Caracterización de la fosfatasa PP4 en respuesta a una lesión en el ADN

Los organismos están continuamente expuestos a la aparición de daños en su genoma, debido a factores tanto exógenos como endógenos. Afortunadamente, las células eucariotas han desarrollado una serie de mecanismos que aseguran la integridad de su material genético, conocidos en conjunto como respuesta a daño en el ADN o DDR (DNA Damage Response). Numerosos estudios han demostrado que las señales del DDR se transmiten principalmente a través de eventos de fosforilación en diversos factores de la ruta mediados por quinasas específicas. Sin embargo, poco se conoce sobre la función que las proteínas fosfatasas juegan en este proceso. La fosfatasa PP4 ha sido involucrada recientemente en la regulación del DDR, siendo su papel principal la desactivación del checkpoint de daño una vez la lesión ha sido reparada. Los resultados recogidos en esta tesis ponen de manifiesto que esta fosfatasa también presenta una función directa en la reparación de roturas en el genoma. Concretamente, esta enzima contrarresta la actividad de la quinasa Rad53 durante la reparación de un corte de doble cadena. Esta función afecta al estado de fosforilación de múltiples factores implicados en el DDR, entre ellos, del adaptador Rad9. La desfosforilación de Rad53 por PP4 estimula la resección del ADN mitigando el efecto negativo que Rad9 ejerce sobre el complejo Sgs1/Dna2. Como consecuencia, la eliminación de la actividad de PP4 afecta a la resección y reparación mediante SSA (single-strand annealing), defectos que se revierten al reducir los altos niveles de fosforilación de Rad53 o al eliminar la unión de Rad9 de la zona de ADN dañada. Estos datos confirman que PP4 controla la fosforilación de Rad53 durante la regeneración de un daño, y demuestran que la atenuación de su actividad quinasa durante las etapas iniciales del proceso de reparación es esencial para potenciar el desarrollo de rutas de reparación que dependen de una resección de largo alcance para su consecución. Además, estos resultados implican que estas vías son incompatibles con una activación excesiva del checkpoint de daño, y sugieren que las quinasas y fosfatasas del DDR han de cooperar durante la respuesta para acoplar la activación de la parada del ciclo celular con la reparación. Este balance de la actividad del checkpoint asegura una respuesta a daño precisa, suficientemente fuerte para activar un arresto en G2/M adecuado, pero no demasiado robusta como para influir negativamente en la reparación, asegurándose de esta forma el mantenimiento de la integridad genómica

Role of protein phosphatases PP1, PP2A, PP4 and Cdc14 in the DNA damage response

Maintenance of genome integrity is fundamental for cellular physiology. Our hereditary information encoded in the DNA is intrinsically susceptible to suffer variations, mostly due to the constant presence of endogenous and environmental genotoxic stresses. Genomic insults must be repaired to avoid loss or inappropriate transmission of the genetic information, a situation that could lead to the appearance of developmental anomalies and tumorigenesis. To safeguard our genome, cells have evolved a series of mechanisms collectively known as the DNA damage response (DDR). This surveillance system regulates multiple features of the cellular response, including the detection of the lesion, a transient cell cycle arrest and the restoration of the broken DNA molecule. While the role of multiple kinases in the DDR has been well documented over the last years, the intricate roles of protein dephosphorylation have only recently begun to be addressed. In this review, we have compiled recent information about the function of protein phosphatases PP1, PP2A, PP4 and Cdc14 in the DDR, focusing mainly on their capacity to regulate the DNA damage checkpoint and the repair mechanism encompassed in the restoration of a DNA lesion

Sgs1’s roles in DNA end resection, HJ dissolution, and crossover suppression require a two-step SUMO regulation dependent on Smc5/6

The RecQ helicase Sgs1 plays critical roles during DNA repair by homologous recombination, fromend resection to Holliday junction (HJ) dissolution. Sgs1 has both pro- and anti-recombinogenic roles, and therefore its activity must be tightly regulated. However, the controls involved in recruitment and activation of Sgs1 at damaged sites are unknown. Here we show a two-step role for Smc5/6 in recruiting and activating Sgs1 through SUMOylation. First, auto-SUMOylation of Smc5/6 subunits leads to recruitment of Sgs1 as part of the STR (Sgs1–Top3–Rmi1) complex, mediated by two SUMO-interacting motifs (SIMs) on Sgs1 that specifically recognize SUMOylated Smc5/6. Second, Smc5/6-dependent SUMOylation of Sgs1 and Top3 is required for the efficient function of STR. Sgs1 mutants impaired in recognition of SUMOylated Smc5/6 (sgs1-SIMΔ) or SUMO-dead alleles (sgs1-KR) exhibit unprocessed HJs at damaged replication forks, increased crossover frequencies during double-strand break repair, and severe impairment in DNA end resection. Smc5/6 is a key regulator of Sgs1’s recombination functions.We thank the Aragon laboratory for discussions and critical reading of the manuscript.We thank the Clinical Sciences Centre Proteomics Facility (P. Cutillas and P. Faull) for help and advice on our proteomic analysis. Work in J.T.-R.’s laboratory is supported by grants BFU2015-71308-P and BFU2013-50245-EXP from the Spanish Ministry of Economy and Competitivity.Work in the Aragon laboratory was supported by the intramural programme of the Medical Research Council UK and the Wellcome Trust (100955)

CIBERER : Spanish national network for research on rare diseases: A highly productive collaborative initiative

Altres ajuts: Instituto de Salud Carlos III (ISCIII); Ministerio de Ciencia e Innovación.CIBER (Center for Biomedical Network Research; Centro de Investigación Biomédica En Red) is a public national consortium created in 2006 under the umbrella of the Spanish National Institute of Health Carlos III (ISCIII). This innovative research structure comprises 11 different specific areas dedicated to the main public health priorities in the National Health System. CIBERER, the thematic area of CIBER focused on rare diseases (RDs) currently consists of 75 research groups belonging to universities, research centers, and hospitals of the entire country. CIBERER's mission is to be a center prioritizing and favoring collaboration and cooperation between biomedical and clinical research groups, with special emphasis on the aspects of genetic, molecular, biochemical, and cellular research of RDs. This research is the basis for providing new tools for the diagnosis and therapy of low-prevalence diseases, in line with the International Rare Diseases Research Consortium (IRDiRC) objectives, thus favoring translational research between the scientific environment of the laboratory and the clinical setting of health centers. In this article, we intend to review CIBERER's 15-year journey and summarize the main results obtained in terms of internationalization, scientific production, contributions toward the discovery of new therapies and novel genes associated to diseases, cooperation with patients' associations and many other topics related to RD research

Caracterización de la fosfatasa PP4 en respuesta a una lesión en el ADN

Tesis llevada a cabo para conseguir el grado de Doctor por la Universidad de Salamanca.--2020-06-22.--Sobresaliente Cum LaudemLos organismos están continuamente expuestos a la aparición de daños en su genoma, debido a factores tanto exógenos como endógenos. Afortunadamente, las células eucariotas han desarrollado una serie de mecanismos que aseguran la integridad de su material genético, conocidos en conjunto como respuesta a daño en el ADN o DDR (DNA Damage Response). Numerosos estudios han demostrado que las señales del DDR se transmiten principalmente a través de eventos de fosforilación en diversos factores de la ruta mediados por quinasas específicas. Sin embargo, poco se conoce sobre la función que las proteínas fosfatasas juegan en este proceso. La fosfatasa PP4 ha sido involucrada recientemente en la regulación del DDR, siendo su papel principal la desactivación del checkpoint de daño una vez la lesión ha sido reparada. Los resultados recogidos en esta tesis ponen de manifiesto que esta fosfatasa también presenta una función directa en la reparación de roturas en el genoma. Concretamente, esta enzima contrarresta la actividad de la quinasa Rad53 durante la reparación de un corte de doble cadena. Esta función afecta al estado de fosforilación de múltiples factores implicados en el DDR, entre ellos, del adaptador Rad9. La desfosforilación de Rad53 por PP4 estimula la resección del ADN mitigando el efecto negativo que Rad9 ejerce sobre el complejo Sgs1/Dna2. Como consecuencia, la eliminación de la actividad de PP4 afecta a la resección y reparación mediante SSA (single-strand annealing), defectos que se revierten al reducir los altos niveles de fosforilación de Rad53 o al eliminar la unión de Rad9 de la zona de ADN dañada. Estos datos confirman que PP4 controla la fosforilación de Rad53 durante la regeneración de un daño, y demuestran que la atenuación de su actividad quinasa durante las etapas iniciales del proceso de reparación es esencial para potenciar el desarrollo de rutas de reparación que dependen de una resección de largo alcance para su consecución. Además, estos resultadosLa realización de este trabajo ha sido posible gracias a la concesión de una ayuda destinada a financiar la contratación predoctoral de personal investigador, concedida por la Junta de Castilla y León y cofinanciada por el Fondo Social Europeo (Orden de bases reguladoras EDU/602/2016 y Orden de resolución EDU/529/2017). Este trabajo ha sido financiado por los proyectos del Ministerio de Ciencia, Innovación y Universidades BFU2013-41216-P y BFU2016-77081-P

PP4 phosphatase enhances recombinational DNA repair by stimulating DNA end resection

Resumen del póster presentado al First CABIMER International Workshop: "Trends in Genome Integrity & Chromosome Dynamics", celebrado en Sevilla (España) del 19 al 21 de febrero de 2020.Eukaryotic cells are constantly threatened by innumerable sources of genotoxic stresses that cause DNA damage. In order to maintain genome integrity in response to a DNA lesion, cells have developed a coordinated signalling network known as the DNA Damage Response (DDR). This surveillance pathway coordinates the expression of genes required for the repair of the DNA break and triggers a checkpoint signal that restrains replication and segregation of damaged DNA. While numerous kinases have been thoroughly studied during the activation of the DDR, the intricate roles of protein phosphatases in the response remain elusive. Here we show that PP4 phosphatase counteracts Rad53 activation during the repair of a DNA break, a process that affects on the phosphorylation state of multiple DDR factors. Active dephosphorylation of Rad53 during the initial stages of the repair pathway stimulates DNA end resection by relieving the negative effect that Rad9 exerts over the Sgs1/Dna2 exonuclease complex. PP4-dependent resection is essential to efficiently enhance recombinational repair pathways that rely on long-range resection for their accomplishment. Importantly, the deficiencies in resection and repair observed in the absence of PP4 activity are bypassed by reducing Rad53 phosphorylation or by disrupting Rad9 tethering to the DNA lesion. These results indicate that PP4 enhances resection/repair by counteracting Rad53-dependent association of Rad9 to the vicinity of the DNA break. We also show evidences suggesting that PP4 steady-state activity in response to a DNA lesion is regulated by the posttranscriptional modifications of its regulatory subunits. Overall, we propose that the control of Rad53 activity during DNA repair is tightly regulated by DDR-kinases and phosphatases cooperating along the damage response to couple checkpoint activation with repair efficiency. This balance in checkpoint activity ensures a precise DNA damage response, strong enough to activate an accurate G2/M cell cycle arrest, but not too robust as to negative influence in the repair of the DNA lesion

The spindle-stabilizing function of Cdc14 is required to promote recombinatorial DNA repair

Póster presentado al XL Congreso de la Sociedad Española de Genética, celebrado en Córdoba del 16 al 18 de septiembre de 2015.Endogenous or exogenous agents that cause genotoxic stress are constantly threatening the genomes of all organisms. In response to a DNA lesion, different processes (collectively known as DDR) are triggered in order to coordinate the repair of the damage with cell cycle progression. While phosphorylation events after DNA damage have been thoroughly studied in the DDR, little is known about the role of dephosphorylation during the response. Previous data coming from our group have revealed the importance of the phosphatase Cdc14 in promoting recombinational DNA repair. Here we show that in response to a DSB (double strand break) induced by the expression of the HO endonuclease, cells block in G2/M with a metaphase spindle aligned along the bud axis. Under this arrest, the DNA break is actively recruited to one of the SPBs (Spindle Pole Bodies). Inactivation of the phosphatase activity causes continuous misalignment of the metaphase spindle, increases oscillatory SPBs movements and impairs DSB-SPB tethering. All these phenotypes can be mimicked in a wild-type strain just by adding the microtubule depolymerizing drug nocodazole or by inactivating Spc110, a component of the SPB and a Cdc14 target during the DDR. Surprisingly, these phenotypes are directly linked to DNA repair, since both nocodazole treatment and lack of Spc110 activity impair DSB repair by HR to the same extent as cdc14-1 mutants. Together, our results point to the function of Cdc14 in DNA repair by promoting SPB stabilization and SPB-DSB interaction, and suggest that the relocation of damage sites to the SPBs plays an important role in a naturally occurring repair process that minimizes genome instability.Peer Reviewe

Cdc14 is released from the nucleolus under DNA damage and is required for DNA repair by homologous recombination

Resumen del póster presentado al XXXIX Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Salamanca del 5 al 8 de septiembre de 2016.Endogenous metabolic products, such as reactive oxygen species, and exogenous genotoxic stress, constantly assault the genetic material in the cell. In response to these sources of DNA damage, cells have developed a coordinated signalling network known as the DDR (DNA damage response), which coordinates cell cycle progression with DNA repair. Recently, multiple lines of evidences have suggested a complex role for several kinases in the regulation of the DDR (including the CDK), but little is known about the phosphatases that revert the effects of these kinases. The serine/threonine phosphatase Cdc14 was firstly identify in S. cerevisiae as an essential cell cycle phosphatase required for Cdk inactivation. This phosphatase is sequestered into the nucleolus during interphase due to its interaction with Net1. During anaphase, Net1 is phosphorylated and Cdc14 is released from the nucleolus to allow cells exit from mitosis. Cdc14 has predisposition to dephosphorylate Cdk targets, therefore is reasonable to think that could be a key candidate to counterbalance the effect imposed by the Cdk in the DDR. In favour of this hypothesis, Cdc14 is required for cell viability under different DNA damage conditions. To determine the role of the phosphatase in the DDR, we used two different recombinational repair pathways: SDSA (Synthensis dependent strand annealing) and dHJ (double Holliday junction repair). We observed that cells lacking Cdc14 activity presented defects in DNA repair in both SDSA and dHJ. Supporting the role of the phosphatase in DNA repair, we observed that Cdc14 was released from the nucleolus after induction of a single DSB or treatment with phleomycin. Accordingly, Net1 was phosphorylated during the execution of the DDR. Finally, by using mass spectrometry in a screen to identify targets of the phosphatase exclusively in DNA damage, we have detected several targets directly implicated in DNA damage repair. Altogether, we have placed Cdc14 at the DNA damage response context by collaborating in the repair throughout the modulation of the homologous recombination repair pathway, and providing new evidences about the role of this phosphatase in the repair of a DNA lesion.Peer Reviewe

The importance of the mitotic spindle integrity in DNA repair

Resumen del trabajo presentado al XXXIX Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Salamanca del 5 al 8 de septiembre de 2016.Maintenance of genome integrity is a vital aspect of our cellular physiology. Our hereditary information encoded in the DNA is constantly threatened by both endogenous and environmental genotoxic stresses that could alter our genomic material. To combat this threat, eukaryotic cells have evolved a series of mechanisms, collectively known as DDR, to survey several features of the cellular response, including the detection of the lesion, a transient cell cycle arrest and the repair of the broken DNA. During the last years it has becoming clearer the biochemical mechanisms operating at each stage of the DDR. However, little is known about the spatial regulation of their components during the DNA damage response and how the relocation of the DNA lesion itself can influence in the repair process. Interestingly, previous studies have indicated that DNA breaks are re-localized from the nucleoplasm to the nuclear periphery, suggesting that nuclear compartmentalization of DNA lesions could comprise another layer in the regulation of the DNA repair pathway. Nevertheless, whether the nuclear periphery harbours an environment that is permissive for DNA repair and its implications in maintaining genome integrity is a subject that still under debate. Remarkably, new data coming from our group indicate that the cell cycle phosphatase Cdc14 is involved in DNA repair by controlling the tethering of a DNA lesion into the spindle pole body (SPB) region of the nuclear envelope. This function is attained by preserving the integrity of the metaphase spindle, a vital requirement to stimulate DSB-SPB interaction and thus DNA repair. Accordingly, disruption of spindle stability impairs both DSB-SPB interaction and DNA repair by homologous recombination. These observations directly connect spindle integrity with DNA repair and reveal that DSBs are preferentially tethered to the SPBs to be restored. Importantly, this new function of Cdc14 could provide a physiological mechanism that spatially regulates the DNA damage response and therefore the fate of the repair process.Peer Reviewe

Cdc14 is required for DNA repair by homologous recombination

Resumen del póster presentado al XL Congreso de la Sociedad Española de Genética, celebrado en Córdoba del 16 al 18 de septiembre de 2015.Endogenous metabolic products, such as reactive oxygen species, and exogenous physical and chemical genotoxic stress, constantly assault the genetic material in the cell. It has been estimated that there are about 105 lesions per cell per day in humans. In response to such a high levels of DNA damage, cells have developed a coordinated signalling network known as the DDR (DNA damage response), which coordinate cell cycle progression with DNA repair. When this system fail or the rate of DNA damage exceeds the capacity of the pathway to repair them, the accumulation of errors can overwhelm the cell and result in early senescence, apoptosis, or cancer. Recently, multiple lines of evidences have suggested a complex role for several kinases in the regulation of the DDR (including the CDK), but little is known about the phosphatases that revert the effects of these kinases. The serine/threonine phosphatase Cdc14 was firstly identify in S. cerevisiae as an essential cell cycle phosphatase required for Cdk inactivation. One special feature of this phosphatase is its predisposition to dephosphorylate targets of the Cdk, therefore is reasonable to think that Cdc14 could be a key candidate to counterbalance the effect imposed by the Cdk in the DDR. In favour of this hypothesis, Cdc14 is required for cell viability under different DNA damage conditions. Cells lacking Cdc14 activity present a slow kinetic of DNA repair in two different recombinatorial repair pathways: SDSA (Synthensis dependent strand annealing) and dHR (double Holliday junction repair). These defects are not associated with a slow resection at the break, indicating a later role of the phosphatase in the repair process. Additionally, Cdc14 is not required for determine the choice repair between NHEJ (Non-homologous end joining) and HR (Homologous recombination). Supporting a function of the phosphatase in DNA repair, Cdc14 is release from the nucleolus after induction of a single DSB, colocalizing at the cut site by microscopy and chromatin immunoprecipitation assays. Finally, by using mass spectrometry in a screen to identify targets of Cdc14 exclusively in DNA damage, we have detected several targets directly implicated in DNA damage repair. Altogether, we have placed Cdc14 at the DNA damage response context by collaborating in the repair throughout the modulation of the HR repair pathway, and providing new evidences about the role of this phosphatase in the repair of a DNA lesion.Peer Reviewe