RESUMEN
Alterations in transcriptional regulators can orchestrate oncogenic gene expression programs in cancer. Here, we show that the BRG1/BRM-associated factor (BAF) chromatin remodeling complex, which is mutated in over 20% of human tumors, interacts with EWSR1, a member of a family of proteins with prion-like domains (PrLD) that are frequent partners in oncogenic fusions with transcription factors. In Ewing sarcoma, we find that the BAF complex is recruited by the EWS-FLI1 fusion protein to tumor-specific enhancers and contributes to target gene activation. This process is a neomorphic property of EWS-FLI1 compared to wild-type FLI1 and depends on tyrosine residues that are necessary for phase transitions of the EWSR1 prion-like domain. Furthermore, fusion of short fragments of EWSR1 to FLI1 is sufficient to recapitulate BAF complex retargeting and EWS-FLI1 activities. Our studies thus demonstrate that the physical properties of prion-like domains can retarget critical chromatin regulatory complexes to establish and maintain oncogenic gene expression programs.
Asunto(s)
Proteínas de Unión a Calmodulina/química , Proteínas de Unión a Calmodulina/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Proteína Proto-Oncogénica c-fli-1/metabolismo , Proteína EWS de Unión a ARN/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Sarcoma de Ewing/genética , Línea Celular Tumoral , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Humanos , Células Madre Mesenquimatosas/metabolismo , Repeticiones de Microsatélite , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas Priónicas/metabolismo , Dominios Proteicos , Sarcoma de Ewing/patologíaRESUMEN
Acquired drug resistance to anticancer targeted therapies remains an unsolved clinical problem. Although many drivers of acquired drug resistance have been identified1-4, the underlying molecular mechanisms shaping tumour evolution during treatment are incompletely understood. Genomic profiling of patient tumours has implicated apolipoprotein B messenger RNA editing catalytic polypeptide-like (APOBEC) cytidine deaminases in tumour evolution; however, their role during therapy and the development of acquired drug resistance is undefined. Here we report that lung cancer targeted therapies commonly used in the clinic can induce cytidine deaminase APOBEC3A (A3A), leading to sustained mutagenesis in drug-tolerant cancer cells persisting during therapy. Therapy-induced A3A promotes the formation of double-strand DNA breaks, increasing genomic instability in drug-tolerant persisters. Deletion of A3A reduces APOBEC mutations and structural variations in persister cells and delays the development of drug resistance. APOBEC mutational signatures are enriched in tumours from patients with lung cancer who progressed after extended responses to targeted therapies. This study shows that induction of A3A in response to targeted therapies drives evolution of drug-tolerant persister cells, suggesting that suppression of A3A expression or activity may represent a potential therapeutic strategy in the prevention or delay of acquired resistance to lung cancer targeted therapy.
Asunto(s)
Citidina Desaminasa , Neoplasias Pulmonares , Humanos , Citidina Desaminasa/deficiencia , Citidina Desaminasa/efectos de los fármacos , Citidina Desaminasa/genética , Citidina Desaminasa/metabolismo , Roturas del ADN de Doble Cadena , Inestabilidad Genómica , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Terapia Molecular Dirigida , Mutación , Resistencia a AntineoplásicosRESUMEN
Numerous DNA repair and signaling proteins function at DNA damage sites to protect the genome. Here, we show that fusion of the promiscuous biotin ligase BirAR118G with RAD18 leads to localized protein biotinylation at DNA damage sites, allowing identification of ZPET (zinc finger protein proximal to RAD eighteen)/ZNF280C as a potential DNA damage response (DDR) protein. ZPET binds ssDNA and localizes to DNA double-strand breaks (DSBs) and stalled replication forks. In vitro, ZPET inhibits MRE11 binding to ssDNA. In cells, ZPET delays MRE11 binding to chromatin after DSB formation and slows DNA end resection through binding ssDNA. ZPET hinders resection independently of 53BP1 and HELB. Cells lacking ZPET displayed enhanced homologous recombination (HR), accelerated replication forks under stress, and increased resistance to DSBs and PARP inhibition. These results not only reveal ZPET as an HR repressor but also suggest that localized protein biotinylation at DNA damage sites is a useful strategy to identify DDR proteins.
Asunto(s)
Biotinilación/métodos , Daño del ADN , Reparación del ADN/genética , Proteínas de Unión al ADN/metabolismo , Recombinación Homóloga/genética , Factores de Transcripción/metabolismo , Ligasas de Carbono-Nitrógeno/genética , Línea Celular , Roturas del ADN de Doble Cadena , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Escherichia coli/genética , Técnicas de Silenciamiento del Gen , Humanos , Proteína Homóloga de MRE11/metabolismo , Unión Proteica , Transporte de Proteínas/genética , Proteínas Represoras/genética , Factores de Transcripción/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
ATR is a key regulator of cell-cycle checkpoints and homologous recombination (HR). Paradoxically, ATR inhibits CDKs during checkpoint responses, but CDK activity is required for efficient HR. Here, we show that ATR promotes HR after CDK-driven DNA end resection. ATR stimulates the BRCA1-PALB2 interaction after DNA damage and promotes PALB2 localization to DNA damage sites. ATR enhances BRCA1-PALB2 binding at least in part by inhibiting CDKs. The optimal interaction of BRCA1 and PALB2 requires phosphorylation of PALB2 at S59, an ATR site, and hypo-phosphorylation of S64, a CDK site. The PALB2-S59A/S64E mutant is defective for localization to DNA damage sites and HR, whereas the PALB2-S59E/S64A mutant partially bypasses ATR for its localization. Thus, HR is a biphasic process requiring both high-CDK and low-CDK periods. As exemplified by the regulation of PALB2 by ATR, ATR promotes HR by orchestrating a "CDK-to-ATR switch" post-resection, directly coupling the checkpoint to HR.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN por Recombinación , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Proteína del Grupo de Complementación N de la Anemia de Fanconi , Células HeLa , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilación , Unión Proteica , Transducción de Señal , Factores de Tiempo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Poly-(ADP-ribose) polymerase (PARP) inhibitors (PARPis) selectively kill BRCA1/2-deficient cells, but their efficacy in BRCA-deficient patients is limited by drug resistance. Here, we used derived cell lines and cells from patients to investigate how to overcome PARPi resistance. We found that the functions of BRCA1 in homologous recombination (HR) and replication fork protection are sequentially bypassed during the acquisition of PARPi resistance. Despite the lack of BRCA1, PARPi-resistant cells regain RAD51 loading to DNA double-stranded breaks (DSBs) and stalled replication forks, enabling two distinct mechanisms of PARPi resistance. Compared with BRCA1-proficient cells, PARPi-resistant BRCA1-deficient cells are increasingly dependent on ATR for survival. ATR inhibitors (ATRis) disrupt BRCA1-independent RAD51 loading to DSBs and stalled forks in PARPi-resistant BRCA1-deficient cells, overcoming both resistance mechanisms. In tumor cells derived from patients, ATRis also overcome the bypass of BRCA1/2 in fork protection. Thus, ATR inhibition is a unique strategy to overcome the PARPi resistance of BRCA-deficient cancers.
Asunto(s)
Recombinación Homóloga/genética , Neoplasias Ováricas/genética , Poli(ADP-Ribosa) Polimerasa-1/antagonistas & inhibidores , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Proteína BRCA1/deficiencia , Proteína BRCA1/genética , Reparación del ADN , ADN de Neoplasias , Resistencia a Antineoplásicos/genética , Femenino , Recombinación Homóloga/efectos de los fármacos , Humanos , Neoplasias Ováricas/tratamiento farmacológico , Células Tumorales CultivadasRESUMEN
APOBEC3A is an antiviral DNA deaminase often induced by virus infection. APOBEC3A is also a source of cancer mutation in viral and nonviral tumor types. It is therefore critical to identify factors responsible for APOBEC3A upregulation. Here, we test the hypothesis that leaked mitochondrial (mt) double-stranded (ds)RNA is recognized as foreign nucleic acid, which triggers innate immune signaling, APOBEC3A upregulation, and DNA damage. Knockdown of an enzyme responsible for degrading mtdsRNA, the exoribonuclease polynucleotide phosphorylase, results in mtdsRNA leakage into the cytosol and induction of APOBEC3A expression. APOBEC3A upregulation by cytoplasmic mtdsRNA requires RIG-I, MAVS, and STAT2 and is likely part of a broader type I interferon response. Importantly, although mtdsRNA-induced APOBEC3A appears cytoplasmic by subcellular fractionation experiments, its induction triggers an overt DNA damage response characterized by elevated nuclear γ-H2AX staining. Thus, mtdsRNA dysregulation may induce APOBEC3A and contribute to observed genomic instability and mutation signatures in cancer.
Asunto(s)
Citidina Desaminasa , Daño del ADN , Neoplasias , ARN Bicatenario , Humanos , ADN , Neoplasias/genética , ARN Bicatenario/genética , ARN Mitocondrial/genética , Citidina Desaminasa/genéticaRESUMEN
The ATR-Chk1 pathway is critical for DNA damage responses and cell-cycle progression. Chk1 inhibition is more deleterious to cycling cells than ATR inhibition, raising questions about ATR and Chk1 functions in the absence of extrinsic replication stress. Here we show that a key role of ATR in S phase is to coordinate RRM2 accumulation and origin firing. ATR inhibitor (ATRi) induces massive ssDNA accumulation and replication catastrophe in a fraction of early S-phase cells. In other S-phase cells, however, ATRi induces moderate ssDNA and triggers a DNA-PK and Chk1-mediated backup pathway to suppress origin firing. The backup pathway creates a threshold such that ATRi selectively kills cells under high replication stress, whereas Chk1 inhibitor induces cell death at a lower threshold. The levels of ATRi-induced ssDNA correlate with ATRi sensitivity in a panel of cell lines, suggesting that ATRi-induced ssDNA could be predictive of ATRi sensitivity in cancer cells.
Asunto(s)
Proteína Quinasa Activada por ADN/fisiología , Proteínas Nucleares/fisiología , Proteínas Quinasas/fisiología , Fase S , Proteínas de la Ataxia Telangiectasia Mutada/fisiología , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Daño del ADN , Replicación del ADN , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Humanos , Fosforilación , Procesamiento Proteico-Postraduccional , Origen de Réplica , Ribonucleósido Difosfato Reductasa/metabolismo , Estrés FisiológicoRESUMEN
Fanconi Anemia (FA) clinical phenotypes are heterogenous and rely on a mutation in one of the 22 FANC genes (FANCA-W) involved in a common interstrand DNA crosslink-repair pathway. A critical step in the activation of FA pathway is the monoubiquitination of FANCD2 and its binding partner FANCI. To better address the clinical phenotype associated with FANCI and the epistatic relationship with FANCD2, we created the first conditional inactivation model for FANCI in mouse. Fanci -/- mice displayed typical FA features such as delayed development in utero, microphtalmia, cellular sensitivity to mitomycin C, occasional limb abnormalities and hematological deficiencies. Interestingly, the deletion of Fanci leads to a strong meiotic phenotype and severe hypogonadism. FANCI was localized in spermatocytes and spermatids and in the nucleus of oocytes. Both FANCI and FANCD2 proteins co-localized with RPA along meiotic chromosomes, albeit at different levels. Consistent with a role in meiotic recombination, FANCI interacted with RAD51 and stimulated D-loop formation, unlike FANCD2. The double knockout Fanci-/- Fancd2-/- also showed epistatic relationship for hematological defects while being not epistatic with respect to generating viable mice in crosses of double heterozygotes. Collectively, this study highlights common and distinct functions of FANCI and FANCD2 during mouse development, meiotic recombination and hematopoiesis.
Asunto(s)
Reparación del ADN , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Anemia de Fanconi/genética , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Anemia de Fanconi/metabolismo , Anemia de Fanconi/patología , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Femenino , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Oocitos/metabolismo , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , Espermatocitos/metabolismoRESUMEN
The KAT5 (Tip60/Esa1) histone acetyltransferase is part of NuA4, a large multifunctional complex highly conserved from yeast to mammals that targets lysines on H4 and H2A (X/Z) tails for acetylation. It is essential for cell viability, being a key regulator of gene expression, cell proliferation, and stem cell renewal and an important factor for genome stability. The NuA4 complex is directly recruited near DNA double-strand breaks (DSBs) to facilitate repair, in part through local chromatin modification and interplay with 53BP1 during the DNA damage response. While NuA4 is detected early after appearance of the lesion, its precise mechanism of recruitment remains to be defined. Here, we report a stepwise recruitment of yeast NuA4 to DSBs first by a DNA damage-induced phosphorylation-dependent interaction with the Xrs2 subunit of the Mre11-Rad50-Xrs2 (MRX) complex bound to DNA ends. This is followed by a DNA resection-dependent spreading of NuA4 on each side of the break along with the ssDNA-binding replication protein A (RPA). Finally, we show that NuA4 can acetylate RPA and regulate the dynamics of its binding to DNA, hence targeting locally both histone and nonhistone proteins for lysine acetylation to coordinate repair.
Asunto(s)
Roturas del ADN de Doble Cadena , ADN de Hongos , Histona Acetiltransferasas , Proteínas de Saccharomyces cerevisiae , Acetilación , ADN de Hongos/química , ADN de Hongos/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas/química , Endodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/química , Exodesoxirribonucleasas/metabolismo , Histona Acetiltransferasas/química , Histona Acetiltransferasas/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/química , Proteína 1 de Unión al Supresor Tumoral P53/metabolismoRESUMEN
Telomere integrity is essential to maintain genome stability, and telomeric dysfunctions are associated with cancer and aging pathologies. In human, the shelterin complex binds TTAGGG DNA repeats and provides capping to chromosome ends. Within shelterin, RAP1 is recruited through its interaction with TRF2, and TRF2 is required for telomere protection through a network of nucleic acid and protein interactions. RAP1 is one of the most conserved shelterin proteins although one unresolved question is how its interaction may influence TRF2 properties and regulate its capacity to bind multiple proteins. Through a combination of biochemical, biophysical and structural approaches, we unveiled a unique mode of assembly between RAP1 and TRF2. The complete interaction scheme between the full-length proteins involves a complex biphasic interaction of RAP1 that directly affects the binding properties of the assembly. These results reveal how a non-DNA binding protein can influence the properties of a DNA-binding partner by mutual conformational adjustments.
Asunto(s)
Proteínas de Unión al ADN/genética , Inestabilidad Genómica , Proteínas de Unión a Telómeros/genética , Proteína 2 de Unión a Repeticiones Teloméricas/genética , Daño del ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Complejos Multiproteicos , Unión Proteica , Complejo Shelterina , Telómero/genética , Proteínas de Unión a Telómeros/química , Proteínas de Unión a Telómeros/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/química , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismoRESUMEN
PALB2 [partner and localizer of BRCA2 (breast cancer early-onset 2)] [corrected] has emerged as a key player in the maintenance of genome integrity. Biallelic mutations in PALB2 cause FA (Fanconi's anaemia) subtype FA-N, a devastating inherited disorder marked by developmental abnormalities, bone marrow failure and childhood cancer susceptibility, whereas monoallelic mutations predispose to breast, ovarian and pancreatic cancer. The tumour suppressor role of PALB2 has been intimately linked to its ability to promote HR (homologous recombination)-mediated repair of DNA double-strand breaks. Because PALB2 lies at the crossroads between FA, HR and cancer susceptibility, understanding its function has become the primary focus of several studies. The present review discusses a current synthesis of the contribution of PALB2 to these pathways. We also provide a molecular description of FA- or cancer-associated PALB2 mutations.
Asunto(s)
Reparación del ADN , Recombinación Homóloga , Neoplasias/fisiopatología , Proteínas Nucleares/fisiología , Proteínas Supresoras de Tumor/fisiología , Animales , Proteína BRCA2/fisiología , Neoplasias de la Mama/genética , Neoplasias de la Mama Masculina/genética , Anemia de Fanconi/genética , Proteína del Grupo de Complementación N de la Anemia de Fanconi , Femenino , Humanos , Masculino , Ratones , Neoplasias/genética , Neoplasias Ováricas/genética , Neoplasias Pancreáticas/genética , Factores de Transcripción/fisiologíaRESUMEN
The partner and localizer of breast cancer 2 susceptibility protein (PALB2) is crucial for the repair of DNA damage by homologous recombination. Here, we report that chromatin-association motif (ChAM), an evolutionarily conserved motif in PALB2, is necessary and sufficient to mediate its chromatin association in both unperturbed and damaged cells. ChAM is distinct from the previously described PALB2 DNA-binding regions. Deletion of ChAM decreases PALB2 and Rad51 accumulation at DNA damage sites and confers cellular hypersensitivity to the genotoxic drug mitomycin C. These results suggest that PALB2 chromatin association via ChAM facilitates PALB2 function in the cellular resistance to DNA damage.
Asunto(s)
Cromatina/metabolismo , Reparación del ADN , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Línea Celular , Secuencia Conservada/genética , Daño del ADN , Evolución Molecular , Proteína del Grupo de Complementación N de la Anemia de Fanconi , Recombinación Homóloga/genética , Humanos , Modelos Biológicos , Datos de Secuencia Molecular , Nucleosomas/metabolismo , Unión Proteica , Eliminación de Secuencia , Relación Estructura-ActividadRESUMEN
PALB2 is essential for BRCA2 anchorage to nuclear structures and for homologous recombinational repair of DNA double-strand breaks. Here, we report that the N-terminal coiled-coil motif of PALB2 regulates its self-association and homologous recombination. Monomeric PALB2 shows higher efficiency to bind DNA and promotes RAD51 filament formation with or without the inhibitory effect of Replication Protein A. Moreover, overexpression of the PALB2 coiled-coil domain severely affects RAD51 loading to DNA damage sites suggesting a competition between PALB2 self-interaction and PALB2-BRCA1 interaction. In the presence of DNA damage, the switch between PALB2-PALB2 and PALB2-BRCA1 interactions allows the activation of HR. Controlling HR via PALB2 self-interactions could be important to prevent aberrant recombination in normal conditions and activate DNA repair when required.
Asunto(s)
Recombinación Homóloga , Proteínas Nucleares/metabolismo , Proteínas Supresoras de Tumor/metabolismo , ADN/metabolismo , Proteína del Grupo de Complementación N de la Anemia de Fanconi , Células HEK293 , Células HeLa , Humanos , Proteínas Nucleares/química , Dominios y Motivos de Interacción de Proteínas , Recombinasa Rad51/análisis , Proteínas Supresoras de Tumor/químicaRESUMEN
In most organisms, the primary function of homologous recombination (HR) is to allow genome protection by the faithful repair of DNA double-strand breaks. The vital step of HR is the search for sequence homology, mediated by the RAD51 recombinase, which is stimulated further by proteins mediators such as the tumor suppressor BRCA2. The biochemical interplay between RAD51 and BRCA2 is unknown in Leishmania or Trypanosoma. Here we show that the Leishmania infantum BRCA2 protein possesses several critical features important for the regulation of DNA recombination at the genetic and biochemical level. A BRCA2 null mutant, generated by gene disruption, displayed genomic instability and gene-targeting defects. Furthermore, cytological studies show that LiRAD51 can no longer localize to the nucleus in this mutant. The Leishmania RAD51 and BRCA2 interact together and the purified proteins bind single-strand DNA. Remarkably, LiBRCA2 is a recombination mediator that stimulates the invasion of a resected DNA double-strand break in an undamaged template by LiRAD51 to form a D-loop structure. Collectively, our data show that LiBRCA2 and LiRAD51 promote HR at the genetic and biochemical level in L. infantum, the causative agent of visceral leishmaniasis.
Asunto(s)
Proteína BRCA2/metabolismo , Recombinación Homóloga , Leishmania infantum/genética , Proteínas Protozoarias/metabolismo , Recombinasa Rad51/metabolismo , Proteína BRCA2/análisis , Proteína BRCA2/genética , Biología Computacional , ADN/metabolismo , Daño del ADN , Silenciador del Gen , Genes BRCA2 , Leishmania infantum/metabolismo , Fenotipo , Unión Proteica , Proteínas Protozoarias/análisis , Proteínas Protozoarias/genéticaRESUMEN
Antiviral DNA cytosine deaminases APOBEC3A and APOBEC3B are major sources of mutations in cancer by catalyzing cytosine-to-uracil deamination. APOBEC3A preferentially targets single-stranded DNAs, with a noted affinity for DNA regions that adopt stem-loop secondary structures. However, the detailed substrate preferences of APOBEC3A and APOBEC3B have not been fully established, and the specific influence of the DNA sequence on APOBEC3A and APOBEC3B deaminase activity remains to be investigated. Here, we find that APOBEC3B also selectively targets DNA stem-loop structures, and they are distinct from those subjected to deamination by APOBEC3A. We develop Oligo-seq, an in vitro sequencing-based method to identify specific sequence contexts promoting APOBEC3A and APOBEC3B activity. Through this approach, we demonstrate that APOBEC3A and APOBEC3B deaminase activity is strongly regulated by specific sequences surrounding the targeted cytosine. Moreover, we identify the structural features of APOBEC3B and APOBEC3A responsible for their substrate preferences. Importantly, we determine that APOBEC3B-induced mutations in hairpin-forming sequences within tumor genomes differ from the DNA stem-loop sequences mutated by APOBEC3A. Together, our study provides evidence that APOBEC3A and APOBEC3B can generate distinct mutation landscapes in cancer genomes, driven by their unique substrate selectivity.
Asunto(s)
Neoplasias , Proteínas , Humanos , Mutación , Neoplasias/genética , Citidina Desaminasa/genética , Citidina Desaminasa/química , ADN , Antígenos de Histocompatibilidad Menor/genética , Antígenos de Histocompatibilidad Menor/química , CitosinaRESUMEN
Circular RNAs are a novel class of RNA transcripts, which regulate important cellular functions in health and disease. Herein, we report on the functional relevance of the circPCMTD1 transcript in acute leukemias. In screening experiments, we found that circPCMTD1 depletion strongly inhibited the proliferative capacity of leukemic cells with BCR-ABL translocations. Mass cytometry experiments identified the aberrant activation of the DNA damage response as an early downstream event of circPCMTD1 depletion. In in vivo experiments, circPCMTD1 targeting prolonged the survival of mice engrafted with leukemic blasts harboring the Philadelphia chromosome. Mechanistically, we found that circPCMTD1 was enriched in the cytoplasm and associated with the ribosomes of the leukemic cells. We detected a cryptic open reading frame within the circPCMTD1 sequence and found that circPCMTD1 could generate a peptide product. The circPCMTD 1-derived peptide interacted with proteins of the BTR complex and enhanced BTR complex formation, thereby increasing tolerance to genotoxic stress.
RESUMEN
Two APOBEC (apolipoprotein-B mRNA editing enzyme catalytic polypeptide-like) DNA cytosine deaminase enzymes (APOBEC3A and APOBEC3B) generate somatic mutations in cancer, driving tumour development and drug resistance. Here we used single cell RNA sequencing to study APOBEC3A and APOBEC3B expression in healthy and malignant mucosal epithelia, validating key observations with immunohistochemistry, spatial transcriptomics and functional experiments. Whereas APOBEC3B is expressed in keratinocytes entering mitosis, we show that APOBEC3A expression is confined largely to terminally differentiating cells and requires Grainyhead-like transcription factor 3 (GRHL3). Thus, in normal tissue, neither deaminase appears to be expressed at high levels during DNA replication, the cell cycle stage associated with APOBEC-mediated mutagenesis. In contrast, we show that in squamous cell carcinoma tissues, there is expansion of GRHL3 expression and activity to a subset of cells undergoing DNA replication and concomitant extension of APOBEC3A expression to proliferating cells. These findings indicate a mechanism for acquisition of APOBEC3A mutagenic activity in tumours.
RESUMEN
The ability of the telomeric DNA-binding protein, TRF2, to stimulate t-loop formation while preventing t-loop deletion is believed to be crucial to maintain telomere integrity in mammals. However, little is known on the molecular mechanisms behind these properties of TRF2. In this report, we show that TRF2 greatly increases the rate of Holliday junction (HJ) formation and blocks the cleavage by various types of HJ resolving activities, including the newly identified human GEN1 protein. By using potassium permanganate probing and differential scanning calorimetry, we reveal that the basic domain of TRF2 induces structural changes to the junction. We propose that TRF2 contributes to t-loop stabilisation by stimulating HJ formation and by preventing resolvase cleavage. These findings provide novel insights into the interplay between telomere protection and homologous recombination and suggest a general model in which TRF2 maintains telomere integrity by controlling the turnover of HJ at t-loops and at regressed replication forks.
Asunto(s)
ADN Cruciforme/metabolismo , Telómero/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo , Bacterias/enzimología , Emparejamiento Base , Secuencia de Bases , Bioensayo , Histidina/metabolismo , Resolvasas de Unión Holliday/metabolismo , Humanos , Datos de Secuencia Molecular , Permanganato de Potasio/farmacología , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Recombinasas/metabolismo , Saccharomyces cerevisiae/enzimología , Proteína 2 de Unión a Repeticiones Teloméricas/químicaRESUMEN
Cancer is now the leading cause of mortality in France. It has been clearly demonstrated that mutations in the genetic information is the initiating event of cancer. DNA damage such as DNA double-strand breaks leads to genomic instability and cancer development. Cells can repair DNA double-strand breaks through several mechanisms. Nevertheless, only homologous recombination repair is faithful and repairs DNA without creating mutations. Here, we review the roles of PALB2 and BRCA2 in homologous recombination and genome stability.