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1.
EMBO J ; 38(20): e101443, 2019 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-31424118

RESUMEN

Cyclins are central engines of cell cycle progression in conjunction with cyclin-dependent kinases (CDKs). Among the different cyclins controlling cell cycle progression, cyclin F does not partner with a CDK, but instead forms via its F-box domain an SCF (Skp1-Cul1-F-box)-type E3 ubiquitin ligase module. Although various substrates of cyclin F have been identified, the vulnerabilities of cells lacking cyclin F are not known. Thus, we assessed viability of cells lacking cyclin F upon challenging them with more than 180 different kinase inhibitors. The screen revealed a striking synthetic lethality between Chk1 inhibition and cyclin F loss. Chk1 inhibition in cells lacking cyclin F leads to DNA replication catastrophe. Replication catastrophe depends on accumulation of the transcription factor E2F1 in cyclin F-depleted cells. We find that SCF-cyclin F controls E2F1 ubiquitylation and degradation during the G2/M phase of the cell cycle and upon challenging cells with Chk1 inhibitors. Thus, Cyclin F restricts E2F1 activity during the cell cycle and upon checkpoint inhibition to prevent DNA replication stress. Our findings pave the way for patient selection in the clinical use of checkpoint inhibitors.


Asunto(s)
Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/antagonistas & inhibidores , Ciclinas/metabolismo , Factor de Transcripción E2F1/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteolisis , Proteínas Ligasas SKP Cullina F-box/metabolismo , Mutaciones Letales Sintéticas , Ciclo Celular/efectos de los fármacos , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Ciclinas/genética , Replicación del ADN , Factor de Transcripción E2F1/genética , Células HeLa , Humanos , Fosforilación , Unión Proteica , Proteínas Ligasas SKP Cullina F-box/genética , Ubiquitinación
2.
Nucleic Acids Res ; 48(22): 12483-12501, 2020 12 16.
Artículo en Inglés | MEDLINE | ID: mdl-33166394

RESUMEN

Efficient S phase entry is essential for development, tissue repair, and immune defences. However, hyperactive or expedited S phase entry causes replication stress, DNA damage and oncogenesis, highlighting the need for strict regulation. Recent paradigm shifts and conflicting reports demonstrate the requirement for a discussion of the G1/S transition literature. Here, we review the recent studies, and propose a unified model for the S phase entry decision. In this model, competition between mitogen and DNA damage signalling over the course of the mother cell cycle constitutes the predominant control mechanism for S phase entry of daughter cells. Mitogens and DNA damage have distinct sensing periods, giving rise to three Commitment Points for S phase entry (CP1-3). S phase entry is mitogen-independent in the daughter G1 phase, but remains sensitive to DNA damage, such as single strand breaks, the most frequently-occurring lesions that uniquely threaten DNA replication. To control CP1-3, dedicated hubs integrate the antagonistic mitogenic and DNA damage signals, regulating the stoichiometric cyclin: CDK inhibitor ratio for ultrasensitive control of CDK4/6 and CDK2. This unified model for the G1/S cell cycle transition combines the findings of decades of study, and provides an updated foundation for cell cycle research.


Asunto(s)
Puntos de Control del Ciclo Celular/genética , Ciclo Celular/genética , División Celular/genética , Replicación del ADN/genética , Daño del ADN/genética , Fase G1/genética , Humanos , Fase S/genética , Transducción de Señal/genética
3.
FASEB J ; 34(8): 10443-10461, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32539222

RESUMEN

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme involved in energy metabolism. Recently, GAPDH has been suggested to have extraglycolytic functions in DNA repair, but the underlying mechanism for the GAPDH response to DNA damage remains unclear. Here, we demonstrate that the tyrosine kinase Src is activated under DNA damage stress and phosphorylates GAPDH at Tyr41. This phosphorylation of GAPDH is essential for its nuclear translocation and DNA repair function. Blocking the nuclear import of GAPDH by suppressing Src signaling or through a GAPDH Tyr41 mutation impairs its response to DNA damage. Nuclear GAPDH is recruited to DNA lesions and associates with DNA polymerase ß (Pol ß) to function in DNA repair. Nuclear GAPDH promotes Pol ß polymerase activity and increases base excision repair (BER) efficiency. Furthermore, GAPDH knockdown dramatically decreases BER efficiency and sensitizes cells to DNA damaging agents. Importantly, the knockdown of GAPDH in colon cancer SW480 cells and xenograft models effectively enhances their sensitivity to the chemotherapeutic drug 5-FU. In summary, our findings provide mechanistic insight into the new function of GAPDH in DNA repair and suggest a potential therapeutic target in chemotherapy.


Asunto(s)
Núcleo Celular/genética , Núcleo Celular/metabolismo , Daño del ADN/genética , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/genética , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/metabolismo , Fosforilación/genética , Familia-src Quinasas/metabolismo , Transporte Activo de Núcleo Celular/genética , Animales , Línea Celular Tumoral , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , ADN/genética , ADN Polimerasa beta/genética , ADN Polimerasa beta/metabolismo , Reparación del ADN/genética , Femenino , Células HEK293 , Xenoinjertos , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Mutación/genética , Transporte de Proteínas/genética , Transducción de Señal/genética , Familia-src Quinasas/genética
4.
FASEB J ; 33(11): 11668-11681, 2019 11.
Artículo en Inglés | MEDLINE | ID: mdl-31348687

RESUMEN

Multiple DNA repair pathways may be involved in the removal of the same DNA lesion caused by endogenous or exogenous agents. Although distinct DNA repair machinery fulfill overlapping roles in the repair of DNA lesions, the mechanisms coordinating different pathways have not been investigated in detail. Here, we show that Ku70, a core protein of nonhomologous end-joining (NHEJ) repair pathway, can directly interact with DNA polymerase-ß (Pol-ß), a central player in the DNA base excision repair (BER), and this physical complex not only promotes the polymerase activity of Pol-ß and BER efficiency but also enhances the classic NHEJ repair. Moreover, we find that DNA damages caused by methyl methanesulfonate (MMS) or etoposide promote the formation of Ku70-Pol-ß complexes at the repair foci. Furthermore, suppression of endogenous Ku70 expression by small interfering RNA reduces BER efficiency and leads to higher sensitivity to MMS and accumulation of the DNA strand breaks. Similarly, Pol-ß knockdown impairs total-NHEJ capacity but only has a slight influence on alternative NHEJ. These results suggest that Pol-ß and Ku70 coordinate 2-way crosstalk between the BER and NHEJ pathways.-Xia, W., Ci, S., Li, M., Wang, M., Dianov, G. L., Ma, Z., Li, L., Hua, K., Alagamuthu, K. K., Qing, L., Luo, L., Edick, A. M., Liu, L., Hu, Z., He, L., Pan, F., Guo, Z. Two-way crosstalk between BER and c-NHEJ repair pathway is mediated by Pol-ß and Ku70.


Asunto(s)
Daño del ADN/genética , Reparación del ADN/genética , Replicación del ADN/genética , Autoantígeno Ku/metabolismo , ADN/metabolismo , Roturas del ADN de Doble Cadena , ADN Polimerasa beta/genética , Proteínas de Unión al ADN/metabolismo , Humanos
5.
Mol Cell ; 45(6): 801-13, 2012 Mar 30.
Artículo en Inglés | MEDLINE | ID: mdl-22361354

RESUMEN

The deubiquitylation enzyme USP7/HAUSP plays a major role in regulating genome stability and cancer prevention by controlling the key proteins involved in the DNA damage response. Despite this important role in controlling other proteins, USP7 itself has not been recognized as a target for regulation. Here, we report that USP7 regulation plays a central role in DNA damage signal transmission. We find that stabilization of Mdm2, and correspondingly p53 downregulation in unstressed cells, is accomplished by a specific isoform of USP7 (USP7S), which is phosphorylated at serine 18 by the protein kinase CK2. Phosphorylation stabilizes USP7S and thus contributes to Mdm2 stabilization and downregulation of p53. After ionizing radiation, dephosphorylation of USP7S by the ATM-dependent protein phosphatase PPM1G leads to USP7S downregulation, followed by Mdm2 downregulation and accumulation of p53. Our findings provide a quantitative transmission mechanism of the DNA damage signal to coordinate a p53-dependent DNA damage response.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Daño del ADN/fisiología , Proteínas de Unión al ADN/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Secuencia de Aminoácidos , Proteínas de la Ataxia Telangiectasia Mutada , Quinasa de la Caseína II/genética , Quinasa de la Caseína II/metabolismo , Puntos de Control del Ciclo Celular , Proteínas de Ciclo Celular/genética , Proteínas de Unión al ADN/genética , Regulación hacia Abajo , Células HeLa/efectos de la radiación , Humanos , Datos de Secuencia Molecular , Fosfoproteínas Fosfatasas/genética , Fosforilación , Proteína Fosfatasa 2C , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas c-mdm2/genética , Radiación Ionizante , Serina/metabolismo , Transducción de Señal , Proteína p53 Supresora de Tumor/genética , Proteínas Supresoras de Tumor/genética , Ubiquitina Tiolesterasa/genética , Peptidasa Específica de Ubiquitina 7
6.
Nucleic Acids Res ; 46(4): 1834-1846, 2018 02 28.
Artículo en Inglés | MEDLINE | ID: mdl-29294106

RESUMEN

ATM (ataxia-telangiectasia mutated) is a central molecule for DNA quality control. Its activation by DNA damage promotes cell-cycle delay, which facilitates DNA repair prior to replication. On the other hand, persistent DNA damage has been implicated in ATM-dependent cell death via apoptosis; however, the mechanisms underlying this process remain elusive. Here we find that, in response to persistent DNA strand breaks, ATM phosphorylates transcription factor Sp1 and initiates its degradation. We show that Sp1 controls expression of the key base excision repair gene XRCC1, essential for DNA strand break repair. Therefore, degradation of Sp1 leads to a vicious cycle that involves suppression of DNA repair and further aggravation of the load of DNA damage. This activates transcription of pro-apoptotic genes and renders cells susceptible to elimination via both apoptosis and natural killer cells. These findings constitute a previously unrecognized 'gatekeeper' function of ATM as a detector of cells with persistent DNA damage.


Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Reparación del ADN , Factor de Transcripción Sp1/metabolismo , Apoptosis , Células Cultivadas , Daño del ADN , Regulación hacia Abajo , Humanos , Células Asesinas Naturales/fisiología , Masculino , Fosforilación , Serina/metabolismo , Factor de Transcripción Sp1/química , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/biosíntesis , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/genética
7.
Mol Cell ; 41(5): 609-15, 2011 Mar 04.
Artículo en Inglés | MEDLINE | ID: mdl-21362556

RESUMEN

DNA base excision repair (BER) is an essential cellular process required for genome stability, and misregulation of BER is linked to premature aging, increased rate of mutagenesis, and cancer. We have now identified the cytoplasmic ubiquitin-specific protease USP47 as the major enzyme involved in deubiquitylation of the key BER DNA polymerase (Pol ß) and demonstrate that USP47 is required for stability of newly synthesized cytoplasmic Pol ß that is used as a source for nuclear Pol ß involved in DNA repair. We further show that knockdown of USP47 causes an increased level of ubiquitylated Pol ß, decreased levels of Pol ß, and a subsequent deficiency in BER, leading to accumulation of DNA strand breaks and decreased cell viability in response to DNA damage. Taken together, these data demonstrate an important role for USP47 in regulating DNA repair and maintaining genome integrity.


Asunto(s)
ADN Polimerasa beta/metabolismo , Reparación del ADN , Regulación Enzimológica de la Expresión Génica , Ubiquitina Tiolesterasa/fisiología , Ubiquitina/química , Dominio Catalítico , Citoplasma/metabolismo , Daño del ADN , Genoma , Glicosilación , Células HeLa , Humanos , Lisina/química , Modelos Biológicos , Ubiquitina Tiolesterasa/química , Proteasas Ubiquitina-Específicas
8.
Nucleic Acids Res ; 45(17): 10042-10055, 2017 Sep 29.
Artículo en Inglés | MEDLINE | ID: mdl-28973444

RESUMEN

Ataxia telangiectasia (A-T) is a syndrome associated with loss of ATM protein function. Neurodegeneration and cancer predisposition, both hallmarks of A-T, are likely to emerge as a consequence of the persistent oxidative stress and DNA damage observed in this disease. Surprisingly however, despite these severe features, a lack of functional ATM is still compatible with early life, suggesting that adaptation mechanisms contributing to cell survival must be in place. Here we address this gap in our knowledge by analysing the process of human fibroblast adaptation to the lack of ATM. We identify profound rearrangement in cellular proteostasis occurring very early on after loss of ATM in order to counter protein damage originating from oxidative stress. Change in proteostasis, however, is not without repercussions. Modulating protein turnover in ATM-depleted cells also has an adverse effect on the DNA base excision repair pathway, the major DNA repair system that deals with oxidative DNA damage. As a consequence, the burden of unrepaired endogenous DNA lesions intensifies, progressively leading to genomic instability. Our study provides a glimpse at the cellular consequences of loss of ATM and highlights a previously overlooked role for proteostasis in maintaining cell survival in the absence of ATM function.


Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/deficiencia , Reparación del ADN/fisiología , Ataxia Telangiectasia/enzimología , Ataxia Telangiectasia/patología , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Proteínas de la Ataxia Telangiectasia Mutada/genética , Supervivencia Celular , Células Cultivadas , Fibroblastos/citología , Fibroblastos/enzimología , Humanos , Chaperonas Moleculares/metabolismo , Oxidación-Reducción , Estrés Oxidativo , Complejo de la Endopetidasa Proteasomal/metabolismo , Biosíntesis de Proteínas , Deficiencias en la Proteostasis , Interferencia de ARN , ARN Interferente Pequeño/genética , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes/metabolismo , Respuesta de Proteína Desplegada
9.
Nucleic Acids Res ; 44(7): 3165-75, 2016 Apr 20.
Artículo en Inglés | MEDLINE | ID: mdl-26773055

RESUMEN

DNA constantly undergoes chemical modification due to endogenous and exogenous mutagens. The DNA base excision repair (BER) pathway is the frontline mechanism handling the majority of these lesions, and primarily involves a DNA incision and subsequent resealing step. It is imperative that these processes are extremely well-coordinated as unrepaired DNA single strand breaks (SSBs) can be converted to DNA double strand breaks during replication thus triggering genomic instability. However, the mechanism(s) governing the BER process are poorly understood. Here we show that accumulation of unrepaired SSBs triggers a p53/Sp1-dependent downregulation of APE1, the endonuclease responsible for the DNA incision during BER. Importantly, we demonstrate that impaired p53 function, a characteristic of many cancers, leads to a failure of the BER coordination mechanism, overexpression of APE1, accumulation of DNA strand breaks and results in genomic instability. Our data provide evidence for a previously unrecognized mechanism for coordination of BER by p53, and its dysfunction in p53-inactivated cells.


Asunto(s)
Reparación del ADN , Inestabilidad Genómica , Proteína p53 Supresora de Tumor/metabolismo , Células Cultivadas , Roturas del ADN de Cadena Simple , ADN-(Sitio Apurínico o Apirimidínico) Liasa/biosíntesis , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , Regulación hacia Abajo , Humanos , Factor de Transcripción Sp1/metabolismo
10.
Proc Natl Acad Sci U S A ; 112(13): 3997-4002, 2015 Mar 31.
Artículo en Inglés | MEDLINE | ID: mdl-25775545

RESUMEN

DNA single-strand breaks (SSBs) arise as a consequence of spontaneous DNA instability and are also formed as DNA repair intermediates. Their repair is critical because they otherwise terminate gene transcription and generate toxic DNA double-strand breaks (DSBs) on replication. To prevent the formation of DSBs, SSB repair must be completed before DNA replication. To accomplish this, cells should be able to detect unrepaired SSBs, and then delay cell cycle progression to allow more time for repair; however, to date there is no evidence supporting the coordination of SSB repair and replication in human cells. Here we report that ataxia-telangiectasia mutated kinase (ATM) plays a major role in restricting the replication of SSB-containing DNA and thus prevents DSB formation. We show that ATM is activated by SSBs and coordinates their repair with DNA replication. SSB-mediated ATM activation is followed by a G1 cell cycle delay that allows more time for repair and thus prevents the replication of damaged DNA and DSB accrual. These findings establish an unanticipated role for ATM in the signaling of DNA SSBs and provide important insight into the molecular defects leading to genetic instability in patients with ataxia-telangiectasia.


Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Ciclo Celular , Roturas del ADN de Doble Cadena , Roturas del ADN de Cadena Simple , Reparación del ADN , Apoptosis , Línea Celular , Ensayo Cometa , ADN/química , Proteínas de Unión al ADN/metabolismo , Fibroblastos/metabolismo , Silenciador del Gen , Genoma , Humanos , Microscopía Fluorescente , Mutación , Fosforilación , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X
11.
Development ; 141(20): 3966-77, 2014 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-25294941

RESUMEN

Initially identified in DNA damage repair, ATM-interactor (ATMIN) further functions as a transcriptional regulator of lung morphogenesis. Here we analyse three mouse mutants, Atmin(gpg6/gpg6), Atmin(H210Q/H210Q) and Dynll1(GT/GT), revealing how ATMIN and its transcriptional target dynein light chain LC8-type 1 (DYNLL1) are required for normal lung morphogenesis and ciliogenesis. Expression screening of ciliogenic genes confirmed Dynll1 to be controlled by ATMIN and further revealed moderately altered expression of known intraflagellar transport (IFT) protein-encoding loci in Atmin mutant embryos. Significantly, Dynll1(GT/GT) embryonic cilia exhibited shortening and bulging, highly similar to the characterised retrograde IFT phenotype of Dync2h1. Depletion of ATMIN or DYNLL1 in cultured cells recapitulated the in vivo ciliogenesis phenotypes and expression of DYNLL1 or the related DYNLL2 rescued the effects of loss of ATMIN, demonstrating that ATMIN primarily promotes ciliogenesis by regulating Dynll1 expression. Furthermore, DYNLL1 as well as DYNLL2 localised to cilia in puncta, consistent with IFT particles, and physically interacted with WDR34, a mammalian homologue of the Chlamydomonas cytoplasmic dynein 2 intermediate chain that also localised to the cilium. This study extends the established Atmin-Dynll1 relationship into a developmental and a ciliary context, uncovering a novel series of interactions between DYNLL1, WDR34 and ATMIN. This identifies potential novel components of cytoplasmic dynein 2 and furthermore provides fresh insights into the molecular pathogenesis of human skeletal ciliopathies.


Asunto(s)
Cilios/fisiología , Regulación del Desarrollo de la Expresión Génica , Pulmón/embriología , Factores de Transcripción/fisiología , Animales , Chlamydomonas/metabolismo , Cilios/metabolismo , Dineínas Citoplasmáticas , Daño del ADN , Dineínas/metabolismo , Marcadores Genéticos , Células HEK293 , Proteínas Hedgehog/metabolismo , Humanos , Ratones , Mutación , Fenotipo , Transducción de Señal , Factores de Transcripción/metabolismo , Transcripción Genética
12.
Nucleic Acids Res ; 43(7): 3667-79, 2015 Apr 20.
Artículo en Inglés | MEDLINE | ID: mdl-25800737

RESUMEN

Genetic instability, provoked by exogenous mutagens, is well linked to initiation of cancer. However, even in unstressed cells, DNA undergoes a plethora of spontaneous alterations provoked by its inherent chemical instability and the intracellular milieu. Base excision repair (BER) is the major cellular pathway responsible for repair of these lesions, and as deficiency in BER activity results in DNA damage it has been proposed that it may trigger the development of sporadic cancers. Nevertheless, experimental evidence for this model remains inconsistent and elusive. Here, we performed a proteomic analysis of BER deficient human cells using stable isotope labelling with amino acids in cell culture (SILAC), and demonstrate that BER deficiency, which induces genetic instability, results in dramatic changes in gene expression, resembling changes found in many cancers. We observed profound alterations in tissue homeostasis, serine biosynthesis, and one-carbon- and amino acid metabolism, all of which have been identified as cancer cell 'hallmarks'. For the first time, this study describes gene expression changes characteristic for cells deficient in repair of endogenous DNA lesions by BER. These expression changes resemble those observed in cancer cells, suggesting that genetically unstable BER deficient cells may be a source of pre-cancerous cells.


Asunto(s)
Reparación del ADN , Inestabilidad Genómica , Neoplasias/genética , Aminoácidos/biosíntesis , Aminoácidos/metabolismo , Secuencia de Bases , Línea Celular , Ensayo Cometa , Cartilla de ADN , Proteínas de Unión al ADN/genética , Metabolismo Energético , Humanos , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X
13.
Mol Cell ; 29(4): 477-87, 2008 Feb 29.
Artículo en Inglés | MEDLINE | ID: mdl-18313385

RESUMEN

Base excision repair (BER) is the major pathway for processing of simple lesions in DNA, including single-strand breaks, base damage, and base loss. The scaffold protein XRCC1, DNA polymerase beta, and DNA ligase IIIalpha play pivotal roles in BER. Although all these enzymes are essential for development, their cellular levels must be tightly regulated because increased amounts of BER enzymes lead to elevated mutagenesis and genetic instability and are frequently found in cancer cells. Here we report that BER enzyme levels are linked to and controlled by the level of DNA lesions. We demonstrate that stability of BER enzymes increases after formation of a repair complex on damaged DNA and that proteins not involved in a repair complex are ubiquitylated by the E3 ubiquitin ligase CHIP and subsequently rapidly degraded. These data identify a molecular mechanism controlling cellular levels of BER enzymes and correspondingly the efficiency and capacity of BER.


Asunto(s)
Daño del ADN , ADN Ligasas/metabolismo , ADN Polimerasa beta/metabolismo , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Secuencia de Aminoácidos , Animales , Cromatina/metabolismo , ADN Ligasa (ATP) , ADN Ligasas/genética , ADN Polimerasa beta/genética , Proteínas de Unión al ADN/genética , Células HeLa , Humanos , Peróxido de Hidrógeno/metabolismo , Sustancias Macromoleculares/metabolismo , Chaperonas Moleculares/metabolismo , Datos de Secuencia Molecular , Oxidantes/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa , Procesamiento Proteico-Postraduccional , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genética , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X , Proteínas de Xenopus
14.
Nucleic Acids Res ; 42(4): 2320-9, 2014 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-24293653

RESUMEN

The ARF tumour suppressor protein, the gene of which is frequently mutated in many human cancers, plays an important role in the cellular stress response by orchestrating up-regulation of p53 protein and consequently promoting cell-cycle delay. Although p53 protein function has been clearly linked to the cellular DNA damage response, the role of ARF protein in this process is unclear. Here, we report that arf gene transcription is induced by DNA strand breaks (SBs) and that ARF protein accumulates in response to persistent DNA damage. We discovered that poly(ADP-ribose) synthesis catalysed by PARP1 at the sites of unrepaired SBs activates ARF transcription through a protein signalling cascade, including the NAD(+)-dependent deacetylase SIRT1 and the transcription factor E2F1. Our data suggest that poly(ADP-ribose) synthesis at the sites of SBs initiates DNA damage signal transduction by reducing the cellular concentration of NAD(+), thus down-regulating SIRT1 activity and consequently activating E2F1-dependent ARF transcription. Our findings suggest a vital role for ARF in DNA damage signalling, and furthermore explain the critical requirement for ARF inactivation in cancer cells, which are frequently deficient in DNA repair and accumulate DNA damage.


Asunto(s)
Roturas del ADN , Poli(ADP-Ribosa) Polimerasas/fisiología , Proteína p14ARF Supresora de Tumor/biosíntesis , Factor de Transcripción E2F1/fisiología , Células HeLa , Humanos , Poli(ADP-Ribosa) Polimerasa-1 , Transducción de Señal , Sirtuina 1/fisiología , Proteína p14ARF Supresora de Tumor/genética
15.
Int J Mol Sci ; 17(6)2016 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-27258260

RESUMEN

Schizophrenia and autism spectrum disorder (ASD) are multi-factorial and multi-symptomatic psychiatric disorders, each affecting 0.5%-1% of the population worldwide. Both are characterized by impairments in cognitive functions, emotions and behaviour, and they undermine basic human processes of perception and judgment. Despite decades of extensive research, the aetiologies of schizophrenia and ASD are still poorly understood and remain a significant challenge to clinicians and scientists alike. Adding to this unsatisfactory situation, patients with schizophrenia or ASD often develop a variety of peripheral and systemic disturbances, one prominent example of which is cancer, which shows a direct (but sometimes inverse) comorbidity in people affected with schizophrenia and ASD. Cancer is a disease characterized by uncontrolled proliferation of cells, the molecular origin of which derives from mutations of a cell's DNA sequence. To counteract such mutations and repair damaged DNA, cells are equipped with intricate DNA repair pathways. Oxidative stress, oxidative DNA damage, and deficient repair of oxidative DNA lesions repair have been proposed to contribute to the development of schizophrenia and ASD. In this article, we summarize the current evidence of cancer comorbidity in these brain disorders and discuss the putative roles of oxidative stress, DNA damage and DNA repair in the aetiopathology of schizophrenia and ASD.


Asunto(s)
Trastorno Autístico/genética , Daño del ADN , Reparación del ADN , Esquizofrenia/genética , Animales , Trastorno del Espectro Autista/diagnóstico , Trastorno del Espectro Autista/etiología , Trastorno del Espectro Autista/genética , Trastorno Autístico/diagnóstico , Trastorno Autístico/etiología , Trastorno Autístico/metabolismo , Comorbilidad , Humanos , Neoplasias/etiología , Neoplasias/metabolismo , Estrés Oxidativo , Riesgo , Esquizofrenia/diagnóstico , Esquizofrenia/etiología , Esquizofrenia/metabolismo
16.
Nucleic Acids Res ; 41(6): 3483-90, 2013 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-23408852

RESUMEN

Base excision repair (BER) is a frontline repair system that is responsible for maintaining genome integrity and thus preventing premature aging, cancer and many other human diseases by repairing thousands of DNA lesions and strand breaks continuously caused by endogenous and exogenous mutagens. This fundamental and essential function of BER not only necessitates tight control of the continuous availability of basic components for fast and accurate repair, but also requires temporal and spatial coordination of BER and cell cycle progression to prevent replication of damaged DNA. The major goal of this review is to critically examine controversial and newly emerging questions about mammalian BER pathways, mechanisms regulating BER capacity, BER responses to DNA damage and their links to checkpoint control of DNA replication.


Asunto(s)
Reparación del ADN , Animales , Roturas del ADN de Doble Cadena , Roturas del ADN de Cadena Simple , Daño del ADN , Replicación del ADN , Inestabilidad Genómica , Humanos , Procesamiento Proteico-Postraduccional , Transducción de Señal
17.
Nucleic Acids Res ; 41(3): 1750-6, 2013 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-23275561

RESUMEN

The E3 ubiquitin ligase Mule/ARF-BP1 plays an important role in the cellular DNA damage response by controlling base excision repair and p53 protein levels. However, how the activity of Mule is regulated in response to DNA damage is currently unknown. Here, we report that the Ser18-containing isoform of the USP7 deubiquitylation enzyme (USP7S) controls Mule stability by preventing its self-ubiquitylation and subsequent proteasomal degradation. We find that in response to DNA damage, downregulation of USP7S leads to self-ubiquitylation and proteasomal degradation of Mule, which eventually leads to p53 accumulation. Cells that are unable to downregulate Mule show reduced ability to upregulate p53 levels in response to DNA damage. We also find that, as Mule inactivation is required for stabilization of base excision repair enzymes, the failure of cells to downregulate Mule after DNA damage results in deficient DNA repair. Our data describe a novel mechanism by which Mule is regulated in response to DNA damage and coordinates cellular DNA damage responses and DNA repair.


Asunto(s)
Reparación del ADN , Transducción de Señal , Ubiquitina Tiolesterasa/fisiología , Ubiquitina-Proteína Ligasas/metabolismo , Línea Celular Tumoral , Daño del ADN , Regulación hacia Abajo , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/fisiología , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor , Ubiquitina Tiolesterasa/antagonistas & inhibidores , Peptidasa Específica de Ubiquitina 7 , Ubiquitinación
18.
Proc Natl Acad Sci U S A ; 109(2): 437-42, 2012 Jan 10.
Artículo en Inglés | MEDLINE | ID: mdl-22203964

RESUMEN

It is of pivotal importance for genome stability that repair DNA polymerases (Pols), such as Pols λ and ß, which all exhibit considerably reduced fidelity when replicating undamaged DNA, are tightly regulated, because their misregulation could lead to mutagenesis. Recently, we found that the correct repair of the abundant and highly miscoding oxidative DNA lesion 7,8-dihydro-8-oxo-2'-deoxyguanine (8-oxo-G) is performed by an accurate repair pathway that is coordinated by the MutY glycosylase homologue (MutYH) and Pol λ in vitro and in vivo. Pol λ is phosphorylated by Cdk2/cyclinA in late S and G2 phases of the cell cycle, promoting Pol λ stability by preventing it from being targeted for proteasomal degradation by ubiquitination. However, it has remained a mystery how the levels of Pol λ are controlled, how phosphorylation promotes its stability, and how the engagement of Pol λ in active repair complexes is coordinated. Here, we show that the E3 ligase Mule mediates the degradation of Pol λ and that the control of Pol λ levels by Mule has functional consequences for the ability of mammalian cells to deal with 8-oxo-G lesions. Furthermore, we demonstrate that phosphorylation of Pol λ by Cdk2/cyclinA counteracts its Mule-mediated degradation by promoting recruitment of Pol λ to chromatin into active 8-oxo-G repair complexes through an increase in Pol λ's affinity to chromatin-bound MutYH. Finally, MutYH appears to promote the stability of Pol λ by binding it to chromatin. In contrast, Pol λ not engaged in active repair on chromatin is subject for proteasomal degradation.


Asunto(s)
Daño del ADN/fisiología , ADN Glicosilasas/metabolismo , ADN Polimerasa beta/metabolismo , Reparación del ADN/fisiología , Desoxiguanosina/análogos & derivados , Ubiquitina-Proteína Ligasas/metabolismo , 8-Hidroxi-2'-Desoxicoguanosina , Western Blotting , Desoxiguanosina/metabolismo , Desoxiguanosina/fisiología , Células HeLa , Humanos , Peróxido de Hidrógeno , Oligonucleótidos/genética , Oxidación-Reducción , Fosforilación , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Supresoras de Tumor , Ubiquitinación
19.
Nucleic Acids Res ; 40(22): 11404-15, 2012 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-23042680

RESUMEN

We examined the mechanism regulating the cellular levels of PNKP, the major kinase/phosphatase involved in the repair of oxidative DNA damage, and find that it is controlled by ATM phosphorylation and ubiquitylation-dependent proteasomal degradation. We discovered that ATM-dependent phosphorylation of PNKP at serines 114 and 126 in response to oxidative DNA damage inhibits ubiquitylation-dependent proteasomal degradation of PNKP, and consequently increases PNKP stability that is required for DNA repair. We have also purified a novel Cul4A-DDB1 ubiquitin ligase complex responsible for PNKP ubiquitylation and identify serine-threonine kinase receptor associated protein (STRAP) as the adaptor protein that provides specificity of the complex to PNKP. Strap(-/-) mouse embryonic fibroblasts subsequently contain elevated cellular levels of PNKP, and show elevated resistance to oxidative DNA damage. These data demonstrate an important role for ATM and the Cul4A-DDB1-STRAP ubiquitin ligase in the regulation of the cellular levels of PNKP, and consequently in the repair of oxidative DNA damage.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Estrés Oxidativo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Ubiquitinación , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas Portadoras/metabolismo , Proteínas Cullin/metabolismo , Daño del ADN , Enzimas Reparadoras del ADN/química , Estabilidad de Enzimas , Células HeLa , Humanos , Ratones , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Ubiquitina-Proteína Ligasas/aislamiento & purificación , Ubiquitina-Proteína Ligasas/metabolismo
20.
Nucleic Acids Res ; 40(2): 701-11, 2012 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-21933813

RESUMEN

APE1 (Ref-1) is an essential human protein involved in DNA damage repair and regulation of transcription. Although the cellular functions and biochemical properties of APE1 are well characterized, the mechanism involved in regulation of the cellular levels of this important DNA repair/transcriptional regulation enzyme, remains poorly understood. Using an in vitro ubiquitylation assay, we have now purified the human E3 ubiquitin ligase UBR3 as a major activity that polyubiquitylates APE1 at multiple lysine residues clustered on the N-terminal tail. We further show that a knockout of the Ubr3 gene in mouse embryonic fibroblasts leads to an up-regulation of the cellular levels of APE1 protein and subsequent genomic instability. These data propose an important role for UBR3 in the control of the steady state levels of APE1 and consequently error free DNA repair.


Asunto(s)
ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Inestabilidad Genómica , Ubiquitina-Proteína Ligasas/metabolismo , Animales , ADN-(Sitio Apurínico o Apirimidínico) Liasa/química , Técnicas de Inactivación de Genes , Células HeLa , Humanos , Lisina/metabolismo , Ratones , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación
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