14A accelerates mitotic entry. Biochemical analyses revealed that Cdc14A acts through key Cdk1-cyclin B1 regulators. We observed that Cdc14A directly bound to and dephosphorylated Cdc25B, inhibiting its catalytic activity. Cdc14A also regulated the activity of Cdc25A at the G2/M transition. Our results indicate that Cdc14A phosphatase prevents premature activation of Cdk1 regulating Cdc25A and Cdc25B at the entry into mitosis.
Asunto(s)
Fase G2/fisiología , Mitosis/fisiología , Monoéster Fosfórico Hidrolasas/metabolismo , Fosfatasas cdc25/metabolismo , Proteína Quinasa CDC2/genética , Proteína Quinasa CDC2/metabolismo , Línea Celular , Activación Enzimática/fisiología , Humanos , Monoéster Fosfórico Hidrolasas/genética , Proteínas Tirosina Fosfatasas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fosfatasas cdc25/genéticaRESUMEN
Eukaryotes ubiquitylate the replication factor PCNA (proliferating-cell nuclear antigen) so that it tolerates DNA damage. Although, in the last few years, the understanding of the evolutionarily conserved mechanism of ubiquitylation of PCNA, and its crucial role in DNA damage tolerance, has progressed impressively, little is known about the deubiquitylation of this sliding clamp in most organisms. In the present review, we will discuss potential molecular mechanisms regulating PCNA deubiquitylation in yeast.
Asunto(s)
Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Daño del ADN , Reparación del ADN , Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Datos de Secuencia Molecular , Mutación , Proteínas de Saccharomyces cerevisiae/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Ubiquitina Tiolesterasa/genética , Ubiquitina Tiolesterasa/metabolismo , UbiquitinaciónRESUMEN
Fidelity in chromosome duplication and segregation is indispensable for maintaining genomic stability and the perpetuation of life. Challenges to genome integrity jeopardize cell survival and are at the root of different types of pathologies, such as cancer. The following three main sources of genomic instability exist: DNA damage, replicative stress, and chromosome segregation defects. In response to these challenges, eukaryotic cells have evolved control mechanisms, also known as checkpoint systems, which sense under-replicated or damaged DNA and activate specialized DNA repair machineries. Cells make use of these checkpoints throughout interphase to shield genome integrity before mitosis. Later on, when the cells enter into mitosis, the spindle assembly checkpoint (SAC) is activated and remains active until the chromosomes are properly attached to the spindle apparatus to ensure an equal segregation among daughter cells. All of these processes are tightly interconnected and under strict regulation in the context of the cell division cycle. The chromosomal instability underlying cancer pathogenesis has recently emerged as a major source for understanding the mitotic processes that helps to safeguard genome integrity. Here, we review the special interconnection between the S-phase and mitosis in the presence of under-replicated DNA regions. Furthermore, we discuss what is known about the DNA damage response activated in mitosis that preserves chromosomal integrity.
Asunto(s)
Inestabilidad Genómica/genética , Inestabilidad Genómica/fisiología , Proteínas de Ciclo Celular/genética , Inestabilidad Cromosómica/genética , Segregación Cromosómica/genética , Cromosomas/genética , Daño del ADN/genética , Reparación del ADN/genética , Replicación del ADN/genética , Humanos , Puntos de Control de la Fase M del Ciclo Celular/genética , Mitosis/genética , Fase S/genética , Huso Acromático/genéticaRESUMEN
DNA damage tolerance plays a key role in protecting cell viability through translesion synthesis and template switching-mediated bypass of genotoxic polymerase-blocking base lesions. Both tolerance pathways critically rely on ubiquitylation of the proliferating-cell nuclear antigen (PCNA) on lysine 164 and have been proposed to operate uncoupled from replication. We report that Ubp10 and Ubp12 ubiquitin proteases differentially cooperate in PCNA deubiquitylation, owing to distinct activities on PCNA-linked ubiquitin chains. Ubp10 and Ubp12 associate with replication forks in a fashion determined by Ubp10 dependency on lagging-strand PCNA residence, and they downregulate translesion polymerase recruitment and template switch events engaging nascent strands. These findings reveal PCNAK164 deubiquitylation as a key mechanism for the modulation of lesion bypass during replication, which might set a framework for establishing strand-differential pathway choices. We propose that damage tolerance is tempered at replication forks to limit the extension of bypass events and sustain chromosome replication rates.
Asunto(s)
Daño del ADN , Replicación del ADN , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Ubiquitinación , ADN de Hongos/biosíntesis , ADN Polimerasa Dirigida por ADN/metabolismo , Mutación/genética , Fase S , Moldes GenéticosRESUMEN
The Saccharomyces cerevisiae protein kinase Rad53 plays a key role in maintaining genomic integrity after DNA damage and is an essential component of the 'intra-S-phase checkpoint'. In budding yeast, alkylating chemicals, such as methyl methanesulfonate (MMS), or depletion of nucleotides by hydroxyurea (HU) stall DNA replication forks and thus activate Rad53 during S-phase. This stabilizes stalled DNA replication forks and prevents the activation of later origins of DNA replication. Here, we report that a reduction in the level of Rad53 kinase causes cells to behave very differently in response to DNA alkylation or to nucleotide depletion. While cells lacking Rad53 are unable to activate the checkpoint response to HU or MMS, so that they rapidly lose viability, a reduction in Rad53 enhances cell survival only after DNA alkylation. This reduction in the level of Rad53 allows S-phase cells to maintain the stability of DNA replication forks upon MMS treatment, but does not prevent the collapse of forks in HU. Our results may have important implications for cancer therapies, as they suggest that partial impairment of the S-phase checkpoint Rad53/Chk2 kinase provides cells with a growth advantage in the presence of drugs that damage DNA.
Asunto(s)
Antineoplásicos Alquilantes/toxicidad , Proteínas de Ciclo Celular/metabolismo , Daño del ADN , Proteínas Serina-Treonina Quinasas/metabolismo , Fase S/efectos de los fármacos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Alelos , Proteínas de Ciclo Celular/genética , Quinasa de Punto de Control 2 , Replicación del ADN , Resistencia a Medicamentos , Eliminación de Gen , Hidroxiurea/toxicidad , Metilmetanosulfonato/toxicidad , Mutación , Proteínas Serina-Treonina Quinasas/genética , Fase S/genética , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transcripción GenéticaRESUMEN
Cdc14 enzymes compose a family of highly conserved phosphatases that are present in a wide range of organisms, including yeast and humans, and that preferentially reverse the phosphorylation of Cyclin-Dependent Kinase (Cdk) substrates. The budding yeast Cdc14 orthologue has essential functions in the control of late mitosis and cytokinesis. In mammals, however, the two Cdc14 homologues, Cdc14A and Cdc14B, do not play a prominent role in controlling late mitotic events, suggesting that some Cdc14 functions are not conserved across species. Moreover, in yeast, Cdc14 is regulated by changes in its subcellular location and by phosphorylation events. In contrast, little is known about the regulation of human Cdc14 phosphatases. Here, we have studied how the human Cdc14A orthologue is regulated during the cell cycle. We found that Cdc14A is phosphorylated on Ser411, Ser453 and Ser549 by Cdk1 early in mitosis and becomes dephosphorylated during late mitotic stages. Interestingly, in vivo and in vitro experiments revealed that, unlike in yeast, Cdk1-mediated phosphorylation of human Cdc14A did not control its catalytic activity but likely modulated its interaction with other proteins in early mitosis. These findings point to differences in Cdk1-mediated mechanisms of regulation between human and yeast Cdc14 orthologues.
Asunto(s)
Aminoácidos/metabolismo , Proteína Quinasa CDC2/metabolismo , Ciclo Celular/fisiología , Monoéster Fosfórico Hidrolasas/metabolismo , Fosforilación/fisiología , Fenómenos Bioquímicos/fisiología , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Línea Celular Tumoral , Citocinesis/fisiología , Proteínas Fúngicas/metabolismo , Células HEK293 , Células HeLa , Humanos , Mitosis/fisiología , Proteínas Tirosina Fosfatasas , Levaduras/metabolismoRESUMEN
Proliferating-cell nuclear antigen (PCNA) is a DNA sliding clamp with an essential function in DNA replication and a key role in tolerance to DNA damage by ensuring the bypass of lesions. In eukaryotes, DNA damage tolerance is regulated by ubiquitylation of lysine 164 of PCNA through a well-known control mechanism; however, the regulation of PCNA deubiquitylation remains poorly understood. Our work is a systematic and functional study on PCNA deubiquitylating enzymes (DUBs) in Schizosaccharomyces pombe. Our study reveals that the deubiquitylation of PCNA in fission yeast cells is a complex process that requires several ubiquitin proteases dedicated to the deubiquitylation of a specific subnuclear fraction of mono- and di-ubiquitylated PCNA or a particular type of poly-ubiquitylated PCNA and that there is little redundancy among these enzymes. To understand how DUB activity regulates the oscillatory pattern of ubiquitylated PCNA in fission yeast, we assembled multiple DUB mutants and found that a quadruple mutation of ubp2(+), ubp12(+), ubp15(+), and ubp16(+) leads to the stable accumulation of mono-, di-, and poly-ubiquitylated forms of PCNA, increases S-phase duration, and sensitizes cells to DNA damage. Our data suggest that the dynamic ubiquitylation and deubiquitylation of PCNA occurs during S-phase to ensure processive DNA replication.
Asunto(s)
Ciclo Celular , Enzimas Desubicuitinizantes/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Procesamiento Proteico-Postraduccional , Schizosaccharomyces/fisiología , Replicación del ADN , Enzimas Desubicuitinizantes/genética , Técnicas de Inactivación de Genes , Schizosaccharomyces/metabolismoRESUMEN
Replication and segregation of the information contained in genomic DNA are strictly regulated processes that eukaryotic cells alternate to divide successfully. Experimental work on yeast has suggested that this alternation is achieved through oscillations in the activity of a serine/threonine kinase complex, CDK, which ensures the timely activation of DNA synthesis. At the same time, this CDK-mediated activation sets up the basis of the mechanism that ensures ploidy maintenance in eukaryotes. DNA synthesis is initiated at discrete sites of the genome called origins of replication on which a prereplicative complex (pre-RC) of different protein subunits is formed during the G1 phase of the cell division cycle. Only after pre-RCs are formed is the genome competent to be replicated. Several lines of evidence suggest that CDK activity prevents the assembly of pre-RCs ensuring single rounds of genome replication during each cell division cycle. This review offers a descriptive discussion of the main molecular events that a unicellular eukaryote such as the budding yeast Saccharomyces cerevisiae undergoes to initiate DNA replication.
Asunto(s)
Proteínas de Ciclo Celular/genética , Ciclo Celular/genética , Replicación del ADN/genética , ADN/biosíntesis , Células Eucariotas/metabolismo , Genes cdc/fisiología , Levaduras/genética , Animales , Células Eucariotas/citología , Evolución Molecular , Humanos , Ploidias , Levaduras/citología , Levaduras/metabolismoRESUMEN
Checkpoint response, tolerance and repair are three major pathways that eukaryotic cells evolved independently to maintain genome stability and integrity. Here, we studied the sensitivity to DNA damage in checkpoint-deficient budding yeast cells and found that checkpoint kinases Mec1 and Rad53 may modulate the balance between error-free and error-prone branches of the tolerance pathway. We have consistently observed that mutation of the RAD53 counterbalances error-free and error-prone branches upon exposure of cells to DNA damage induced either by MMS alkylation or by UV-radiation. We have also found that the potential Mec1/Rad53 balance modulation is independent from Rad6/Rad18-mediated PCNA ubiquitylation, as mec1Δ or rad53Δ mutants show no defects in the modification of the sliding clamp, therefore, we infer that it is likely exerted by acting on TLS polymerases and/or template switching targets.
Asunto(s)
Alquilantes/farmacología , Proteínas de Ciclo Celular/genética , Quinasa de Punto de Control 2/genética , Regulación Fúngica de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/genética , Metilmetanosulfonato/farmacología , Proteínas Serina-Treonina Quinasas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efectos de los fármacos , Proteínas de Ciclo Celular/metabolismo , Quinasa de Punto de Control 2/metabolismo , Daño del ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Mutación , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efectos de la radiación , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Rayos UltravioletaRESUMEN
The activity of Cdk1-cyclin B1 mitotic complexes is regulated by the balance between the counteracting activities of Wee1/Myt1 kinases and Cdc25 phosphatases. These kinases and phosphatases must be strictly regulated to ensure proper mitotic timing. One masterpiece of this regulatory network is Cdk1, which promotes Cdc25 activity and suppresses inhibitory Wee1/Myt1 kinases through direct phosphorylation. The Cdk1-dependent phosphorylation of Wee1 primes phosphorylation by additional kinases such as Plk1, triggering Wee1 degradation at the onset of mitosis. Here we report that Cdc14A plays an important role in the regulation of Wee1 stability. Depletion of Cdc14A results in a significant reduction in Wee1 protein levels. Cdc14A binds to Wee1 at its amino-terminal domain and reverses CDK-mediated Wee1 phosphorylation. In particular, we found that Cdc14A inhibits Wee1 degradation through the dephosphorylation of Ser-123 and Ser-139 residues. Thus the lack of phosphorylation of these two residues prevents the interaction with Plk1 and the consequent efficient Wee1 degradation at the onset of mitosis. These data support the hypothesis that Cdc14A counteracts Cdk1-cyclin B1 activity through Wee1 dephosphorylation.
Asunto(s)
Proteínas de Ciclo Celular , Mitosis/genética , Proteínas Nucleares , Monoéster Fosfórico Hidrolasas , Fosforilación , Proteínas Tirosina Quinasas , Proteína Quinasa CDC2/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Ciclina B1/metabolismo , Regulación de la Expresión Génica , Células HCT116 , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Estabilidad Proteica , Proteínas Tirosina Fosfatasas , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteolisis , Proteínas Proto-Oncogénicas/metabolismo , Fosfatasas cdc25/metabolismo , Quinasa Tipo Polo 1RESUMEN
Cdc14 belongs to a dual-specificity phosphatase family highly conserved through evolution that preferentially reverses CDK (Cyclin dependent kinases) -dependent phosphorylation events. In the yeast Saccharomyces cerevisiae, Cdc14 is an essential regulator of late mitotic events and exit from mitosis by counteracting CDK activity at the end of mitosis. However, many studies have shown that Cdc14 is dispensable for exiting mitosis in all other model systems analyzed. In fission yeast, the Cdc14 homologue Flp1/Clp1 regulates the stability of the mitotic inducer Cdc25 at the end of mitosis to ensure Cdk1 inactivation before cytokinesis. We have recently reported that human Cdc14A, the Cdc14 isoform located at the centrosomes during interphase, down-regulates Cdc25 activity at the G2/M transition to prevent premature activation of Cdk1-Cyclin B1 complexes and untimely entry into mitosis. Here we speculate about new molecular mechanisms for Cdc14A and discuss the current evidence suggesting that Cdc14 phosphatase plays a role in cell cycle control in higher eukaryotes.
Asunto(s)
Proteína Quinasa CDC2/antagonistas & inhibidores , División Celular , Fase G2 , Genes cdc , Monoéster Fosfórico Hidrolasas/fisiología , Centrosoma/metabolismo , Humanos , Modelos Genéticos , Monoéster Fosfórico Hidrolasas/genética , Fosforilación , Proteínas Tirosina FosfatasasRESUMEN
Kinases and phosphatases regulate messenger RNA synthesis through post-translational modification of the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II (ref. 1). In yeast, the phosphatase Cdc14 is required for mitotic exit(2,3) and for segregation of repetitive regions(4). Cdc14 is also a subunit of the silencing complex RENT (refs 5,6), but no roles in transcriptional repression have been described. Here we report that inactivation of Cdc14 causes silencing defects at the intergenic spacer sequences of ribosomal genes during interphase and at Y' repeats in subtelomeric regions during mitosis. We show that the role of Cdc14 in silencing is independent of the RENT deacetylase subunit Sir2. Instead, Cdc14 acts directly on RNA polymerase II by targeting CTD phosphorylation at Ser 2 and Ser 5. We also find that the role of Cdc14 as a CTD phosphatase is conserved in humans. Finally, telomere segregation defects in cdc14 mutants(4) correlate with the presence of subtelomeric Y' elements and can be rescued by transcriptional inhibition of RNA polymerase II.
Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Silenciador del Gen/fisiología , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/metabolismo , ARN Polimerasa II/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Telómero/metabolismo , Transcripción Genética/fisiología , Proteínas de Ciclo Celular/antagonistas & inhibidores , ADN Espaciador Ribosómico/genética , Interfase/genética , Mitosis/genética , Fosforilación/genética , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , ARN Polimerasa II/antagonistas & inhibidores , Secuencias Repetitivas de Ácidos Nucleicos , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Supresión Genética/fisiología , Telómero/enzimologíaRESUMEN
To maintain genomic integrity cells have to respond properly to a variety of exogenous and endogenous factors that produce genome injuries and interfere with DNA replication. DNA integrity checkpoints coordinate this response by slowing cell cycle progression to provide time for the cell to repair the damage, stabilizing replication forks and stimulating DNA repair to restore the original DNA sequence and structure. In addition, there are also mechanisms of damage tolerance, such as translesion synthesis (TLS), which are important for survival after DNA damage. TLS allows replication to continue without removing the damage, but results in a higher frequency of mutagenesis. Here, we investigate the functional contribution of the Dot1 histone methyltransferase and the Rad53 checkpoint kinase to TLS regulation in Saccharomyces cerevisiae. We demonstrate that the Dot1-dependent status of H3K79 methylation modulates the resistance to the alkylating agent MMS, which depends on PCNA ubiquitylation at lysine 164. Strikingkly, either the absence of DOT1, which prevents full activation of Rad53, or the expression of an HA-tagged version of RAD53, which produces low amounts of the kinase, confer increased MMS resistance. However, the dot1Δ rad53-HA double mutant is hypersensitive to MMS and shows barely detectable amounts of activated kinase. Furthermore, moderate overexpression of RAD53 partially suppresses the MMS resistance of dot1Δ. In addition, we show that MMS-treated dot1Δ and rad53-HA cells display increased number of chromosome-associated Rev1 foci. We propose that threshold levels of Rad53 activity exquisitely modulate the tolerance to alkylating damage at least by controlling the abundance of the key TLS factor Rev1 bound to chromatin.
Asunto(s)
Alquilantes/metabolismo , Proteínas de Ciclo Celular/metabolismo , Daño del ADN , N-Metiltransferasa de Histona-Lisina/genética , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Ciclo Celular/genética , Quinasa de Punto de Control 2 , Reparación del ADN , Replicación del ADN , ADN de Hongos/genética , ADN de Hongos/metabolismo , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Metilmetanosulfonato/metabolismo , Mutagénesis , Proteínas Nucleares/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
The Cdc14p-like phosphatase Flp1p (also known as Clp1p) is regulated by cell cycle-dependent changes in its subcellular localization. Flp1p is restricted to the nucleolus and spindle pole body until prophase, when it is dispersed throughout the nucleus, mitotic spindle, and medial ring. Once released, Flp1p antagonizes Cdc2p/cyclin activity by reverting Cdc2p-phosphorylation sites on Cdc25p. On replication stress, ataxia-telangiectasia mutated/ATM/Rad3-related kinase Rad3p activates Cds1p, which phosphorylates key proteins ensuring the stability of stalled DNA replication forks. Here, we show that replication stress induces changes in the subcellular localization of Flp1p in a checkpoint-dependent manner. Active Cds1p checkpoint kinase is required to release Flp1p into the nucleus. Consistently, a Flp1p mutant (flp1-9A) lacking all potential Cds1p phosphorylation sites fails to relocate in response to replication blocks and, similarly to cells lacking flp1 (Deltaflp1), presents defects in checkpoint response to replication stress. Deltaflp1 cells accumulate reduced levels of a less active Cds1p kinase in hydroxyurea (HU), indicating that nuclear Flp1p regulates Cds1p full activation. Consistently, Deltaflp1 and flp1-9A have an increased percentage of Rad22p-recombination foci during HU treatment. Together, our data show that by releasing Flp1p into the nucleus Cds1p checkpoint kinase modulates its own full activation during replication stress.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Nucléolo Celular/enzimología , Replicación del ADN , Fosfoproteínas Fosfatasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/citología , Schizosaccharomyces/enzimología , Proteínas 14-3-3/metabolismo , Proteína Quinasa CDC2/metabolismo , Nucléolo Celular/efectos de los fármacos , Quinasa de Punto de Control 2 , Replicación del ADN/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Hidroxiurea/farmacología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Cinética , Mutación/genética , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Proteínas Recombinantes de Fusión/metabolismo , Recombinación Genética/genética , Fase S/efectos de los fármacos , Schizosaccharomyces/efectos de los fármacosRESUMEN
The Schizosaccharomyces pombe Flp1p serine-threonine phosphatase is required for the degradation of the mitotic inducer Cdc25p at the end of mitosis. Cdc25p degradation prevents Cdc2p-tyrosine 15 dephosphorylation and, thus, contributes to the timely inactivation of mitotic CDK-associated kinase activity. Both RING- and HECT-type protein-ubiquitin ligases are involved in Cdc25p destabilization. Flp1p function is required for Cdc25p ubiquitination via anaphase-promoting complex/cyclosome or APC/C (RING-type) and the absence of Pub1p (HECT-type) stabilizes the mitotic inducer. In the present report, we study the functional relationship of Flp1p with Pub1p and Pub2p HECT-type-protein ubiquitin ligases. We show that Flp1p is required for the rapid degradation of Cdc25p while Pub1p is responsible for the long-term destabilization of the mitotic inducer. Accordingly, flp1 and pub1 mutants have a strong genetic interaction, correlating defects in the coordination of mitosis and cytokinesis with the stabilization of hyperactive Cdc25p. However, we also show that Flp1 and Pub2p proteins functionally interact in vivo suggesting that both proteins belong to the same regulatory network in S. pombe cells. Thus Flp1p appears to have an important role in integrating HECT- and RING-type ubiquitin ligases in cell cycle control.
Asunto(s)
Ligasas de Carbono-Nitrógeno/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimología , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Ligasas de Carbono-Nitrógeno/genética , Proteínas de Ciclo Celular/genética , Citocinesis/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Enzimológica de la Expresión Génica/genética , Regulación Fúngica de la Expresión Génica/genética , Genes cdc/fisiología , Mitosis/genética , Proteínas Tirosina Fosfatasas/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Complejos de Ubiquitina-Proteína Ligasa/genética , Ubiquitinación/genética , ras-GRF1/genética , ras-GRF1/metabolismoRESUMEN
Human Cdc14A is an evolutionary conserved dual-specificity protein phosphatase that reverses the modifications effected by cyclin-dependent kinases and plays an important role in centrosome duplication and mitotic regulation. Few substrates of Cdc14A have been identified, some of them with homologues in yeast that, in turn, are substrates of the Saccharomyces cerevisiae Cdc14 homologue, a protein phosphatase essential for yeast cell viability owing its role in mitotic exit regulation. Identification of the physiological substrates of human Cdc14A is an immediate goal in order to elucidate which cellular processes it regulates. Here, we show that human Cdc14A can dephosphorylate Cdc25A in vitro. Specifically, the Cdk1/Cyclin-B1-dependent phosphate groups on Ser115 and Ser320 of Cdc25A were found to be removed by Cdc14A. Cdc25A is an important cell cycle-regulatory protein involved in several cell cycle transitions and checkpoint responses and whose function and own regulation depend on complex phosphorylation/dephosphorylation-mediated processes. Importantly, we also show that the upregulation of Cdc14A phosphatase affects Cdc25A protein levels in human cells. Our results suggest that Cdc14A may be involved in the cell cycle regulation of Cdc25A stability.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Fosfoserina/metabolismo , Fosfatasas cdc25/química , Fosfatasas cdc25/metabolismo , Secuencia de Aminoácidos , Ciclina B/metabolismo , Ciclina B1 , Humanos , Espectrometría de Masas , Datos de Secuencia Molecular , Fosforilación , Proteínas Tirosina FosfatasasRESUMEN
Budding and fission yeast Cdc14 homologues, a conserved family of serine-threonine phosphatases, play a role in the inactivation of mitotic cyclin-dependent kinases (CDKs) by molecularly distinct mechanisms. Saccharomyces cerevisiae Cdc14 protein phosphatase inactivates CDKs by promoting mitotic cyclin degradation and the accumulation of a CDK inhibitor to allow budding yeast cells to exit from mitosis. Schizosaccharomyces pombe Flp1 phosphatase down-regulates CDK/cyclin activity, controlling the degradation of the Cdc25 tyrosine phosphatase for fission yeast cells to undergo cytokinesis. In the present work, we show that human Cdc14 homologues (hCdc14A and hCdc14B) rescued flp1-deficient fission yeast strains, indicating functional homology. We also show that hCdc14A and B interacted in vivo with S. pombe Cdc25 and that hCdc14A dephosphorylated this mitotic inducer both in vitro and in vivo. Our results support a Cdc14 conserved inhibitory mechanism acting on S. pombe Cdc25 protein and suggest that human cells may regulate Cdc25 in a similar manner to inactivate Cdk1-mitotic cyclin complexes.