RESUMEN
Ataxia-telangiectasia mutated (ATM) functions as a key initiator and coordinator of DNA damage and cellular stress responses. ATM signaling pathways contain many downstream targets that regulate multiple important cellular processes, including DNA damage repair, apoptosis, cell cycle arrest, oxidative sensing, and proliferation. Over the past few decades, associations between germline ATM pathogenic variants and cancer risk have been reported, particularly for breast and pancreatic cancers. In addition, given that ATM plays a critical role in repairing double-strand breaks, inhibiting other DNA repair pathways could be a synthetic lethal approach. Based on this rationale, several DNA damage response inhibitors are currently being tested in ATM-deficient cancers. In this review, we discuss the current knowledge related to the structure of the ATM gene, function of ATM kinase, clinical significance of ATM germline pathogenic variants in patients with hereditary cancers, and ongoing efforts to target ATM for the benefit of cancer patients.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Predisposición Genética a la Enfermedad , Neoplasias/etiología , Neoplasias/metabolismo , Animales , Apoptosis/genética , Proteínas de la Ataxia Telangiectasia Mutada/química , Ciclo Celular/genética , Roturas del ADN de Doble Cadena , Daño del ADN , Reparación del ADN , Manejo de la Enfermedad , Regulación de la Expresión Génica , Mutación de Línea Germinal , Humanos , Neoplasias/diagnóstico , Neoplasias/terapia , Oxidación-Reducción , Estrés Oxidativo , Medicina de Precisión , Transducción de SeñalRESUMEN
DNA double-strand breaks (DSBs) can lead to mutations, chromosomal rearrangements, genome instability, and cancer. Central to the sensing of DSBs is the ATM (Ataxia-telangiectasia mutated) kinase, which belongs to the phosphatidylinositol 3-kinase-related protein kinase (PIKK) family. In response to DSBs, ATM is activated by the MRN (Mre11-Rad50-Nbs1) protein complex through a poorly understood process that also requires double-stranded DNA. Previous studies indicate that the FxF/Y motif of Nbs1 directly binds to ATM, and is required to retain active ATM at sites of DNA damage. Here, we report the 2.5 Å resolution cryo-EM structures of human ATM and its complex with the Nbs1 FxF/Y motif. In keeping with previous structures of ATM and its yeast homolog Tel1, the dimeric human ATM kinase adopts a symmetric, butterfly-shaped structure. The conformation of the ATM kinase domain is most similar to the inactive states of other PIKKs, suggesting that activation may involve an analogous realigning of the N and C lobes along with relieving the blockage of the substrate-binding site. We also show that the Nbs1 FxF/Y motif binds to a conserved hydrophobic cleft within the Spiral domain of ATM, suggesting an allosteric mechanism of activation. We evaluate the importance of these structural findings with mutagenesis and biochemical assays.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/química , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de Ciclo Celular/metabolismo , Roturas del ADN de Doble Cadena , Proteínas Nucleares/metabolismo , Células HEK293 , Humanos , Mutación/genética , Fosfatidilinositol 3-Quinasa/metabolismo , Fosforilación , Unión ProteicaRESUMEN
Despite a considerable amount of data, the molecular and cellular bases of the toxicity due to metal exposure remain unknown. Recent mechanistic models from radiobiology have emerged, pointing out that the radiation-induced nucleo-shuttling of the ATM protein (RIANS) initiates the recognition and the repair of DNA double-strand breaks (DSB) and the final response to genotoxic stress. In order to document the role of ATM-dependent DSB repair and signalling after metal exposure, we applied twelve different metal species representing nine elements (Al, Cu, Zn Ni, Pd, Cd, Pb, Cr, and Fe) to human skin, mammary, and brain cells. Our findings suggest that metals may directly or indirectly induce DSB at a rate that depends on the metal properties and concentration, and tissue type. At specific metal concentration ranges, the nucleo-shuttling of ATM can be delayed which impairs DSB recognition and repair and contributes to toxicity and carcinogenicity. Interestingly, as observed after low doses of ionizing radiation, some phenomena equivalent to the biological response observed at high metal concentrations may occur at lower concentrations. A general mechanistic model of the biological response to metal exposure based on the nucleo-shuttling of ATM is proposed to describe the metal-induced stress response and to define quantitative endpoints for toxicity and carcinogenicity.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/química , Roturas del ADN de Doble Cadena/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Metales/química , Aluminio/farmacología , Proteínas de la Ataxia Telangiectasia Mutada/efectos de los fármacos , Proteínas de la Ataxia Telangiectasia Mutada/efectos de la radiación , Cadmio/farmacología , Cromo/farmacología , Cobre/farmacología , Reparación del ADN/efectos de la radiación , Humanos , Hierro/farmacología , Plomo/farmacología , Metales/farmacología , Metales/toxicidad , Níquel/farmacología , Paladio/farmacología , Zinc/farmacologíaRESUMEN
Human checkpoint kinase ataxia telangiectasia-mutated (ATM) plays a key role in initiation of the DNA damage response following DNA double-strand breaks. ATM inhibition is a promising approach in cancer therapy, but, so far, detailed insights into the binding modes of known ATM inhibitors have been hampered due to the lack of high-resolution ATM structures. Using cryo-EM, we have determined the structure of human ATM to an overall resolution sufficient to build a near-complete atomic model and identify two hitherto unknown zinc-binding motifs. We determined the structure of the kinase domain bound to ATPγS and to the ATM inhibitors KU-55933 and M4076 at 2.8 Å, 2.8 Å and 3.0 Å resolution, respectively. The mode of action and selectivity of the ATM inhibitors can be explained by structural comparison and provide a framework for structure-based drug design.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Proteínas de la Ataxia Telangiectasia Mutada/química , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Sitios de Unión , Dominio Catalítico , Microscopía por Crioelectrón , Humanos , Modelos Moleculares , Morfolinas/química , Morfolinas/metabolismo , Mutación , Neoplasias/genética , Conformación Proteica , Pironas/química , Pironas/metabolismoRESUMEN
The protein kinase ataxia telangiectasia mutated (ATM) is a master regulator of double-strand DNA break (DSB) signalling and stress responses. For three decades, ATM has been investigated extensively to elucidate its roles in the DNA damage response (DDR) and in the pathogenesis of ataxia telangiectasia (A-T), a human neurodegenerative disease caused by loss of ATM. Although hundreds of proteins have been identified as ATM phosphorylation targets and many important roles for this kinase have been identified, it is still unclear how ATM deficiency leads to the early-onset cerebellar degeneration that is common in all individuals with A-T. Recent studies suggest the existence of links between ATM deficiency and other cerebellum-specific neurological disorders, as well as the existence of broader similarities with more common neurodegenerative disorders. In this Review, we discuss recent structural insights into ATM regulation, and possible aetiologies of A-T phenotypes, including reactive oxygen species, mitochondrial dysfunction, alterations in transcription, R-loop metabolism and alternative splicing, defects in cellular proteostasis and metabolism, and potential pathogenic roles for hyper-poly(ADP-ribosyl)ation.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Ataxia Telangiectasia/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Ataxia Telangiectasia/genética , Ataxia Telangiectasia/patología , Proteínas de la Ataxia Telangiectasia Mutada/química , Proteínas de la Ataxia Telangiectasia Mutada/deficiencia , Reparación del ADN , Homeostasis , Humanos , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/patología , Oxidación-Reducción , Fosforilación , Poli(ADP-Ribosa) Polimerasas/metabolismo , ARN/metabolismoRESUMEN
Ataxia telangiectasia and Rad3 related (ATR) activation after replication stress involves a cascade of reactions, including replication protein A (RPA) complex loading onto single-stranded DNA and ATR activator loading onto chromatin. The contribution of histone modifications to ATR activation, however, is unclear. Here, we report that H3K14 trimethylation responds to replication stress by enhancing ATR activation. First, we confirmed that H3K14 monomethylation, dimethylation, and trimethylation all exist in mammalian cells, and that both SUV39H1 and SETD2 methyltransferases can catalyze H3K14 trimethylation in vivo and in vitro. Interestingly, SETD2-mediated H3K14 trimethylation markedly increases in response to replication stress induced with hydroxyurea, a replication stress inducer. Under these conditions, SETD2-mediated H3K14me3 recruited the RPA complex to chromatin via a direct interaction with RPA70. The increase in H3K14me3 levels was abolished, and RPA loading was attenuated when SETD2 was depleted or H3K14 was mutated. Rather, the cells were sensitive to replication stress such that the replication forks failed to restart, and cell-cycle progression was delayed. These findings help us understand how H3K14 trimethylation links replication stress with ATR activation.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Replicación del ADN , ADN/biosíntesis , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Proteína de Replicación A/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada/química , Proteínas de la Ataxia Telangiectasia Mutada/genética , ADN/química , ADN/genética , N-Metiltransferasa de Histona-Lisina/química , N-Metiltransferasa de Histona-Lisina/genética , Histonas/química , Histonas/genética , Humanos , Metilación , Metiltransferasas/química , Metiltransferasas/genética , Metiltransferasas/metabolismo , Proteína de Replicación A/química , Proteína de Replicación A/genética , Proteínas Represoras/química , Proteínas Represoras/genética , Proteínas Represoras/metabolismoRESUMEN
ATM is among of the most critical initiators and coordinators of the DNA-damage response. ATM canonical and non-canonical signaling pathways involve hundreds of downstream targets that control many important cellular processes such as DNA damage repair, apoptosis, cell cycle arrest, metabolism, proliferation, oxidative sensing, among others. Of note, ATM is often considered a major tumor suppressor because of its ability to induce apoptosis and cell cycle arrest. However, in some advanced stage tumor cells, ATM signaling is increased and confers remarkable advantages for cancer cell survival, resistance to radiation and chemotherapy, biosynthesis, proliferation, and metastasis. This review focuses on addressing major characteristics, signaling pathways and especially the diverse roles of ATM in cellular homeostasis and cancer development.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/genética , Carcinogénesis/genética , Animales , Proteínas de la Ataxia Telangiectasia Mutada/química , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Carcinogénesis/metabolismo , Reparación del ADN , Humanos , Transducción de SeñalRESUMEN
ATR checkpoint signaling is crucial for cellular responses to DNA replication impediments. Using an optogenetic platform, we show that TopBP1, the main activator of ATR, self-assembles extensively to yield micrometer-sized condensates. These opto-TopBP1 condensates are functional entities organized in tightly packed clusters of spherical nano-particles. TopBP1 condensates are reversible, occasionally fuse, and co-localize with TopBP1 partner proteins. We provide evidence that TopBP1 condensation is a molecular switch that amplifies ATR activity to phosphorylate checkpoint kinase 1 (Chk1) and slow down replication forks. Single amino acid substitutions of key residues in the intrinsically disordered ATR activation domain disrupt TopBP1 condensation and consequently ATR/Chk1 signaling. In physiologic salt concentration and pH, purified TopBP1 undergoes liquid-liquid phase separation in vitro. We propose that the actuation mechanism of ATR signaling is the assembly of TopBP1 condensates driven by highly regulated multivalent and cooperative interactions.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada , Proteínas Portadoras , Núcleo Celular , Proteínas de Unión al ADN , Mutación Missense , Proteínas Nucleares , Transducción de Señal , Sustitución de Aminoácidos , Animales , Proteínas de la Ataxia Telangiectasia Mutada/química , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Núcleo Celular/química , Núcleo Celular/genética , Núcleo Celular/metabolismo , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/química , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Células HeLa , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Células Sf9 , SpodopteraRESUMEN
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
Proper repair of damaged DNA is critical for the maintenance of genome stability. A complex composed of Integrator subunit 3 (Ints3), single-stranded DNA-binding protein 1 (SSB1), and SSB-interacting protein 1 (SSBIP1) is required for efficient homologous recombination-dependent repair of double-strand breaks (DSBs) and ataxia-telangiectasia mutated (ATM)-dependent signaling pathways. It is known that in this complex the Ints3 N-terminal domain scaffolds SSB1 and SSBIP1. However, the molecular basis for the function of the Ints3 C-terminal domain remains unclear. Here, we present the crystal structure of the Ints3 C-terminal domain, uncovering a HEAT-repeat superhelical fold. Using structure and mutation analysis, we show that the C-terminal domain exists as a stable dimer. A basic groove and a cluster of conserved residues on two opposite sides of the dimer bind single-stranded RNA/DNA (ssRNA/ssDNA) and Integrator complex subunit 6 (Ints6), respectively. Dimerization is required for nucleic acid binding, but not for Ints6 binding. Additionally, in vitro experiments using HEK 293T cells demonstrate that Ints6 interaction is critical for maintaining SSB1 protein level. Taken together, our findings establish the structural basis of a multifunctional Ints3 C-terminal module, allowing us to propose a novel mode of nucleic acid recognition by helical repeat protein and paving the way for future mechanistic studies.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/química , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/metabolismo , Roturas del ADN de Doble Cadena , Células HEK293 , Humanos , Unión Proteica , Multimerización de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , ProteolisisRESUMEN
Genomic integrity is most threatened by double-strand breaks, which, if left unrepaired, lead to carcinogenesis or cell death. The cell generates a network of protein-protein signaling interactions that emanate from the DNA damage which are now recognized as a rich basis for anti-cancer therapy development. Deciphering the structures of signaling proteins has been an uphill task owing to their large size and complex domain organization. Recent advances in mammalian protein expression/purification and cryo-EM-based structure determination have led to significant progress in our understanding of these large multidomain proteins. This review is an overview of the structural principles that underlie some of the key signaling proteins that function at the double-strand break site. We also discuss some plausible ideas that could be considered for future structural approaches to visualize and build a more complete understanding of protein dynamics at the break site.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN/genética , Transducción de Señal/genética , Ácido Anhídrido Hidrolasas/química , Ácido Anhídrido Hidrolasas/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada/química , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Daño del ADN/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Humanos , Proteína Homóloga de MRE11/química , Proteína Homóloga de MRE11/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Procesamiento Proteico-Postraduccional/genética , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/química , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Coordinated by ataxia-telangiectasia-mutated (ATM) and ATM and Rad3-related (ATR), two highly conserved kinases, DNA damage repair ensures genome integrity and survival in all organisms. The Arabidopsis thaliana (A. thaliana) orthologues are well characterized and exhibit typical mammalian characteristics. We mutated the Physcomitrellapatens (P. patens) PpATM and PpATR genes by deleting functionally important domains using gene targeting. Both mutants showed growth abnormalities, indicating that these genes, particularly PpATR, are important for normal vegetative development. ATR was also required for repair of both direct and replication-coupled double-strand breaks (DSBs) and dominated the transcriptional response to direct DSBs, whereas ATM was far less important, as shown by assays assessing resistance to DSB induction and SuperSAGE-based transcriptomics focused on DNA damage repair genes. These characteristics differed significantly from the A. thaliana genes but resembled those in yeast (Saccharomyces cerevisiae). PpATR was not important for gene targeting, pointing to differences in the regulation of gene targeting and direct DSB repair. Our analysis suggests that ATM and ATR functions can be substantially diverged between plants. The differences in ATM and ATR reflect the differences in DSB repair pathway choices between A. thaliana and P. patens, suggesting that they represent adaptations to different demands for the maintenance of genome stability.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/genética , Bryopsida/genética , Proteínas de Plantas/genética , Reparación del ADN por Recombinación , Proteínas de la Ataxia Telangiectasia Mutada/química , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Bryopsida/crecimiento & desarrollo , Roturas del ADN de Doble Cadena , Mutación , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Dominios ProteicosRESUMEN
The ATR kinase plays a key role in the DNA damage response by activating essential signaling pathways of DNA damage repair, especially in response to replication stress. Because DNA damage and replication stress are major sources of genomic instability, selective ATR inhibition has been recognized as a promising new approach in cancer therapy. We now report the identification and preclinical evaluation of the novel, clinical ATR inhibitor BAY 1895344. Starting from quinoline 2 with weak ATR inhibitory activity, lead optimization efforts focusing on potency, selectivity, and oral bioavailability led to the discovery of the potent, highly selective, orally available ATR inhibitor BAY 1895344, which exhibited strong monotherapy efficacy in cancer xenograft models that carry certain DNA damage repair deficiencies. Moreover, combination treatment of BAY 1895344 with certain DNA damage inducing chemotherapy resulted in synergistic antitumor activity. BAY 1895344 is currently under clinical investigation in patients with advanced solid tumors and lymphomas (NCT03188965).
Asunto(s)
Antineoplásicos/administración & dosificación , Antineoplásicos/farmacocinética , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Morfolinas/administración & dosificación , Morfolinas/farmacocinética , Pirazoles/administración & dosificación , Pirazoles/farmacocinética , Administración Oral , Animales , Antineoplásicos/química , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Proteínas de la Ataxia Telangiectasia Mutada/química , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Disponibilidad Biológica , Carboplatino/administración & dosificación , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Inhibidores del Citocromo P-450 CYP2C8/química , Inhibidores del Citocromo P-450 CYP2C8/farmacología , Reparación del ADN/efectos de los fármacos , Perros , Descubrimiento de Drogas , Ensayos de Selección de Medicamentos Antitumorales , Estabilidad de Medicamentos , Femenino , Humanos , Ratones SCID , Microsomas Hepáticos/efectos de los fármacos , Morfolinas/química , Pirazoles/química , Ratas Wistar , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Cells confront DNA damage in every cell cycle. Among the most deleterious types of DNA damage are DNA double-strand breaks (DSBs), which can cause cell lethality if unrepaired or cancers if improperly repaired. In response to DNA DSBs, cells activate a complex DNA damage checkpoint (DDC) response that arrests the cell cycle, reprograms gene expression, and mobilizes DNA repair factors to prevent the inheritance of unrepaired and broken chromosomes. Here we examine the DDC, induced by DNA DSBs, in the budding yeast model system and in mammals.
Asunto(s)
Puntos de Control del Ciclo Celular/genética , Reparación del ADN por Unión de Extremidades , ADN/genética , Reparación del ADN por Recombinación , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Animales , Proteínas de la Ataxia Telangiectasia Mutada/química , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Quinasa de Punto de Control 2/genética , Quinasa de Punto de Control 2/metabolismo , ADN/química , ADN/metabolismo , Roturas del ADN de Doble Cadena , Humanos , Modelos Moleculares , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Estructura Secundaria de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Homologous recombination (HR) is a faithful repair mechanism for double stranded DNA breaks. Two highly homologous master kinases, the tumour suppressors ATM and ATR (Tel1 and Mec1 in yeast), coordinate cell cycle progression with repair during HR. Despite their importance, our molecular understanding of these apical coordinators has been limited, in part due to their large sizes. With the recent development in cryo-electron microscopy, significant advances have been made in structural characterisation of these proteins in the last two years. These structures, combined with new biochemical studies, now provide a more detailed understanding of how a low basal activity is maintained and how activation may occur. In this review, we summarize recent advances in the structural and molecular understanding of these key components in HR, compare the common and distinct features of these kinases and suggest aspects of structural components that are likely to be involved in regulating its activity.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/química , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Modelos Moleculares , Conformación Proteica , Secuencia de Aminoácidos , Proteínas de la Ataxia Telangiectasia Mutada/genética , Sitios de Unión , Dominio Catalítico , Microscopía por Crioelectrón , Activación Enzimática , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Inestabilidad Genómica , Humanos , Unión Proteica , Relación Estructura-ActividadRESUMEN
The RAS-RAF-MEK-ERK signaling axis is frequently activated in human cancers. Physiological concentrations of ATP prevent formation of RAF kinase-domain (RAFKD) dimers that are critical for activity. Here we present a 2.9-Å-resolution crystal structure of human BRAFKD in complex with MEK and the ATP analog AMP-PCP, revealing interactions between BRAF and ATP that induce an inactive, monomeric conformation of BRAFKD. We also determine how 14-3-3 relieves the negative regulatory effect of ATP through a 2.5-Å-resolution crystal structure of the BRAFKD-14-3-3 complex, in which dimeric 14-3-3 enforces a dimeric BRAFKD assembly to increase BRAF activity. Our data suggest that most oncogenic BRAF mutations alter interactions with ATP and counteract the negative effects of ATP binding by lowering the threshold for RAF dimerization and pathway activation. Our study establishes a framework for rationalizing oncogenic BRAF mutations and provides new avenues for improved RAF-inhibitor discovery.
Asunto(s)
Proteínas 14-3-3/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas 14-3-3/química , Adenosina Trifosfato/análogos & derivados , Proteínas de la Ataxia Telangiectasia Mutada/química , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Cristalografía por Rayos X , Humanos , Simulación de Dinámica Molecular , Conformación Proteica , Multimerización de Proteína , Proteínas Proto-Oncogénicas B-raf/químicaRESUMEN
Homologous recombination (HR) is a pathway to faithfully repair DNA double-strand breaks (DSBs). At the core of this pathway is a DNA recombinase, which, as a nucleoprotein filament on ssDNA, pairs with homologous DNA as a template to repair the damaged site. In eukaryotes Rad51 is the recombinase capable of carrying out essential steps including strand invasion, homology search on the sister chromatid and strand exchange. Importantly, a tightly regulated process involving many protein factors has evolved to ensure proper localisation of this DNA repair machinery and its correct timing within the cell cycle. Dysregulation of any of the proteins involved can result in unchecked DNA damage, leading to uncontrolled cell division and cancer. Indeed, many are tumour suppressors and are key targets in the development of new cancer therapies. Over the past 40 years, our structural and mechanistic understanding of homologous recombination has steadily increased with notable recent advancements due to the advances in single particle cryo electron microscopy. These have resulted in higher resolution structural models of the signalling proteins ATM (ataxia telangiectasia mutated), and ATR (ataxia telangiectasia and Rad3-related protein), along with various structures of Rad51. However, structural information of the other major players involved, such as BRCA1 (breast cancer type 1 susceptibility protein) and BRCA2 (breast cancer type 2 susceptibility protein), has been limited to crystal structures of isolated domains and low-resolution electron microscopy reconstructions of the full-length proteins. Here we summarise the current structural understanding of homologous recombination, focusing on key proteins in recruitment and signalling events as well as the mediators for the Rad51 recombinase.
Asunto(s)
Daño del ADN , Mapas de Interacción de Proteínas , Reparación del ADN por Recombinación , Animales , Proteínas de la Ataxia Telangiectasia Mutada/química , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteína BRCA1/química , Proteína BRCA1/metabolismo , Proteína BRCA2/química , Proteína BRCA2/metabolismo , ADN/química , ADN/genética , Humanos , Modelos Moleculares , Conformación Proteica , Recombinasa Rad51/química , Recombinasa Rad51/metabolismoRESUMEN
Tel1 (ATM in humans) is a large kinase that resides in the cell in an autoinhibited dimeric state and upon activation orchestrates the cellular response to DNA damage. We report the structure of an endogenous Tel1 dimer from Chaetomium thermophilum. Major parts are at 2.8 Å resolution, including the kinase active site with ATPγS bound, and two different N-terminal solenoid conformations are at 3.4 Å and 3.6 Å, providing a side-chain model for 90% of the Tel1 polypeptide. We show that the N-terminal solenoid has DNA binding activity, but that its movements are not coupled to kinase activation. Although ATPγS and catalytic residues are poised for catalysis, the kinase resides in an autoinhibited state. The PIKK regulatory domain acts as a pseudo-substrate, blocking direct access to the site of catalysis. The structure allows mapping of human cancer mutations and defines mechanisms of autoinhibition at near-atomic resolution.
Asunto(s)
Adenosina Trifosfato/análogos & derivados , Proteínas de la Ataxia Telangiectasia Mutada/química , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Chaetomium/enzimología , Adenosina Trifosfato/metabolismo , Dominio Catalítico , Chaetomium/química , ADN/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Homeostasis , Modelos Moleculares , Unión Proteica , Conformación Proteica , Dominios Proteicos , Multimerización de ProteínaAsunto(s)
Quinasa de Punto de Control 2/metabolismo , Ácido Graso Sintasas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/química , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Quinasa de Punto de Control 2/química , Activación Enzimática , Ácido Graso Sintasas/química , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Unión Proteica , Conformación Proteica , Proteínas de Schizosaccharomyces pombe/química , Relación Estructura-ActividadRESUMEN
Although oxaliplatin is an effective chemotherapeutic drug for treatment of colorectal cancer (CRC), tumor cells can develop mechanisms to evade oxaliplatin-induced cell death and show high tolerance and acquired resistance to this drug. Heterogeneous nuclear ribonucleoprotein L (hnRNP L) has been proved to play a critical role in DNA repair during IgH class switch recombination (CSR) in B lymphocytes, while, its role in CRC and chemotherapeutic resistance remain unknown. Our study aims to uncover an unidentified mechanism of regulating DNA double-strand breaks (DSBs) by hnRNP L in CRC cells treated by oxaliplatin. In present study, we observed that knockdown of hnRNP L enhanced the level of DNA breakage and sensitivity of CRC cells to oxaliplatin. The expression of key DNA repair factors (BRCA1, 53BP1, and ATM) was unaffected by hnRNP L knockdown, thereby excluding the likelihood of hnRNP L mediation via mRNA regulation. Moreover, we observed that phosphorylation level of ATM changed oppositely to 53BP1 and BRCA1 in the CRC cells (SW620 and HCT116) which exhibit synergistic effect by oxaliplatin plus hnRNP L impairment. And similar phenomenon was observed in the foci formation of these critical repair factors. We also found that hnRNP L binds directly with these DNA repair factors through its RNA-recognition motifs (RRMs). Analysis of cell death indicated that the RRMs of hnRNP L are required for cell survival under incubation with oxaliplatin. In conclusion, hnRNP L is critical for the recruitment of the DNA repair factors in oxaliplatin-induced DSBs. Targeting hnRNP L is a promising new clinical approach that could enhance the effectiveness of current chemotherapeutic treatment in patients with resistance to oxaliplatin.