RESUMEN
The tumour suppressor complex BRCA1-BARD1 functions in the repair of DNA double-stranded breaks by homologous recombination. During this process, BRCA1-BARD1 facilitates the nucleolytic resection of DNA ends to generate a single-stranded template for the recruitment of another tumour suppressor complex, BRCA2-PALB2, and the recombinase RAD51. Here, by examining purified wild-type and mutant BRCA1-BARD1, we show that both BRCA1 and BARD1 bind DNA and interact with RAD51, and that BRCA1-BARD1 enhances the recombinase activity of RAD51. Mechanistically, BRCA1-BARD1 promotes the assembly of the synaptic complex, an essential intermediate in RAD51-mediated DNA joint formation. We provide evidence that BRCA1 and BARD1 are indispensable for RAD51 stimulation. Notably, BRCA1-BARD1 mutants with weakened RAD51 interactions show compromised DNA joint formation and impaired mediation of homologous recombination and DNA repair in cells. Our results identify a late role of BRCA1-BARD1 in homologous recombination, an attribute of the tumour suppressor complex that could be targeted in cancer therapy.
Asunto(s)
Proteína BRCA1/metabolismo , Emparejamiento Base , Emparejamiento Cromosómico , Recombinasa Rad51/metabolismo , Reparación del ADN por Recombinación , Homología de Secuencia de Ácido Nucleico , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Secuencia de Aminoácidos , Proteína BRCA1/genética , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Proteína del Grupo de Complementación N de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación N de la Anemia de Fanconi/metabolismo , Genes BRCA1 , Genes BRCA2 , Humanos , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Mutación , Unión Proteica , Recombinasa Rad51/genética , Reparación del ADN por Recombinación/genética , Moldes Genéticos , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/genética , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
The tumor suppressor BRCA2 is thought to facilitate the handoff of ssDNA from replication protein A (RPA) to the RAD51 recombinase during DNA break and replication fork repair by homologous recombination. However, we find that RPA-RAD51 exchange requires the BRCA2 partner DSS1. Biochemical, structural, and in vivo analyses reveal that DSS1 allows the BRCA2-DSS1 complex to physically and functionally interact with RPA. Mechanistically, DSS1 acts as a DNA mimic to attenuate the affinity of RPA for ssDNA. A mutation in the solvent-exposed acidic domain of DSS1 compromises the efficacy of RPA-RAD51 exchange. Thus, by targeting RPA and mimicking DNA, DSS1 functions with BRCA2 in a two-component homologous recombination mediator complex in genome maintenance and tumor suppression. Our findings may provide a paradigm for understanding the roles of DSS1 in other biological processes.
Asunto(s)
Proteína BRCA2/metabolismo , Recombinación Homóloga , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteína de Replicación A/metabolismo , Sustitución de Aminoácidos , Proteína BRCA2/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/terapia , Línea Celular , Femenino , Células HeLa , Humanos , Modelos Biológicos , Imitación Molecular , Mutagénesis Sitio-Dirigida , Resonancia Magnética Nuclear Biomolecular , Complejo de la Endopetidasa Proteasomal/genética , Subunidades de Proteína , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína de Replicación A/química , Proteína de Replicación A/genéticaRESUMEN
BRCA2 is a multi-faceted protein critical for the proper regulation of homology-directed repair of DNA double-strand breaks. Elucidating the mechanistic features of BRCA2 is crucial for understanding homologous recombination and how patient-derived mutations impact future cancer risk. Eight centrally located BRC repeats in BRCA2 mediate binding and regulation of RAD51 on resected DNA substrates. Herein, we dissect the biochemical and cellular features of the BRC repeats tethered to the DNA binding domain of BRCA2. To understand how the BRC repeats and isolated domains of BRCA2 contribute to RAD51 binding, we analyzed both the biochemical and cellular properties of these proteins. In contrast to the individual BRC repeat units, we find that the BRC5-8 region potentiates RAD51-mediated DNA strand pairing and provides complementation functions exceeding those of BRC repeats 1-4. Furthermore, BRC5-8 can efficiently repair nuclease-induced DNA double-strand breaks and accelerate the assembly of RAD51 repair complexes upon DNA damage. These findings highlight the importance of the BRC5-8 domain in stabilizing the RAD51 filament and promoting homology-directed repair under conditions of cellular DNA damage.
Asunto(s)
Secuencias de Aminoácidos , Proteína BRCA2/metabolismo , Daño del ADN , Dominios y Motivos de Interacción de Proteínas , Recombinasa Rad51/metabolismo , Proteína BRCA2/química , Unión ProteicaRESUMEN
Mutations in PTEN-induced putative kinase 1 (PINK1) gene are associated to early-onset recessive forms of Parkinson disease. PINK1 function is related to mitochondria homeostasis, but the molecular pathways in which PINK1 is involved are largely unknown. Here, we report the identification of the embryonic ectoderm development polycomb histone-methylation modulator (EED/WAIT1) as a PINK1-interacting and -regulated protein. The PINK1:EED/WAIT1 physical interaction was mediated by the PINK1 kinase domain and the EED/WAIT1 40 amino acid ending with tryptophan and aspartate (WD40)-repeat region, and PINK1 phosphorylated EED/WAIT1 in vitro. PINK1 associated with EED/WAIT1 in cells and relocated EED/WAIT1 to the mitochondria. This interaction reduced the trimethylation of lysine 27 from histone H3, which affected polycomb-regulated gene transcription during RA differentiation of SH-SY5Y human neuroblastoma cells. Our findings unveil a pathway by which PINK1 regulates histone methylation and gene expression through the polycomb repressor complex.
Asunto(s)
Regulación de la Expresión Génica , Histonas/metabolismo , Complejo Represivo Polycomb 2/metabolismo , Proteínas Quinasas/metabolismo , Animales , Antineoplásicos/farmacología , Sitios de Unión/genética , Células COS , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Línea Celular Tumoral , Chlorocebus aethiops , Células HEK293 , Humanos , Immunoblotting , Lisina/metabolismo , Metilación , Mitocondrias/metabolismo , Neuroblastoma/genética , Neuroblastoma/patología , Fosforilación , Complejo Represivo Polycomb 2/genética , Unión Proteica , Proteínas Quinasas/genética , Transporte de Proteínas , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tretinoina/farmacología , Técnicas del Sistema de Dos HíbridosRESUMEN
BRCA2 is a tumor suppressor protein responsible for safeguarding the cellular genome from replication stress and genotoxicity, but the specific mechanism(s) by which this is achieved to prevent early oncogenesis remains unclear. Here, we provide evidence that BRCA2 acts as a critical suppressor of head-on transcription-replication conflicts (HO-TRCs). Using Okazaki-fragment sequencing (Ok-seq) and computational analysis, we identified origins (dormant origins) that are activated near the transcription termination sites (TTS) of highly expressed, long genes in response to replication stress. Dormant origins are a source for HO-TRCs, and drug treatments that inhibit dormant origin firing led to a reduction in HO-TRCs, R-loop formation, and DNA damage. Using super-resolution microscopy, we showed that HO-TRC events track with elongating RNA polymerase II, but not with transcription initiation. Importantly, RNase H2 is recruited to sites of HO-TRCs in a BRCA2-dependent manner to help alleviate toxic R-loops associated with HO-TRCs. Collectively, our results provide a mechanistic basis for how BRCA2 shields against genomic instability by preventing HO-TRCs through both direct and indirect means occurring at predetermined genomic sites based on the pre-cancer transcriptome.
Asunto(s)
Proteína BRCA2 , Replicación del ADN , ARN Polimerasa II , Ribonucleasa H , Humanos , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Ribonucleasa H/metabolismo , Ribonucleasa H/genética , ARN Polimerasa II/metabolismo , Transcripción Genética , Terminación de la Transcripción Genética , Daño del ADN , Origen de Réplica , Estructuras R-Loop , Línea Celular TumoralRESUMEN
The BRCA2 germline missense variant, R3052W, resides in the DNA binding domain and has been previously classified as a pathogenic allele. In this study, we sought to determine how R3052W alters the cellular functions of BRCA2 in the DNA damage response. The BRCA2 R3052W mutated protein exacerbates genome instability, is unable to rescue homology-directed repair, and fails to complement cell survival following exposure to PARP inhibitors and crosslinking drugs. Surprisingly, despite anticipated defects in DNA binding or RAD51-mediated DNA strand exchange, the BRCA2 R3052W protein mislocalizes to the cytoplasm precluding its ability to perform any DNA repair functions. Rather than acting as a simple loss-of-function mutation, R3052W behaves as a dominant negative allele, likely by sequestering RAD51 in the cytoplasm.
RESUMEN
Pathogenic mutations in the BRCA2 tumor suppressor gene predispose to breast, ovarian, pancreatic, prostate, and other cancers. BRCA2 maintains genome stability through homology-directed repair (HDR) of DNA double-strand breaks (DSBs) and replication fork protection. Nonsense or frameshift mutations leading to truncation of the BRCA2 protein are typically considered pathogenic; however, missense mutations resulting in single amino acid substitutions can be challenging to functionally interpret. The majority of missense mutations in BRCA2 have been classified as Variants of Uncertain Significance (VUS) with unknown functional consequences. In this study, we identified three BRCA2 VUS located within the BRC repeat region to determine their impact on canonical HDR and fork protection functions. We provide evidence that S1221P and T1980I, which map to conserved residues in the BRC2 and BRC7 repeats, compromise the cellular response to chemotherapeutics and ionizing radiation, and display deficits in fork protection. We further demonstrate biochemically that S1221P and T1980I disrupt RAD51 binding and diminish the ability of BRCA2 to stabilize RAD51-ssDNA complexes. The third variant, T1346I, located within the spacer region between BRC2 and BRC3 repeats, is fully functional. We conclude that T1346I is a benign allele, whereas S1221P and T1980I are hypomorphic disrupting the ability of BRCA2 to fully engage and stabilize RAD51 nucleoprotein filaments. Our results underscore the importance of correctly classifying BRCA2 VUS as pathogenic variants can impact both future cancer risk and guide therapy selection during cancer treatment.
Asunto(s)
Proteína BRCA2 , Recombinasa Rad51 , Proteína BRCA2/química , Reparación del ADN , ADN de Cadena Simple , Mutación Missense , Nucleoproteínas/metabolismo , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismoRESUMEN
DNA fiber combing is a versatile technique that provides insight into replication fork dynamics at single-molecule resolution. DNA fibers are bound to silanized coverslips and combed, which straightens and aligns the fibers along a single axis. Here, we present a DNA fiber combing protocol that does not use commercial kits; we detail the steps to prepare all materials, reagents, and silanized coverslips. We describe the use of DLD-1 cells, but the protocol is amenable to other cell types.
Asunto(s)
Replicación del ADN , ADN , Animales , Indicadores y Reactivos , Mamíferos/genéticaRESUMEN
Pathological mutations in homology-directed repair (HDR) genes impact both future cancer risk and therapeutic options for patients. HDR is a high-fidelity DNA repair pathway for resolving DNA double-strand breaks throughout the genome. BRCA2 is an essential protein that mediates the loading of RAD51 onto resected DNA breaks, a key step in HDR. Germline mutations in BRCA2 are associated with an increased risk for breast, ovarian, prostate, and pancreatic cancer. Clinical findings of germline or somatic BRCA2 mutations in tumors suggest treatment with platinum agents or PARP inhibitors. However, when genetic analysis reveals a variant of uncertain significance (VUS) in the BRCA2 gene, precision medicine-based decisions become complex. VUS are genetic changes with unknown pathological impact. Current statistics indicate that between 10-20% of BRCA sequencing results are VUS, and of these, more than 50% are missense mutations. Functional assays to determine the pathological outcome of VUS are urgently needed to provide clinical guidance regarding cancer risk and treatment options. In this review, we provide a brief overview of BRCA2 functions in HDR, describe how BRCA2 VUS are currently assessed in the clinic, and how genetic and biochemical functional assays could be integrated into the clinical decision process. We suggest a multi-step workflow composed of robust and accurate functional assays to correctly evaluate the potential pathogenic or benign nature of BRCA2 VUS. Success in this precision medicine endeavor will offer actionable information to patients and their physicians.
Asunto(s)
Proteína BRCA2/genética , Toma de Decisiones Clínicas/métodos , Pruebas Genéticas/métodos , Síndrome de Cáncer de Mama y Ovario Hereditario/genética , Proteína BRCA2/metabolismo , Femenino , Prueba de Complementación Genética/métodos , Síndrome de Cáncer de Mama y Ovario Hereditario/diagnóstico , Síndrome de Cáncer de Mama y Ovario Hereditario/terapia , Humanos , Mutación , Flujo de TrabajoRESUMEN
Homologous Recombination (HR) is a high-fidelity process with a range of biologic functions from generation of genetic diversity to repair of DNA double-strand breaks (DSBs). In mammalian cells, BRCA2 facilitates the polymerization of RAD51 onto ssDNA to form a presynaptic nucleoprotein filament. This filament can then strand invade a homologous dsDNA to form the displacement loop (D-loop) structure leading to the eventual DSB repair. Here, we have found that RAD51 in stoichiometric excess over ssDNA can cause D-loop disassembly in vitro; furthermore, we show that this RAD51 activity is countered by BRCA2. These results demonstrate that BRCA2 may have a previously unexpected activity: regulation of HR at a post-synaptic stage by modulating RAD51-mediated D-loop dissociation. Our in vitro results suggest a mechanistic underpinning of homeostasis between RAD51 and BRCA2, which is an important factor of HR in mammalian cells.
Asunto(s)
Proteína BRCA2/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN , ADN/metabolismo , Recombinación Homóloga , Recombinasa Rad51/metabolismo , Proteína BRCA2/química , ADN/química , Humanos , Conformación de Ácido Nucleico , Recombinasa Rad51/químicaRESUMEN
DNA double-strand break repair by homologous recombination entails the resection of DNA ends to reveal ssDNA tails, which are used to invade a homologous DNA template. CtIP and its yeast ortholog Sae2 regulate the nuclease activity of MRE11 in the initial stage of resection. Deletion of CtIP in the mouse or SAE2 in yeast engenders a more severe phenotype than MRE11 nuclease inactivation, indicative of a broader role of CtIP/Sae2. Here, we provide biochemical evidence that CtIP promotes long-range resection via the BLM-DNA2 pathway. Specifically, CtIP interacts with BLM and enhances its helicase activity, and it enhances DNA cleavage by DNA2. Thus, CtIP influences multiple aspects of end resection beyond MRE11 regulation.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Reparación del ADN por Recombinación , Animales , Proteínas Portadoras/genética , Proteínas de Ciclo Celular/genética , ADN Helicasas/genética , ADN Helicasas/metabolismo , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Células HEK293 , Humanos , Proteína Homóloga de MRE11/genética , Proteína Homóloga de MRE11/metabolismo , Ratones , Enzimas Multifuncionales/genética , Enzimas Multifuncionales/metabolismo , Unión Proteica , Multimerización de Proteína , RecQ Helicasas/genética , RecQ Helicasas/metabolismo , Células Sf9 , Spodoptera , Regulación hacia ArribaRESUMEN
The PINK1 gene is mutated in the germ line of patients with hereditary early-onset Parkinson disease, and PINK1 prosurvival function at neuronal mitochondria has been related with the etiology of this disease. However, the expression and function of PINK1 protein in nonneuronal tissues has not been determined yet. Here, we have analyzed PINK1 protein expression and subcellular distribution in normal and neoplastic human tissues and investigated the function of PINK1 in breast carcinoma cells. PINK1 protein, as stained by a specific anti-PINK1 monoclonal antibody, was widely expressed in human tissues, displaying high expression in epithelial tissues and in the central nervous system and lower expression in tissues of mesenchymal origin. The subcellular distribution of PINK1 was cytoplasmic granular or cytoplasmic diffuse in most tissues. In breast, PINK1 was also associated with the plasma membrane. Human neoplastic tissues ranged from high PINK1 expression in carcinomas to low expression in sarcomas. In neoplastic tissues, PINK1 displayed a diffuse cytoplasmic localization, with an additional membranous localization in breast carcinoma and squamous carcinoma of lung. In the human breast carcinoma Michigan Cancer Foundation-7 cell line, ectopic expression of cytoplasmic or mitochondrial-targeted PINK1 inhibited apoptosis triggered by hydrogen peroxide and suppressed cell growth in soft agar, whereas PINK1 silencing increased hydrogen peroxide-induced apoptosis. Together, our findings indicate that the physiologic functions of PINK1 go beyond its regulatory role of mitochondria-mediated cell survival in neurons.