RESUMEN
DNA double-strand breaks (DSBs) are cytotoxic lesions that threaten genome integrity and cell viability. Typically, cells repair DSBs by either nonhomologous end joining (NHEJ) or homologous recombination (HR). The relative use of these two pathways depends on many factors, including cell cycle stage and the nature of the DNA ends. A critical determinant of repair pathway selection is the initiation of 5'â3' nucleolytic degradation of DNA ends, a process referred to as DNA end resection. End resection is essential to create single-stranded DNA overhangs, which serve as the substrate for the Rad51 recombinase to initiate HR and are refractory to NHEJ repair. Here, we review recent insights into the mechanisms of end resection, how it is regulated, and the pathological consequences of its dysregulation.
Asunto(s)
Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN , ADN , Reparación del ADN por Unión de Extremidades/genética , Reparación del ADN/genética , Proteínas de Unión al ADN/metabolismo , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Recombinación Homóloga/genéticaRESUMEN
The mTOR pathway integrates both extracellular and intracellular signals and serves as a central regulator of cell metabolism, growth, survival, and stress responses. Neurotropic viruses, such as herpes simplex virus-1 (HSV-1), also rely on cellular AKT-mTORC1 signaling to achieve viral latency. Here, we define a novel genotoxic response whereby spatially separated signals initiated by extracellular neurotrophic factors and nuclear DNA damage are integrated by the AKT-mTORC1 pathway. We demonstrate that endogenous DNA double-strand breaks (DSBs) mediated by Topoisomerase 2ß-DNA cleavage complex (TOP2ßcc) intermediates are required to achieve AKT-mTORC1 signaling and maintain HSV-1 latency in neurons. Suppression of host DNA-repair pathways that remove TOP2ßcc trigger HSV-1 reactivation. Moreover, perturbation of AKT phosphorylation dynamics by downregulating the PHLPP1 phosphatase led to AKT mis-localization and disruption of DSB-induced HSV-1 reactivation. Thus, the cellular genome integrity and environmental inputs are consolidated and co-opted by a latent virus to balance lifelong infection with transmission.
Asunto(s)
ADN-Topoisomerasas de Tipo II/genética , Herpesvirus Humano 1/genética , Proteínas Nucleares/genética , Proteínas Proto-Oncogénicas c-akt/genética , Latencia del Virus/genética , Animales , Roturas del ADN de Doble Cadena , Daño del ADN/genética , Reparación del ADN por Unión de Extremidades/genética , Reparación del ADN/genética , Enzimas Reparadoras del ADN/genética , Proteínas de Unión al ADN/genética , Herpesvirus Humano 1/patogenicidad , Humanos , Proteína Homóloga de MRE11/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Neuronas/metabolismo , Neuronas/virología , Fosforilación , Ratas , Transducción de Señal/genética , Serina-Treonina Quinasas TOR/genéticaRESUMEN
Cyclosporin A (CsA) induces DNA double-strand breaks in LIG4 syndrome fibroblasts, specifically upon transit through S-phase. The basis underlying this has not been described. CsA-induced genomic instability may reflect a direct role of Cyclophilin A (CYPA) in DNA repair. CYPA is a peptidyl-prolyl cis-trans isomerase (PPI). CsA inhibits the PPI activity of CYPA. Using an integrated approach involving CRISPR/Cas9-engineering, siRNA, BioID, co-immunoprecipitation, pathway-specific DNA repair investigations as well as protein expression interaction analysis, we describe novel impacts of CYPA loss and inhibition on DNA repair. We characterise a direct CYPA interaction with the NBS1 component of the MRE11-RAD50-NBS1 complex, providing evidence that CYPA influences DNA repair at the level of DNA end resection. We define a set of genetic vulnerabilities associated with CYPA loss and inhibition, identifying DNA replication fork protection as an important determinant of viability. We explore examples of how CYPA inhibition may be exploited to selectively kill cancers sharing characteristic genomic instability profiles, including MYCN-driven Neuroblastoma, Multiple Myeloma and Chronic Myelogenous Leukaemia. These findings propose a repurposing strategy for Cyclophilin inhibitors.
Asunto(s)
Ácido Anhídrido Hidrolasas , Proteínas de Ciclo Celular , Ciclofilina A , Reparación del ADN , Replicación del ADN , Proteínas de Unión al ADN , Proteína Homóloga de MRE11 , Proteínas Nucleares , Humanos , Proteína Homóloga de MRE11/metabolismo , Proteína Homóloga de MRE11/genética , Ciclofilina A/metabolismo , Ciclofilina A/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Ácido Anhídrido Hidrolasas/metabolismo , Ácido Anhídrido Hidrolasas/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Enzimas Reparadoras del ADN/metabolismo , Enzimas Reparadoras del ADN/genética , Roturas del ADN de Doble Cadena , ADN Ligasa (ATP)/metabolismo , ADN Ligasa (ATP)/genética , Inestabilidad GenómicaRESUMEN
The MRE11-RAD50-NBS1 (MRN) complex plays essential roles in the cellular response to DNA double-strand breaks (DSBs), which are the most cytotoxic DNA lesions, and is a target of various modifications and controls. Recently, lysine 48-linked ubiquitination of NBS1, resulting in premature disassembly of the MRN complex from DSB sites, was observed in cells lacking RECQL4 helicase activity. However, the role and control of this ubiquitination during the DSB response in cells with intact RECQL4 remain unknown. Here, we showed that USP2 counteracts this ubiquitination and stabilizes the MRN complex during the DSB response. By screening deubiquitinases that increase the stability of the MRN complex in RECQL4-deficient cells, USP2 was identified as a new deubiquitinase that acts at DSB sites to counteract NBS1 ubiquitination. We determined that USP2 is recruited to DSB sites in a manner dependent on ATM, a major checkpoint kinase against DSBs, and stably interacts with NBS1 and RECQL4 in immunoprecipitation experiments. Phosphorylation of two critical residues in the N terminus of USP2 by ATM is required for its recruitment to DSBs and its interaction with RECQL4. While inactivation of USP2 alone does not substantially influence the DSB response, we found that inactivation of USP2 and USP28, another deubiquitinase influencing NBS1 ubiquitination, results in premature disassembly of the MRN complex from DSB sites as well as defects in ATM activation and homologous recombination repair abilities. These results suggest that deubiquitinases counteracting NBS1 ubiquitination are essential for the stable maintenance of the MRN complex and proper cellular response to DSBs.
Asunto(s)
Proteínas de Ciclo Celular , Roturas del ADN de Doble Cadena , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Enzimas Desubicuitinizantes/genética , ADN , Reparación del ADN , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Proteína Homóloga de MRE11/genética , Ubiquitinación , Humanos , Línea Celular Tumoral , Ubiquitina Tiolesterasa/metabolismo , Proteínas de Unión al ADN/metabolismo , Ácido Anhídrido Hidrolasas/metabolismoRESUMEN
The Mre11-Rad50-Nbs1 complex (MRN) is important for repairing DNA double-strand breaks (DSBs) by homologous recombination (HR). The endonuclease activity of MRN is critical for resecting 5'-ended DNA strands at DSB ends, producing 3'-ended single-strand DNA, a prerequisite for HR. This endonuclease activity is stimulated by Ctp1, the Schizosaccharomyces pombe homolog of human CtIP. Here, with purified proteins, we show that Ctp1 phosphorylation stimulates MRN endonuclease activity by inducing the association of Ctp1 with Nbs1. The highly conserved extreme C terminus of Ctp1 is indispensable for MRN activation. Importantly, a polypeptide composed of the conserved 15 amino acids at the C terminus of Ctp1 (CT15) is sufficient to stimulate Mre11 endonuclease activity. Furthermore, the CT15 equivalent from CtIP can stimulate human MRE11 endonuclease activity, arguing for the generality of this stimulatory mechanism. Thus, we propose that Nbs1-mediated recruitment of CT15 plays a pivotal role in the activation of the Mre11 endonuclease by Ctp1/CtIP.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Exodesoxirribonucleasas/metabolismo , Péptidos/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Secuencia de Aminoácidos , Quinasa de la Caseína II/metabolismo , Secuencia Conservada , Roturas del ADN de Doble Cadena , FosforilaciónRESUMEN
DNA is susceptible to various factors in vitro and in vivo and experience different forms of damage,among which double-strand break(DSB)is a deleterious form.To maintain the stability of genetic information,organisms have developed multiple mechanisms to repair DNA damage.Among these mechanisms,homologous recombination(HR)is praised for the high accuracy.The MRE11-RAD50-NBS1(MRN)complex plays an important role in HR and is conserved across different species.The knowledge on the MRN complex mainly came from the previous studies in Saccharomyces cerevisiae and Caenorhabditis elegans,while studies in the last decades have revealed the role of mammalian MRN complex in DNA repair of higher animals.In this review,we first introduces the MRN complex regarding the composition,structure,and roles in HR.In addition,we discuss the human diseases such as ataxia-telangiectasia-like disorder,Nijmegen breakage syndrome,and Nijmegen breakage syndrome-like disorder that are caused by dysfunctions in the MRN complex.Furthermore,we summarize the mouse models established to study the clinical phenotypes of the above diseases.
Asunto(s)
Ácido Anhídrido Hidrolasas , Proteínas de Ciclo Celular , Enzimas Reparadoras del ADN , Proteínas de Unión al ADN , Proteína Homóloga de MRE11 , Proteínas Nucleares , Humanos , Ácido Anhídrido Hidrolasas/metabolismo , Ácido Anhídrido Hidrolasas/genética , Proteína Homóloga de MRE11/metabolismo , Proteína Homóloga de MRE11/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Enzimas Reparadoras del ADN/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Animales , Reparación del ADN , Ataxia Telangiectasia/genética , Ataxia Telangiectasia/metabolismo , Síndrome de Nijmegen/metabolismo , Síndrome de Nijmegen/genéticaRESUMEN
The proper cellular response to DNA double-strand breaks (DSBs) is critical for maintaining the integrity of the genome. RecQL4, a DNA helicase of which mutations are associated with Rothmund-Thomson syndrome (RTS), is required for the DNA DSB response. However, the mechanism by which RecQL4 performs these essential roles in the DSB response remains unknown. Here, we show that RecQL4 and its helicase activity are required for maintaining the stability of the Mre11-Rad50-Nbs1 (MRN) complex on DSB sites during a DSB response. We found using immunocytochemistry and live-cell imaging that the MRN complex is prematurely disassembled from DSB sites in a manner dependent upon Skp2-mediated ubiquitination of Nbs1 in RecQL4-defective cells. This early disassembly of the MRN complex could be prevented by altering the ubiquitination site of Nbs1 or by expressing a deubiquitinase, Usp28, which sufficiently restored homologous recombination repair and ATM, a major checkpoint kinase against DNA DSBs, activation abilities in RTS, and RecQL4-depleted cells. These results suggest that the essential role of RecQL4 in the DSB response is to maintain the stability of the MRN complex on DSB sites and that defects in the DSB response in cells of patients with RTS can be recovered by controlling the stability of the MRN complex.
Asunto(s)
Ácido Anhídrido Hidrolasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Proteína Homóloga de MRE11/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , RecQ Helicasas/metabolismo , Ácido Anhídrido Hidrolasas/genética , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Células HEK293 , Humanos , Proteína Homóloga de MRE11/genética , Complejos Multiproteicos/genética , Proteínas Nucleares/genética , RecQ Helicasas/genéticaRESUMEN
The metal binding at protein-protein interfaces is still uncharted territory in intermolecular interactions. To date, only a few protein complexes binding Zn(II) in an intermolecular manner have been deeply investigated. The most notable example of such interfaces is located in the highly conserved Rad50 protein, part of the Mre11-Rad50-Nbs1 (MRN) complex, where Zn(II) is required for homodimerization (Zn(Rad50)2). The high stability of Zn(Rad50)2 is conserved not only for the protein derived from the thermophilic archaeon Pyrococcus furiosus (logK12 = 20.95 for 130-amino-acid-long fragment), which was the first one studied, but also for the human paralog studied here (logK12 = 19.52 for a 183-amino-acid-long fragment). As we reported previously, the extremely high stability results from the metal-coupled folding process where particular Rad50 protein fragments play a critical role. The sequence-structure-stability analysis based on human Rad50 presented here separates the individual structural components that increase the stability of the complex, pointing to amino acid residues far away from the Zn(II) binding site as being largely responsible for the complex stabilization. The influence of the individual components is very well reflected by the previously published crystal structure of the human Rad50 zinc hook (PDB: 5GOX). In addition, we hereby report the effect of phosphorylation of the zinc hook domain, which exerts a destabilizing effect on the domain. This study identifies factors governing the stability of metal-mediated protein-protein interactions and illuminates their molecular basis.
Asunto(s)
Proteínas de Unión al ADN , Pyrococcus furiosus , Ácido Anhídrido Hidrolasas/metabolismo , Aminoácidos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Humanos , Fosforilación , Zinc/metabolismoRESUMEN
The MRE11-RAD50-NBS1 (MRN) complex has been studied in multiple cancers. The identification of MRN complex mutations in mismatch repair (MMR)-defective cancers has sparked interest in its role in colorectal cancer (CRC). To date, there is evidence indicating a relationship of MRN expression with reduced progression-free survival, although the significance of the MRN complex in the clinical setting remains controversial. In this review, we present an overview of the function of the MRN complex, its role in cancer progression, and current evidence in colorectal cancer. The evidence indicates that the MRN complex has potential utilisation as a biomarker and as a putative treatment target to improve outcomes of colorectal cancer.
Asunto(s)
Proteínas de Ciclo Celular/genética , Neoplasias Colorrectales/genética , Enzimas Reparadoras del ADN/genética , Proteínas de Unión al ADN/genética , Proteína Homóloga de MRE11/genética , Proteínas Nucleares/genética , Ácido Anhídrido Hidrolasas , Neoplasias Colorrectales/patología , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Complejos Multiproteicos/genética , PronósticoRESUMEN
BACKGROUND: The MRE11/RAD50/NBS1 (MRN) complex plays an essential role in detecting and repairing double-stranded breaks, and thus the potential roles of MRE11, RAD50 and NBS1 proteins in the pathogenesis of various cancers is the subject of investigation. This study was aimed at assessing the three-protein panel of MRN complex subunits as a potential radiosensitivity marker and evaluating the prognostic and clinicopathological implications of MRN expression in rectal cancer. METHODS: Samples from 265 rectal cancer patients treated with surgery and adjuvant chemoradiotherapy, including samples from 55 patients who were treated with neoadjuvant radiotherapy between 2000 and 2011, were analyzed. Expression of MRN complex proteins in tissue samples was determined by immunohistochemistry. Univariate and multivariate analyses were carried out to identify clinicopathological characteristics that are associated with the MRN three-protein panel expression in rectal cancer samples. RESULTS: In Kaplan-Meier survival analyses, we found that high level expression of MRN complex proteins in postoperative samples was associated with poor disease-free (p = 0.021) and overall (P = 0.002) survival. Interestingly, high MRN expression also correlated with poor disease-free (P = 0.047) and overall (P = 0.024) survival in the neoadjuvant radiotherapy subgroup. In multivariate analysis, combined MRN expression (hazard ratio = 2.114, 95% confidence interval 1.096-4.078, P = 0.026) and perineural invasion (hazard ratio = 2.160, 95% confidence interval 1.209-3.859, P = 0.009) were significantly associated with a worse disease-free survival. CONCLUSIONS: Expression levels of MRN complex proteins significantly predict disease-free survival in rectal cancer patients, including those treated with neoadjuvant radiotherapy, and may have value in the management of these patients.
Asunto(s)
Proteínas de Ciclo Celular/genética , Enzimas Reparadoras del ADN/genética , Proteínas de Unión al ADN/genética , Proteína Homóloga de MRE11/genética , Proteínas Nucleares/genética , Neoplasias del Recto/radioterapia , Ácido Anhídrido Hidrolasas , Adulto , Anciano , Anciano de 80 o más Años , Roturas del ADN de Doble Cadena/efectos de la radiación , Supervivencia sin Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Complejos Multiproteicos/genética , Terapia Neoadyuvante/efectos adversos , Pronóstico , Neoplasias del Recto/genética , Neoplasias del Recto/patología , Análisis de Matrices TisularesRESUMEN
Chromosomal double strand breaks provoke an extensive reaction in neighboring chromatin, characterized by phosphorylation of histone H2AX on serine 139 of its C-terminal tail (to form "γH2AX"). The γH2AX response contributes to the repair of double strand breaks encountered in a variety of different contexts, including those induced by ionizing radiation, physiologically programmed breaks that characterize normal immune cell development and the pathological exposure of DNA ends triggered by telomere dysfunction. γH2AX also participates in the evolutionarily conserved process of sister chromatid recombination, a homologous recombination pathway involved in the suppression of genomic instability during DNA replication and directly implicated in tumor suppression. At a biochemical level, the γH2AX response provides a compelling example of how the "histone code" is adapted to the regulation of double strand break repair. Here, we review progress in research aimed at understanding how γH2AX contributes to double strand break repair in mammalian cells.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN/genética , Histonas/fisiología , Animales , Sitios de Unión , Cromatina/fisiología , Humanos , Péptidos y Proteínas de Señalización Intracelular/fisiología , Modelos Genéticos , Proteína 1 de Unión al Supresor Tumoral P53RESUMEN
DNA double strand breaks (DSBs) constitute one of the most dangerous forms of DNA damage. In actively replicating cells, these breaks are first recognized by specialized proteins that initiate a signal transduction cascade that modulates the cell cycle and results in the repair of the breaks by homologous recombination (HR). Protein signaling in response to double strand breaks involves phosphorylation and ubiquitination of chromatin and a variety of associated proteins. Here we review the emerging structural principles that underlie how post-translational protein modifications control protein signaling that emanates from these DNA lesions.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN/genética , Transducción de Señal/genética , Animales , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Recombinación Homóloga/genética , Recombinación Homóloga/fisiología , Humanos , Modelos Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Estructura Terciaria de Proteína/fisiología , Transducción de Señal/fisiologíaRESUMEN
Conjunctival melanoma (CM), a rare and fatal malignant ocular tumor, lacks proper diagnostic biomarkers and therapy. Herein, we revealed the novel application of propafenone, an FDA-approved antiarrhythmic medication, which was identified effective in inhibiting CM cells viability and homologous recombination pathway. Detailed structure-activity relationships generated D34 as one of the most promising derivatives, which strongly suppressed the proliferation, viability, and migration of CM cells at submicromolar concentrations. Mechanically, D34 had the potential to increase γ-H2AX nuclear foci and aggravated DNA damage by suppressing homologous recombination pathway and its factors, particularly the complex of MRE11-RAD50-NBS1. D34 bound to human recombinant MRE11 protein and inhibited its endonuclease activity. Moreover, D34 dihydrochloride significantly suppressed tumor growth in the CRMM1 NCG xenograft model without obvious toxicity. Our finding shows that propafenone derivatives modulating the MRE11-RAD50-NBS1 complex will most likely provide an approach for CM targeted therapy, especially for improving chemo- and radio-sensitivity for CM patients.
Asunto(s)
Antineoplásicos , Melanoma , Humanos , Propafenona , Enzimas Reparadoras del ADN/metabolismo , Proteínas Nucleares/metabolismo , Reposicionamiento de Medicamentos , Enfermedades Raras , Proteínas de Ciclo Celular/metabolismo , Daño del ADN , Recombinación Homóloga , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Melanoma/tratamiento farmacológico , Reparación del ADNRESUMEN
During a normal topoisomerase II (TOP2) reaction, the enzyme forms a covalent enzyme DNA intermediate consisting of a 5' phosphotyrosyl linkage between the enzyme and DNA. While the enzyme typically rejoins the transient breakage after strand passage, a variety of conditions including drugs targeting TOP2 can inhibit DNA resealing, leading to enzyme-mediated DNA damage. A critical aspect of the repair of TOP2-mediated damage is the removal of the TOP2 protein covalently bound to DNA. While proteolysis plays a role in repairing this damage, nucleolytic enzymes must remove the phosphotyrosyl-linked peptide bound to DNA. The MRN complex has been shown to participate in the removal of TOP2 protein from DNA following cellular treatment with TOP2 poisons. In this report we used an optimized ICE (In vivo Complex of Enzyme) assay to measure covalent TOP2/DNA complexes. In agreement with previous independent reports, we find that the absence or inhibition of the MRE11 endonuclease results in elevated levels of both TOP2α and TOP2ß covalent complexes. We also examined levels of TOP2 covalent complexes in cells treated with the proteasome inhibitor MG132. Although MRE11 inhibition plus MG132 was not synergistic in etoposide-treated cells, ectopic overexpression of MRE11 resulted in removal of TOP2 even in the presence of MG132. We also found that VCP/p97 inhibition led to elevated TOP2 covalent complexes and prevented the removal of TOP2 covalent complexes by MRE11 overexpression. Our results demonstrate the existence of multiple pathways for proteolytic processing of TOP2 prior to nucleolytic processing, and that MRE11 can process TOP2 covalent complexes even when the proteasome is inhibited. The interactions between VCP/p97 and proteolytic processing of TOP2 covalent complexes merit additional investigation.
RESUMEN
Genome stability can be threatened by both endogenous and exogenous agents. Organisms have evolved numerous mechanisms to repair DNA damage, including homologous recombination (HR) and non-homologous end joining (NHEJ). Among the factors associated with DNA repair, the MRE11-RAD50-NBS1 (MRN) complex (MRE11-RAD50-XRS2 in Saccharomyces cerevisiae) plays important roles not only in DNA damage recognition and signaling but also in subsequent HR or NHEJ repair. Upon detecting DNA damage, the MRN complex activates signaling molecules, such as the protein kinase ataxia-telangiectasia mutated (ATM), to trigger a broad DNA damage response, including cell cycle arrest. The nuclease activity of the MRN complex is responsible for DNA end resection, which guides DNA repair to HR in the presence of sister chromatids. The MRN complex is also involved in NHEJ, and has a species-specific role in hairpin repair. This review focuses on the structure of the MRN complex and its function in DNA damage repair.
Asunto(s)
Daño del ADN , Reparación del ADN/fisiología , Ácido Anhídrido Hidrolasas/química , Ácido Anhídrido Hidrolasas/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/química , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Reparación del ADN por Unión de Extremidades , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Recombinación Homóloga , Humanos , Proteína Homóloga de MRE11/química , Proteína Homóloga de MRE11/metabolismo , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de ProteínasRESUMEN
The Mre11-Rad50-Nbs1/Xrs2 protein complex plays a pivotal role in the detection and repair of DNA double strand breaks. Through traditional and emerging structural biology techniques, various functional structural states of this complex have been visualized; however, relatively little is known about the transitions between these states. Indeed, it is these structural transitions that are important for Mre11-Rad50-mediated DNA unwinding at a break and the activation of downstream repair signaling events. Here, we present a brief overview of the current understanding of the structure of the core Mre11-Rad50 complex. We then highlight our recent studies emphasizing the contributions of solution state NMR spectroscopy and other biophysical techniques in providing insight into the structures and dynamics associated with Mre11-Rad50 functions.
Asunto(s)
Proteínas de Ciclo Celular , Reparación del ADN , Proteínas de Unión al ADN , Proteína Homóloga de MRE11 , Proteínas de Ciclo Celular/genética , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/genética , Proteína Homóloga de MRE11/genéticaRESUMEN
The MRE11-RAD50-NBS1 (MRN) complex supports the synthesis of damage-induced long non-coding RNA (dilncRNA) by RNA polymerase II (RNAPII) from DNA double-strand breaks (DSBs) by an unknown mechanism. Here, we show that recombinant human MRN and native RNAPII are sufficient to reconstitute a minimal functional transcriptional apparatus at DSBs. MRN recruits and stabilizes RNAPII at DSBs. Unexpectedly, transcription is promoted independently from MRN nuclease activities. Rather, transcription depends on the ability of MRN to melt DNA ends, as shown by the use of MRN mutants and specific allosteric inhibitors. Single-molecule FRET assays with wild-type and mutant MRN show a tight correlation between the ability to melt DNA ends and to promote transcription. The addition of RPA enhances MRN-mediated transcription, and unpaired DNA ends allow MRN-independent transcription by RNAPII. These results support a model in which MRN generates single-strand DNA ends that favor the initiation of transcription by RNAPII.
Asunto(s)
Ácido Anhídrido Hidrolasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteína Homóloga de MRE11/metabolismo , Proteínas Nucleares/metabolismo , Desnaturalización de Ácido Nucleico , ARN Polimerasa II/metabolismo , ARN Largo no Codificante/biosíntesis , Transcripción Genética , Ácido Anhídrido Hidrolasas/genética , Proteínas de Ciclo Celular/genética , Roturas del ADN de Doble Cadena , Daño del ADN , Reparación del ADN , Proteínas de Unión al ADN/genética , Células HeLa , Humanos , Proteína Homóloga de MRE11/genética , Mutación , Proteínas Nucleares/genética , ARN Polimerasa II/genética , ARN Largo no Codificante/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
IQ motif containing GTPase-activating protein 3 (IQGAP3) has been implicated in diverse cellular processes, including neuronal morphogenesis, cell proliferation and motility, and epithelial-mesenchymal transition. However, its role in cancer radioresistance is completely unknown. Here, we report that IQGAP3 is overproduced in lung cancer patients and correlates with poor clinical outcomes. Functionally, we demonstrate that depletion of IQGAP3 impairs oncogenesis and overcomes radioresistance in lung cancer in vitro and in vivo. Mechanistically, we uncover that IQGAP3 interacts with Rad17 and controls its expression to activate the ATM/Chk2 and ATR/Chk1 signaling pathways by recruiting the Mre11-Rad50-Nbs1 (MRN) complex in response to DNA damage. Moreover, Rad17 is identified as the major downstream effector that mediates the functions of IQGAP3 in lung cancer. Clinically, IQGAP3 overexpression positively correlates with Rad17 upregulation in human lung cancer tissues. Collectively, these data support key role for IQGAP3 in promoting lung cancer radioresistance by interacting with Rad17 and suggest that targeting IQGAP3 may be an attractive strategy for lung cancer radiotherapy.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Neoplasias Pulmonares/patología , Tolerancia a Radiación , Regulación hacia Arriba , Células A549 , Ácido Anhídrido Hidrolasas/metabolismo , Animales , Línea Celular Tumoral , Proteínas de Unión al ADN/metabolismo , Transición Epitelial-Mesenquimal , Femenino , Proteínas Activadoras de GTPasa/genética , Eliminación de Gen , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/terapia , Proteína Homóloga de MRE11/metabolismo , Ratones , Trasplante de Neoplasias , Proteínas Nucleares/metabolismo , Pronóstico , Transducción de SeñalRESUMEN
The MRE11-RAD50-NBS1 (MRN) protein complex is one of the primary vehicles for repairing DNA double strand breaks and maintaining the genomic stability within the cell. The role of the MRN complex to recognize and process DNA double-strand breaks as well as signal other damage response factors is critical for maintaining proper cellular function. Mutations in any one of the components of the MRN complex that effect function or expression of the repair machinery could be detrimental to the cell and may initiate and/or propagate disease. Here, we discuss, in a structural and biochemical context, mutations in each of the three MRN components that have been associated with diseases such as ataxia telangiectasia-like disorder (ATLD), Nijmegen breakage syndrome (NBS), NBS-like disorder (NBSLD) and certain types of cancers. Overall, deepening our understanding of disease-causing mutations of the MRN complex at the structural and biochemical level is foundational to the future aim of treating diseases associated with these aberrations.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteína Homóloga de MRE11/genética , Mutación , Proteínas Nucleares/genética , Animales , Roturas del ADN de Doble Cadena , Reparación del ADN , HumanosRESUMEN
The canonical Wnt/ß-catenin signalling pathway plays a crucial role in a variety of functions including cell proliferation and differentiation, tumorigenic processes and radioresistance in cancer cells. The Mre11-Rad50-Nbs1 (MRN) complex has a pivotal role in sensing and repairing DNA damage. However, it remains unclear whether a connection exists between Wnt/ß-catenin signalling and the MRN complex in the repair of cisplatin-induced DNA interstrand cross-links (ICLs). Here, we report that (1) cisplatin exposure results in a significant increase in the levels of MRN complex subunits in human tumour cells; (2) cisplatin treatment stimulates Wnt/ß-catenin signalling through increased ß-catenin expression; (3) the functional perturbation of Wnt/ß-catenin signalling results in aberrant cell cycle dynamics and the activation of DNA damage response and apoptosis; (4) a treatment with CHIR99021, a potent and selective GSK3ß inhibitor, augments cisplatin-induced cell death in cancer cells. On the other hand, inactivation of the Wnt/ß-catenin signalling with FH535 promotes cell survival. Consistently, the staining pattern of γH2AX-foci is significantly reduced in the cells exposed simultaneously to cisplatin and FH535; and (5) inhibition of Wnt/ß-catenin signalling impedes cisplatin-induced phosphorylation of Chk1, abrogates the G2/M phase arrest and impairs recombination-based DNA repair. Our data further show that Wnt signalling positively regulates the expression of ß-catenin, Mre11 and FANCD2 at early time points, but declining thereafter due to negative feedback regulation. These results support a model wherein Wnt/ß-catenin signalling and MRN complex crosstalk during DNA ICL repair, thereby playing an important role in the maintenance of genome stability.