RESUMEN
G-quadruplexes represent unique roadblocks to DNA replication, which tends to stall at these secondary structures. Although G-quadruplexes can be found throughout the genome, telomeres, due to their G-richness, are particularly predisposed to forming these structures and thus represent difficult-to-replicate regions. Here, we demonstrate that exonuclease 1 (EXO1) plays a key role in the resolution of, and replication through, telomeric G-quadruplexes. When replication forks encounter G-quadruplexes, EXO1 resects the nascent DNA proximal to these structures to facilitate fork progression and faithful replication. In the absence of EXO1, forks accumulate at stabilized G-quadruplexes and ultimately collapse. These collapsed forks are preferentially repaired via error-prone end joining as depletion of EXO1 diverts repair away from error-free homology-dependent repair. Such aberrant repair leads to increased genomic instability, which is exacerbated at chromosome termini in the form of dysfunction and telomere loss.
Asunto(s)
Enzimas Reparadoras del ADN/fisiología , Replicación del ADN , Exodesoxirribonucleasas/fisiología , G-Cuádruplex , Telómero/química , Aminoquinolinas/farmacología , Línea Celular , Reparación del ADN por Unión de Extremidades , Reparación del ADN , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , G-Cuádruplex/efectos de los fármacos , Técnicas de Inactivación de Genes , Células HeLa , Humanos , Neoplasias/metabolismo , Neoplasias/mortalidad , Ácidos Picolínicos/farmacología , PronósticoRESUMEN
Various intracellular pattern recognition receptors (PRRs) recognize cytosolic pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). Cyclic GMP-AMP synthase (cGAS), a cytosolic PRR, recognizes cytosolic nucleic acids including dsDNAs. The recognition of dsDNA by cGAS generates cyclic GMP-AMP (GAMP). The cGAMP is then recognized by STING generating type 1 IFNs and NF-κB-mediated generation of pro-inflammatory cytokines and molecules. Thus, cGAS-STING signaling mediated recognition of cytosolic dsDNA causing the induction of type 1 IFNs plays a crucial role in innate immunity against cytosolic pathogens, PAMPs, and DAMPs. The overactivation of this system may lead to the development of autoinflammation and autoimmune diseases. The article opens with the introduction of different PRRs involved in the intracellular recognition of dsDNA and gives a brief introduction of cGAS-STING signaling. The second section briefly describes cGAS as intracellular PRR required to recognize intracellular nucleic acids (dsDNA and CDNs) and the formation of cGAMP. The cGAMP acts as a second messenger to activate STING- and TANK-binding kinase 1-mediated generation of type 1 IFNs and the activation of NF-κB. The third section of the article describes the role of cGAS-STING signaling in the induction of autoinflammation and various autoimmune diseases. The subsequent fourth section describes both chemical compounds developed and the endogenous negative regulators of cGAS-STING signaling required for its regulation. Therapeutic targeting of cGAS-STING signaling could offer new ways to treat inflammatory and autoimmune diseases.
Asunto(s)
Enfermedades Autoinmunes/etiología , Inflamación/etiología , Proteínas de la Membrana/fisiología , Animales , ADN/metabolismo , Exodesoxirribonucleasas/fisiología , Trampas Extracelulares/fisiología , Humanos , Interferón Tipo I/fisiología , Proteínas de la Membrana/antagonistas & inhibidores , Nucleótidos Cíclicos/fisiología , Nucleotidiltransferasas/antagonistas & inhibidores , Nucleotidiltransferasas/fisiología , Fosfoproteínas/fisiología , Transducción de Señal/fisiologíaRESUMEN
KEOPS complex is one of the most conserved protein complexes in eukaryotes. It plays important roles in both telomere uncapping and tRNA N6-threonylcarbamoyladenosine (t6A) modification in budding yeast. But whether KEOPS complex plays any roles in DNA repair remains unknown. Here, we show that KEOPS complex plays positive roles in both DNA damage response and homologous recombination-mediated DNA repair independently of its t6A synthesis function. Additionally, KEOPS displays DNA binding activity in vitro, and is recruited to the chromatin at DNA breaks in vivo, suggesting a direct role of KEOPS in DSB repair. Mechanistically, KEOPS complex appears to promote DNA end resection through facilitating the association of Exo1 and Dna2 with DNA breaks. Interestingly, inactivation of both KEOPS and Mre11/Rad50/Xrs2 (MRX) complexes results in synergistic defect in DNA resection, revealing that KEOPS and MRX have some redundant functions in DNA resection. Thus we uncover a t6A-independent role of KEOPS complex in DNA resection, and propose that KEOPS might be a DSB sensor to assist cells in maintaining chromosome stability.
Asunto(s)
ADN Helicasas/fisiología , ADN de Hongos , Exodesoxirribonucleasas/fisiología , Recombinación Homóloga , Reparación del ADN por Recombinación , Proteínas de Saccharomyces cerevisiae/fisiología , Unión Competitiva , Cromatina/química , ADN/química , Roturas del ADN de Doble Cadena , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/metabolismo , Metaloendopeptidasas/metabolismo , Mutación , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Telómero/metabolismo , Factores de Transcripción/metabolismoRESUMEN
The Fragile X-related disorders (FXDs) are Repeat Expansion Diseases resulting from an expansion of a CGG-repeat tract at the 5' end of the FMR1 gene. The mechanism responsible for this unusual mutation is not fully understood. We have previously shown that mismatch repair (MMR) complexes, MSH2/MSH3 (MutSß) and MSH2/MSH6 (MutSα), together with Polß, a DNA polymerase important for base excision repair (BER), are important for expansions in a mouse model of these disorders. Here we show that MLH1/MLH3 (MutLγ), a protein complex that can act downstream of MutSß in MMR, is also required for all germ line and somatic expansions. However, exonuclease I (EXO1), which acts downstream of MutL proteins in MMR, is not required. In fact, a null mutation in Exo1 results in more extensive germ line and somatic expansions than is seen in Exo1+/+ animals. Furthermore, mice homozygous for a point mutation (D173A) in Exo1 that eliminates its nuclease activity but retains its native conformation, shows a level of expansion that is intermediate between Exo1+/+ and Exo1-/- animals. Thus, our data suggests that expansion of the FX repeat in this mouse model occurs via a MutLγ-dependent, EXO1-independent pathway, with EXO1 protecting against expansion both in a nuclease-dependent and a nuclease-independent manner. Our data thus have implications for the expansion mechanism and add to our understanding of the genetic factors that may be modifiers of expansion risk in humans.
Asunto(s)
Enzimas Reparadoras del ADN/genética , Exodesoxirribonucleasas/genética , Síndrome del Cromosoma X Frágil/genética , Proteínas MutL/genética , Animales , Reparación de la Incompatibilidad de ADN/genética , Reparación de la Incompatibilidad de ADN/fisiología , Reparación del ADN , Enzimas Reparadoras del ADN/fisiología , Modelos Animales de Enfermedad , Exodesoxirribonucleasas/fisiología , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Inestabilidad Genómica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Homólogo 1 de la Proteína MutL/metabolismo , Proteínas MutL/metabolismo , Mutación , Expansión de Repetición de Trinucleótido/genéticaRESUMEN
N6-methyladenosine (m6A) is the most abundant internal modification of eukaryotic mRNA. This modification has previously been shown to alter the export kinetics for mRNAs though the molecular details surrounding this phenomenon remain poorly understood. Recruitment of the TREX mRNA export complex to mRNA is driven by transcription, 5' capping and pre-mRNA splicing. Here we identify a fourth mechanism in human cells driving the association of TREX with mRNA involving the m6A methylase complex. We show that the m6A complex recruits TREX to m6A modified mRNAs and this process is essential for their efficient export. TREX also stimulates recruitment of the m6A reader protein YTHDC1 to the mRNA and the m6A complex influences the interaction of TREX with YTHDC1. Together our studies reveal a key role for TREX in the export of m6A modified mRNAs.
Asunto(s)
Adenosina/análogos & derivados , Exodesoxirribonucleasas/metabolismo , Fosfoproteínas/metabolismo , Transporte de ARN/fisiología , Transporte Activo de Núcleo Celular , Adenosina/metabolismo , Adenosina/fisiología , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Exodesoxirribonucleasas/fisiología , Células HEK293 , Humanos , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Fosfoproteínas/fisiología , Empalme del ARN/fisiología , Factores de Empalme de ARN/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismoRESUMEN
Mitochondrial nucleases play important roles in accurate maintenance and correct metabolism of mtDNA, the own genetic materials of mitochondria that are passed exclusively from mother to child. MGME1 is a highly conserved DNase that was discovered recently. Mutations in MGME1-coding gene lead to severe mitochondrial syndromes characterized by external ophthalmoplegia, emaciation, and respiratory failure in humans. Unlike many other nucleases that are distributed in multiple cellular organelles, human MGME1 is a mitochondria-specific nuclease; therefore, it can serve as an ideal target for treating related syndromes. Here, we report one HsMGME1-Mn2+ complex and three different HsMGME1-DNA complex structures. In combination with in vitro cleavage assays, our structures reveal the detailed molecular basis for substrate DNA binding and/or unwinding by HsMGME1. Besides the conserved two-cation-assisted catalytic mechanism, structural analysis of HsMGME1 and comparison with homologous proteins also clarified substrate binding and cleavage directionalities of the DNA double-strand break repair complexes RecBCD and AddAB.
Asunto(s)
División del ADN , Exodesoxirribonucleasas/química , Exodesoxirribonucleasas/metabolismo , Secuencia de Aminoácidos , Roturas del ADN de Doble Cadena , Fragmentación del ADN , Reparación del ADN/genética , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Escherichia coli , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/fisiología , Humanos , Modelos Moleculares , Conformación Proteica , Relación Estructura-ActividadRESUMEN
BACKGROUND: Mutations in the three-prime repair exonuclease 1 (TREX1) gene have been associated with neurological diseases, including Retinal Vasculopathy with Cerebral Leukoencephalopathy (RVCL). However, the endogenous expression of TREX1 in human brain has not been studied. METHODS: We produced a rabbit polyclonal antibody (pAb) to TREX1 to characterize TREX1 by Western blotting (WB) of cell lysates from normal controls and subjects carrying an RVCL frame-shift mutation. Dual staining was performed to determine cell types expressing TREX1 in human brain tissue. TREX1 distribution in human brain was further evaluated by immunohistochemical analyses of formalin-fixed, paraffin-embedded samples from normal controls and patients with RVCL and ischemic stroke. RESULTS: After validating the specificity of our anti-TREX1 rabbit pAb, WB analysis was utilized to detect the endogenous wild-type and frame-shift mutant of TREX1 in cell lysates. Dual staining in human brain tissues from patients with RVCL and normal controls localized TREX1 to a subset of microglia and macrophages. Quantification of immunohistochemical staining of the cerebral cortex revealed that TREX1+ microglia were primarily in the gray matter of normal controls (22.7 ± 5.1% and 5.5 ± 1.9% of Iba1+ microglia in gray and white matter, respectively) and commonly in association with the microvasculature. In contrast, in subjects with RVCL, the TREX1+ microglia were predominantly located in the white matter of normal appearing cerebral cortex (11.8 ± 3.1% and 38.9 ± 5.8% of Iba1+ microglia in gray and white matter, respectively). The number of TREX1+ microglia was increased in ischemic brain lesions in central nervous system of RVCL and stroke patients. CONCLUSIONS: TREX1 is expressed by a subset of microglia in normal human brain, often in close proximity to the microvasculature, and increases in the setting of ischemic lesions. These findings suggest a role for TREX1+ microglia in vessel homeostasis and response to ischemic injury.
Asunto(s)
Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Encéfalo/metabolismo , Exodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/fisiología , Microglía/metabolismo , Fosfoproteínas/metabolismo , Fosfoproteínas/fisiología , Anciano , Animales , Anticuerpos/metabolismo , Encéfalo/patología , Exodesoxirribonucleasas/genética , Femenino , Mutación del Sistema de Lectura , Células HEK293 , Células HeLa , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/genética , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/patología , Homeostasis/fisiología , Humanos , Macrófagos/metabolismo , Masculino , Persona de Mediana Edad , Fosfoproteínas/genética , Conejos/inmunología , Enfermedades de la Retina/genética , Enfermedades de la Retina/patología , Enfermedades Vasculares/genética , Enfermedades Vasculares/patologíaRESUMEN
Replication of mammalian mitochondrial DNA (mtDNA) is an essential process that requires high fidelity and control at multiple levels to ensure proper mitochondrial function. Mutations in the mitochondrial genome maintenance exonuclease 1 (MGME1) gene were recently reported in mitochondrial disease patients. Here, to study disease pathophysiology, we generated Mgme1 knockout mice and report that homozygous knockouts develop depletion and multiple deletions of mtDNA. The mtDNA replication stalling phenotypes vary dramatically in different tissues of Mgme1 knockout mice. Mice with MGME1 deficiency accumulate a long linear subgenomic mtDNA species, similar to the one found in mtDNA mutator mice, but do not develop progeria. This finding resolves a long-standing debate by showing that point mutations of mtDNA are the main cause of progeria in mtDNA mutator mice. We also propose a role for MGME1 in the regulation of replication and transcription termination at the end of the control region of mtDNA.
Asunto(s)
ADN Mitocondrial/genética , Exodesoxirribonucleasas/fisiología , Eliminación de Gen , Progeria/genética , Animales , Replicación del ADN , Exodesoxirribonucleasas/genética , Femenino , Fibroblastos/metabolismo , Biblioteca de Genes , Células HeLa , Homocigoto , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/metabolismo , Fenotipo , Mutación Puntual , Motilidad Espermática , Distribución Tisular , Transcripción GenéticaRESUMEN
ADP-ribosyltransferases promote repair of DNA single strand breaks and disruption of this pathway by Poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi) is toxic to cells with defects in homologous recombination (HR). Here, we show that this relationship is conserved in the simple eukaryote Dictyostelium and exploit this organism to define mechanisms that drive resistance of the HR-deficient cells to PARPi. Dictyostelium cells disrupted in exonuclease I, a critical factor for HR, are sensitive to PARPi. Deletion of exo1 prevents the accumulation of Rad51 in chromatin induced by PARPi, resulting in DNA damage being channelled through repair by non-homologous end-joining (NHEJ). Inactivation of NHEJ supresses the sensitivity of exo1- cells to PARPi, indicating this pathway drives synthetic lethality and that in its absence alternative repair mechanisms promote cell survival. This resistance is independent of alternate-NHEJ and is instead achieved by re-activation of HR. Moreover, inhibitors of Mre11 restore sensitivity of dnapkcs-exo1- cells to PARPi, indicating redundancy between nucleases that initiate HR can drive PARPi resistance. These data inform on mechanism of PARPi resistance in HR-deficient cells and present Dictyostelium as a convenient genetic model to characterize these pathways.
Asunto(s)
ADP Ribosa Transferasas/fisiología , Dictyostelium/enzimología , Resistencia a Medicamentos/fisiología , Recombinación Homóloga/fisiología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Poli(ADP-Ribosa) Polimerasas/fisiología , Proteínas Protozoarias/fisiología , Benzamidas/farmacología , Células Clonales , Quinasa 8 Dependiente de Ciclina/deficiencia , Quinasa 8 Dependiente de Ciclina/genética , Quinasa 8 Dependiente de Ciclina/fisiología , Daño del ADN , Dictyostelium/efectos de los fármacos , Dictyostelium/genética , Exodesoxirribonucleasas/deficiencia , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/fisiología , Eliminación de Gen , Indoles/farmacología , Ftalazinas/farmacología , Piperazinas/farmacología , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/genética , Quinazolinas/farmacología , Recombinasa Rad51/deficiencia , Recombinasa Rad51/fisiología , Proteínas Recombinantes/metabolismoRESUMEN
Human exonuclease 1 (hExo1) is a member of the RAD2/XPG structure-specific 5'-nuclease superfamily. Its dominant, processive 5'-3' exonuclease and secondary 5'-flap endonuclease activities participate in various DNA repair, recombination, and replication processes. A single active site processes both recessed ends and 5'-flap substrates. By initiating enzyme reactions in crystals, we have trapped hExo1 reaction intermediates that reveal structures of these substrates before and after their exo- and endonucleolytic cleavage, as well as structures of uncleaved, unthreaded, and partially threaded 5' flaps. Their distinctive 5' ends are accommodated by a small, mobile arch in the active site that binds recessed ends at its base and threads 5' flaps through a narrow aperture within its interior. A sequence of successive, interlocking conformational changes guides the two substrate types into a shared reaction mechanism that catalyzes their cleavage by an elaborated variant of the two-metal, in-line hydrolysis mechanism. Coupling of substrate-dependent arch motions to transition-state stabilization suppresses inappropriate or premature cleavage, enhancing processing fidelity. The striking reduction in flap conformational entropy is catalyzed, in part, by arch motions and transient binding interactions between the flap and unprocessed DNA strand. At the end of the observed reaction sequence, hExo1 resets without relinquishing DNA binding, suggesting a structural basis for its processivity.
Asunto(s)
Enzimas Reparadoras del ADN/química , Enzimas Reparadoras del ADN/metabolismo , Exodesoxirribonucleasas/química , Exodesoxirribonucleasas/metabolismo , Biocatálisis , Dominio Catalítico/fisiología , Cristalografía por Rayos X , ADN/química , Reparación del ADN , Enzimas Reparadoras del ADN/fisiología , Proteínas de Unión al ADN/química , Endonucleasas/metabolismo , Exodesoxirribonucleasas/fisiología , Humanos , Hidrólisis , Conformación Proteica , Especificidad por Sustrato/fisiologíaRESUMEN
RecJ nucleases specifically degrade single-stranded (ss) DNA in the 5' to 3' direction. Archaeal RecJ is different from bacterial RecJ in sequence, domain organization, and substrate specificity. The RecJ from archaea Pyrococcus furiosus (PfuRecJ) also hydrolyzes RNA strands in the 3' to 5' direction. Like eukaryotic Cdc45 protein, archaeal RecJ forms a complex with MCM helicase and GINS. Here, we report the crystal structures of PfuRecJ and the complex of PfuRecJ and two CMPs. PfuRecJ bind one or two divalent metal ions in its crystal structure. A channel consisting of several positively charged residues is identified in the complex structure, and might be responsible for binding substrate ssDNA and/or releasing single nucleotide products. The deletion of the complex interaction domain (CID) increases the values of kcat/Km of 5' exonuclease activity on ssDNA and 3' exonuclease activity on ssRNA by 5- and 4-fold, respectively, indicating that the CID functions as a regulator of enzymatic activity. The DHH domain of PfuRecJ interacts with the C-terminal beta-sheet domain of the GINS51 subunit in the tetrameric GINS complex. The relationship of archaeal and bacterial RecJs, as well as eukaryotic Cdc45, is discussed based on biochemical and structural results.
Asunto(s)
Proteínas Bacterianas/química , Exodesoxirribonucleasas/química , Pyrococcus furiosus/enzimología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Proteínas Bacterianas/fisiología , Cationes , Proteínas de Ciclo Celular , Secuencia Conservada , Cristalografía por Rayos X , Reparación del ADN , Replicación del ADN , ADN Bacteriano/metabolismo , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Evolución Molecular , Exodesoxirribonucleasas/fisiología , Modelos Moleculares , Complejos Multiproteicos/metabolismo , Fosfodiesterasa I/metabolismo , Unión Proteica , Conformación Proteica , Dominios Proteicos , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Defects of the intracellular enzyme 3' repair exonuclease 1 (Trex1) cause the rare autoimmune condition Aicardi-Goutières syndrome and are associated with systemic lupus erythematosus. Trex1(-/-) mice develop type I IFN-driven autoimmunity, resulting from activation of the cytoplasmic DNA sensor cyclic GMP-AMP synthase by a nucleic acid substrate of Trex1 that remains unknown. To identify cell types responsible for initiation of autoimmunity, we generated conditional Trex1 knockout mice. Loss of Trex1 in dendritic cells was sufficient to cause IFN release and autoimmunity, whereas Trex1-deficient keratinocytes and microglia produced IFN but did not induce inflammation. In contrast, B cells, cardiomyocytes, neurons, and astrocytes did not show any detectable response to the inactivation of Trex1. Thus, individual cell types differentially respond to the loss of Trex1, and Trex1 expression in dendritic cells is essential to prevent breakdown of self-tolerance ensuing from aberrant detection of endogenous DNA.
Asunto(s)
Autoinmunidad , Células Dendríticas/fisiología , Exodesoxirribonucleasas/fisiología , Fosfoproteínas/fisiología , Animales , Antígenos CD19/fisiología , Linfocitos B/fisiología , Encéfalo/inmunología , Exodesoxirribonucleasas/deficiencia , Interferón Tipo I/biosíntesis , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosfoproteínas/deficienciaRESUMEN
Exonuclease 1 (Exo1) is a 5'â3' exonuclease and 5'-flap endonuclease that plays a critical role in multiple eukaryotic DNA repair pathways. Exo1 processing at DNA nicks and double-strand breaks creates long stretches of single-stranded DNA, which are rapidly bound by replication protein A (RPA) and other single-stranded DNA binding proteins (SSBs). Here, we use single-molecule fluorescence imaging and quantitative cell biology approaches to reveal the interplay between Exo1 and SSBs. Both human and yeast Exo1 are processive nucleases on their own. RPA rapidly strips Exo1 from DNA, and this activity is dependent on at least three RPA-encoded single-stranded DNA binding domains. Furthermore, we show that ablation of RPA in human cells increases Exo1 recruitment to damage sites. In contrast, the sensor of single-stranded DNA complex 1-a recently identified human SSB that promotes DNA resection during homologous recombination-supports processive resection by Exo1. Although RPA rapidly turns over Exo1, multiple cycles of nuclease rebinding at the same DNA site can still support limited DNA processing. These results reveal the role of single-stranded DNA binding proteins in controlling Exo1-catalyzed resection with implications for how Exo1 is regulated during DNA repair in eukaryotic cells.
Asunto(s)
Enzimas Reparadoras del ADN/fisiología , Proteínas de Unión al ADN/fisiología , Exodesoxirribonucleasas/fisiología , Biocatálisis , Daño del ADN , Humanos , Saccharomyces cerevisiae/metabolismoRESUMEN
The maintenance of genome stability is critical for the suppression of diverse human pathologies that include developmental disorders, premature aging, infertility and predisposition to cancer. The DNA damage response (DDR) orchestrates the appropriate cellular responses following the detection of lesions to prevent genomic instability. The MRE11 complex is a sensor of DNA double strand breaks (DSBs) and plays key roles in multiple aspects of the DDR, including DNA end resection that is critical for signaling and DNA repair. The MRE11 complex has been shown to function both upstream and in concert with the 5'-3' exonuclease EXO1 in DNA resection, but it remains unclear to what extent EXO1 influences DSB responses independently of the MRE11 complex. Here we examine the genetic relationship of the MRE11 complex and EXO1 during mammalian development and in response to DNA damage. Deletion of Exo1 in mice expressing a hypomorphic allele of Nbs1 leads to severe developmental impairment, embryonic death and chromosomal instability. While EXO1 plays a minimal role in normal cells, its loss strongly influences DNA replication, DNA repair, checkpoint signaling and damage sensitivity in NBS1 hypomorphic cells. Collectively, our results establish a key role for EXO1 in modulating the severity of hypomorphic MRE11 complex mutations.
Asunto(s)
Proteínas de Ciclo Celular/genética , Enzimas Reparadoras del ADN/fisiología , Reparación del ADN , Desarrollo Embrionario , Exodesoxirribonucleasas/fisiología , Proteínas Nucleares/genética , Alelos , Animales , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Camptotecina/toxicidad , Células Cultivadas , Inestabilidad Cromosómica , Roturas del ADN de Doble Cadena , Enzimas Reparadoras del ADN/genética , Replicación del ADN , Proteínas de Unión al ADN , Desarrollo Embrionario/genética , Exodesoxirribonucleasas/genética , Puntos de Control de la Fase G2 del Ciclo Celular , Eliminación de Gen , Genes Letales , Ratones , MutaciónRESUMEN
Werner syndrome (WS) is a human chromosomal instability disorder associated with cancer predisposition and caused by mutations in the WRN gene. WRN helicase activity is crucial in limiting breakage at common fragile sites (CFS), which are the preferential targets of genome instability in precancerous lesions. However, the precise function of WRN in response to mild replication stress, like that commonly used to induce breaks at CFS, is still missing. Here, we establish that WRN plays a role in mediating CHK1 activation under moderate replication stress. We provide evidence that phosphorylation of CHK1 relies on the ATR-mediated phosphorylation of WRN, but not on WRN helicase activity. Analysis of replication fork dynamics shows that loss of WRN checkpoint mediator function as well as of WRN helicase activity hamper replication fork progression, and lead to new origin activation to allow recovery from replication slowing upon replication stress. Furthermore, bypass of WRN checkpoint mediator function through overexpression of a phospho-mimic form of CHK1 restores fork progression and chromosome stability to the wild-type levels. Together, these findings are the first demonstration that WRN regulates the ATR-checkpoint activation upon mild replication stress, preventing chromosome fragility.
Asunto(s)
Replicación del ADN , Exodesoxirribonucleasas/fisiología , RecQ Helicasas/fisiología , Afidicolina/farmacología , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Puntos de Control del Ciclo Celular/genética , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Genoma , Células HEK293 , Humanos , Mutación , Proteínas Quinasas/genética , RecQ Helicasas/genética , RecQ Helicasas/metabolismo , Transducción de Señal , Estrés Fisiológico/genética , Helicasa del Síndrome de WernerRESUMEN
The expansion of CAG·CTG repeat tracts is responsible for several neurodegenerative diseases, including Huntington disease and myotonic dystrophy. Understanding the molecular mechanism of CAG·CTG repeat tract expansion is therefore important if we are to develop medical interventions limiting expansion rates. Escherichia coli provides a simple and tractable model system to understand the fundamental properties of these DNA sequences, with the potential to suggest pathways that might be conserved in humans or to highlight differences in behavior that could signal the existence of human-specific factors affecting repeat array processing. We have addressed the genetics of CAG·CTG repeat expansion in E. coli and shown that these repeat arrays expand via an orientation-independent mechanism that contrasts with the orientation dependence of CAG·CTG repeat tract contraction. The helicase Rep contributes to the orientation dependence of repeat tract contraction and limits repeat tract expansion in both orientations. However, RuvAB-dependent fork reversal, which occurs in a rep mutant, is not responsible for the observed increase in expansions. The frequency of repeat tract expansion is controlled by both the 5'-3' exonuclease RecJ and the 3'-5' exonuclease ExoI, observations that suggest the importance of both 3'and 5' single-strand ends in the pathway of CAG·CTG repeat tract expansion. We discuss the relevance of our results to two competing models of repeat tract expansion.
Asunto(s)
Proteínas de Escherichia coli/fisiología , Escherichia coli/genética , Exodesoxirribonucleasas/fisiología , Replicación del ADN , ADN Bacteriano/genética , Escherichia coli/enzimología , Inestabilidad Genómica , Expansión de Repetición de Trinucleótido , Repeticiones de TrinucleótidosRESUMEN
TREX-2 is a five protein complex, conserved from yeast to humans, involved in linking mRNA transcription and export. The centrin 2 subunit of TREX-2 is also a component of the centrosome and is additionally involved in a distinctly different process of nuclear protein export. While centrin 2 is a known multifunctional protein, the roles of other human TREX-2 complex proteins other than mRNA export are not known. In this study, we found that human TREX-2 member PCID2 but not ENY2 is involved in some of the same cellular processes as those of centrin 2 apart from the classical TREX-2 function. PCID2 is present at the centrosome in a subset of HeLa cells and this localization is centrin 2 dependent. Furthermore, the presence of PCID2 at the centrosome is prevalent throughout the cell cycle as determined by co-staining with cyclins E, A and B. PCID2 but not ENY2 is also involved in protein export. Surprisingly, siRNA knockdown of PCID2 delayed the rate of nuclear protein export, a mechanism distinct from the effects of centrin 2, which when knocked down inhibits export. Finally we showed that co-depletion of centrin 2 and PCID2 leads to blocking rather than delaying nuclear protein export, indicating the dominance of the centrin 2 phenotype. Together these results represent the first discovery of specific novel functions for PCID2 other than mRNA export and suggest that components of the TREX-2 complex serve alternative shared roles in the regulation of nuclear transport and cell cycle progression.
Asunto(s)
Proteínas de Unión al Calcio/fisiología , Proteínas de Ciclo Celular/fisiología , Núcleo Celular/metabolismo , Centrosoma/metabolismo , Exodesoxirribonucleasas/fisiología , Proteínas Nucleares/fisiología , Fosfoproteínas/fisiología , Factores de Transcripción/fisiología , Transporte Activo de Núcleo Celular/efectos de los fármacos , Transporte Activo de Núcleo Celular/genética , Proteínas de Unión al Calcio/metabolismo , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/efectos de los fármacos , Centrosoma/efectos de los fármacos , Exodesoxirribonucleasas/antagonistas & inhibidores , Células HeLa , Humanos , Proteínas Nucleares/metabolismo , Fosfoproteínas/antagonistas & inhibidores , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/fisiología , ARN Interferente Pequeño/farmacología , Distribución Tisular/efectos de los fármacos , Distribución Tisular/genética , Células Tumorales CultivadasRESUMEN
In this study, we report the effects of deleting the principal mitotic cyclin, Clb2, in different repair deficient contexts on sensitivity to the alkylating DNA damaging agent, methyl methanesulphonate (MMS). A yeast clb2 mutant is sensitive to MMS and displays synergistic effect when combined with inactivation of numerous genes involved in DNA recombination and replication. In contrast, clb2 has basically no additional effect with deletion of the RecQ helicase SGS1, the exonuclease EXO1 and the protein kinase RAD53 suggesting that Clb2 functions in these pathways. In addition, clb2 increases the viability of the mec1 kinase deficient mutant, suggesting Mec1 inhibits a deleterious Clb2 activity. Interestingly, we found that the rescue by EXO1 deletion of rad53K227 mutant, deficient in checkpoint activation, requires Sgs1, suggesting a role for Rad53, independent of its checkpoint function, in regulating an ordered recruitment of Sgs1 and Exo1 to fork structure. Overall, our data suggest that Clb2 affects recombinant structure of replication fork blocked by alkylating DNA damage at numerous steps and could regulate Sgs1 and Exo1 activity. In addition, we found novel requirement of Sgs1 DNA helicase and Exonuclease 1 when replication forks breaks in the presence of alkylation damage. Models for the functional interactions of mitotic cyclin Clb2, Sgs1 and Exo1 with replication fork stabilization are proposed.
Asunto(s)
Alquilantes/farmacología , Ciclina B/genética , Roturas del ADN de Doble Cadena/efectos de los fármacos , Exodesoxirribonucleasas/fisiología , Metilmetanosulfonato/farmacología , RecQ Helicasas/fisiología , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/genética , Alquilación , Daño del ADN , Replicación del ADN/efectos de los fármacos , Replicación del ADN/genética , Epistasis Genética/efectos de los fármacos , Eliminación de Gen , Mitosis/efectos de los fármacos , Mitosis/genética , Organismos Modificados Genéticamente , Saccharomyces cerevisiae/efectos de los fármacosRESUMEN
The three-prime repair exonuclease 1 (TREX1) is the most abundant exonuclease in mammalian cells. Mutations in Trex1 gene are being linked to the development of Aicardi-Goutières syndrome, an inflammatory disease of the brain, and systemic lupus erythematosus. In clinical cases and in a Trex1-deficient murine model, chronic production of type I IFN plays a pathogenic role. In this study, we demonstrate that Trex1(-/-) mice present inflammatory signatures in many different organs, including the brain. Trex1 is highly induced in macrophages in response to proinflammatory stimuli, including TLR7 and TLR9 ligands. Our findings show that, in the absence of Trex1, macrophages displayed an exacerbated proinflammatory response. More specifically, following proinflammatory stimulation, Trex1(-/-) macrophages exhibited an increased TNF-α and IFN-α production, higher levels of CD86, and increased Ag presentation to CD4(+) T cells, as well as an impaired apoptotic T cell clearance. These results evidence an unrevealed function of the Trex1 as a negative regulator of macrophage inflammatory activation and demonstrate that macrophages play a major role in diseases associated with Trex1 mutations, which contributes to the understanding of inflammatory signature in these diseases.
Asunto(s)
Exodesoxirribonucleasas/fisiología , Inflamación/inmunología , Activación de Macrófagos/fisiología , Fosfoproteínas/fisiología , Animales , Presentación de Antígeno , Apoptosis , Antígeno B7-2/biosíntesis , Antígeno B7-2/genética , Química Encefálica , Exodesoxirribonucleasas/deficiencia , Exodesoxirribonucleasas/inmunología , Regulación de la Expresión Génica/inmunología , Humanos , Inflamación/metabolismo , Interferón-alfa/biosíntesis , Interferón-alfa/genética , Células Jurkat , Células L , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Fagocitosis , Fosfoproteínas/deficiencia , Fosfoproteínas/inmunología , Proteínas Recombinantes/farmacología , Linfocitos T/inmunología , Linfocitos T/patología , Receptor Toll-Like 9/fisiología , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Double-strand breaks (DSBs) in budding yeast trigger activation of DNA damage checkpoints, allowing repair to occur. Although resection is necessary for initiating damage-induced cell cycle arrest in G2, no role has been assigned to it in the activation of G1 checkpoint. Here we demonstrate for the first time that the resection proteins Sgs1 and Exo1 are required for efficient G1 checkpoint activation. We find in G1 arrested cells that histone H2A phosphorylation in response to ionizing radiation is independent of Sgs1 and Exo1. In contrast, these proteins are required for damage-induced recruitment of Rfa1 to the DSB sites, phosphorylation of the Rad53 effector kinase, cell cycle arrest and RNR3 expression. Checkpoint activation in G1 requires the catalytic activity of Sgs1, suggesting that it is DNA resection mediated by Sgs1 that stimulates the damage response pathway rather than protein-protein interactions with other DDR proteins. Together, these results implicate DNA resection, which is thought to be minimal in G1, as necessary for activation of the G1 checkpoint.