RESUMEN

DNA double strand breaks (DSBs) are critical for the efficacy of radiotherapy as they lead to cell death if not repaired. DSBs caused by ionizing radiation (IR) initiate histone modifications and accumulate DNA repair proteins, including 53BP1, which forms distinct foci at damage sites and serves as a marker for DSBs. DSB repair primarily occurs through Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR). NHEJ directly ligates DNA ends, employing proteins such as DNA-PKcs, while HR, involving proteins such as Rad54, uses a sister chromatid template for accurate repair and functions in the S and G2 phases of the cell cycle. Both pathways are crucial, as illustrated by the IR sensitivity in cells lacking DNA-PKcs or Rad54. We generated mouse embryonic stem (mES) cells which are knockout (KO) for DNA-PKcs and Rad54 to explore the combined role of HR and NHEJ in DSB repair. We found that cells lacking both DNA-PKcs and Rad54 are hypersensitive to X-ray radiation, coinciding with impaired 53BP1 focus resolution and a more persistent G2 phase cell cycle block. Additionally, mES cells deficient in DNA-PKcs or both DNA-PKcs and Rad54 exhibit an increased nuclear size approximately 18-24 h post-irradiation. To further explore the role of Rad54 in the absence of DNA-PKcs, we generated DNA-PKcs KO mES cells expressing GFP-tagged wild-type (WT) or ATPase-defective Rad54 to track the Rad54 foci over time post-irradiation. Cells lacking DNA-PKcs and expressing ATPase-defective Rad54 exhibited a similar phenotypic response to IR as those lacking both DNA-PKcs and Rad54. Despite a strong G2 phase arrest, live-cell imaging showed these cells eventually progress through mitosis, forming micronuclei. Additionally, mES cells lacking DNA-PKcs showed increased Rad54 foci over time post-irradiation, indicating an enhanced reliance on HR for DSB repair without DNA-PKcs. Our findings underscore the essential roles of HR and NHEJ in maintaining genomic stability post-IR in mES cells. The interplay between these pathways is crucial for effective DSB repair and cell cycle progression, highlighting potential targets for enhancing radiotherapy outcomes.

Asunto(s)

Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Recombinación Homóloga , Células Madre Embrionarias de Ratones , Radiación Ionizante , Animales , Ratones , Reparación del ADN por Unión de Extremidades/efectos de la radiación , Roturas del ADN de Doble Cadena/efectos de la radiación , Células Madre Embrionarias de Ratones/metabolismo , Células Madre Embrionarias de Ratones/efectos de la radiación , Células Madre Embrionarias de Ratones/citología , Recombinación Homóloga/efectos de la radiación , Proteína Quinasa Activada por ADN/metabolismo , Proteína Quinasa Activada por ADN/genética , ADN Helicasas/metabolismo , ADN Helicasas/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/genética , Proteínas Nucleares

RESUMEN

53BP1 is a well-established DNA damage repair factor that has recently emerged to critically regulate gene expression for tumor suppression and neural development. However, its precise function and regulatory mechanisms remain unclear. Here, we showed that phosphorylation of 53BP1 at serine 25 by ATM is required for neural progenitor cell proliferation and neuronal differentiation in cortical brain organoids. Dynamic phosphorylation of 53BP1-serine 25 controls 53BP1 target genes governing neuronal differentiation and function, cellular response to stress, and apoptosis. Mechanistically, ATM and RNF168 govern 53BP1's binding to gene loci to directly affect gene regulation, especially at genes for neuronal differentiation and maturation. 53BP1 serine 25 phosphorylation effectively impedes its binding to bivalent or H3K27me3-occupied promoters, especially at genes regulating H3K4 methylation, neuronal functions, and cell proliferation. Beyond 53BP1, ATM-dependent phosphorylation displays wide-ranging effects, regulating factors in neuronal differentiation, cytoskeleton, p53 regulation, as well as key signaling pathways such as ATM, BDNF, and WNT during cortical organoid differentiation. Together, our data suggest that the interplay between 53BP1 and ATM orchestrates essential genetic programs for cell morphogenesis, tissue organization, and developmental pathways crucial for human cortical development.

Asunto(s)

Proteínas de la Ataxia Telangiectasia Mutada , Organoides , Proteína 1 de Unión al Supresor Tumoral P53 , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/genética , Organoides/metabolismo , Humanos , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/genética , Fosforilación , Daño del ADN , Corteza Cerebral/metabolismo , Corteza Cerebral/citología , Células-Madre Neurales/metabolismo , Diferenciación Celular/genética , Proliferación Celular , Reparación del ADN , Neurogénesis/genética , Neuronas/metabolismo , Transducción de Señal

RESUMEN

Osimertinib is a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (EGFR-TKI) approved for patients with EGFR T790M resistance mutations as first- or second-line treatment of EGFR-positive patients. Resistance to Osimertinib will inevitably develop, and the underlying mechanisms are largely unknown. In this study, we discovered that acquired resistance to Osimertinib is associated with abnormal DNA damage response (DDR) in lung adenocarcinoma cells. We discovered that the polycomb protein Lethal(3) Malignant Brain Tumor-Like Protein 1 (L3MBTL1) regulates chromatin structure, thereby contributing to DDR and Osimertinib resistance. EGFR oncogene inhibition reduced L3MBTL1 ubiquitination while stabilizing its expression in Osimertinib-resistant cells. L3MBTL1 reduction and treatment with Osimertinib significantly inhibited DDR and proliferation of Osimertinib-resistant lung cancer cells in vitro and in vivo. L3MBTL1 binds throughout the genome and plays an important role in EGFR-TKI resistance. It also competes with 53BP1 for H4K20Me2 and inhibits the development of drug resistance in Osimertinib-resistant lung cancer cells in vitro and in vivo. Our findings suggest that L3MBTL1 inhibition is a novel approach to overcoming EGFR-TKI-acquired resistance.

Asunto(s)

Acrilamidas , Adenocarcinoma del Pulmón , Compuestos de Anilina , Daño del ADN , Resistencia a Antineoplásicos , Epigénesis Genética , Receptores ErbB , Neoplasias Pulmonares , Humanos , Acrilamidas/farmacología , Resistencia a Antineoplásicos/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Compuestos de Anilina/farmacología , Compuestos de Anilina/uso terapéutico , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/tratamiento farmacológico , Adenocarcinoma del Pulmón/patología , Adenocarcinoma del Pulmón/metabolismo , Daño del ADN/efectos de los fármacos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/metabolismo , Epigénesis Genética/efectos de los fármacos , Animales , Línea Celular Tumoral , Receptores ErbB/metabolismo , Receptores ErbB/genética , Ratones , Proteínas del Grupo Polycomb/metabolismo , Proteínas del Grupo Polycomb/genética , Ratones Desnudos , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/genética , Inhibidores de Proteínas Quinasas/farmacología , Proliferación Celular/efectos de los fármacos , Ubiquitinación/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Indoles , Pirimidinas

RESUMEN

Although previous studies have reported that pre-mRNA splicing factors (SFs) are involved in the repair of DNA double-strand breaks (DSBs) via homologous recombination (HR), their exact role in promoting HR remains poorly understood. Here, we showed that SART1, an SF upregulated in several types of cancer, promotes DSB end resection, an essential first step of HR. The resection-promoting function of SART1 requires phosphorylation at threonine 430 and 695 by ATM/ATR. SART1 is recruited to DSB sites in a manner dependent on transcription and its RS domain. SART1 is epistatic with BRCA1, a major HR factor, in the promotion of resection, especially transcription-associated resection in the G2 phase. SART1 and BRCA1 accumulate at DSB sites in an interdependent manner, and epistatically counteract the resection blockade posed by 53BP1 and RIF1. Furthermore, chromosome analysis demonstrated that SART1 and BRCA1 epistatically suppressed genomic alterations caused by DSB misrepair in the G2 phase. Collectively, these results indicate that SART1 and BRCA1 cooperatively facilitate resection of DSBs arising in transcriptionally active genomic regions in the G2 phase, thereby promoting faithful repair by HR, and suppressing genome instability.

Asunto(s)

Proteína BRCA1 , Roturas del ADN de Doble Cadena , Reparación del ADN por Recombinación , Proteína BRCA1/metabolismo , Proteína BRCA1/genética , Humanos , Factores de Empalme Serina-Arginina/metabolismo , Factores de Empalme Serina-Arginina/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/genética , Fosforilación , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/genética , Línea Celular Tumoral , Proteínas de Unión a Telómeros/metabolismo , Proteínas de Unión a Telómeros/genética , Epistasis Genética , Fase G2/genética

RESUMEN

53BP1 nucleates the anti-end resection machinery at DNA double-strand breaks, thereby countering BRCA1 activity. Loss of 53BP1 leads to DNA end processing and homologous recombination in BRCA1-deficient cells. Consequently, BRCA1-mutant tumors, typically sensitive to PARP inhibitors (PARPi), become resistant in the absence of 53BP1. Here, we demonstrate that the 'leaky' DNA end resection in the absence of 53BP1 results in increased micronuclei and cytoplasmic double-stranded DNA, leading to activation of the cGAS-STING pathway and pro-inflammatory signaling. This enhances CD8+ T cell infiltration, activates macrophages and natural killer cells, and impedes tumor growth. Loss of 53BP1 correlates with a response to immune checkpoint blockade (ICB) and improved overall survival. Immunohistochemical assessment of 53BP1 in two malignancies, high grade serous ovarian cancer and pancreatic ductal adenocarcinoma, which are refractory to ICBs, reveals that lower 53BP1 levels correlate with an increased adaptive and innate immune response. Finally, BRCA1-deficient tumors that develop resistance to PARPi due to the loss of 53BP1 are susceptible to ICB. Therefore, we conclude that 53BP1 is critical for tumor immunogenicity and underpins the response to ICB. Our results support including 53BP1 expression as an exploratory biomarker in ICB trials for malignancies typically refractory to immunotherapy.

Asunto(s)

Proteínas de la Membrana , Nucleotidiltransferasas , Neoplasias Ováricas , Neoplasias Pancreáticas , Proteína 1 de Unión al Supresor Tumoral P53 , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/genética , Femenino , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Humanos , Animales , Neoplasias Ováricas/inmunología , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Neoplasias Ováricas/metabolismo , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/metabolismo , Ratones , Línea Celular Tumoral , Roturas del ADN de Doble Cadena , Proteína BRCA1/metabolismo , Proteína BRCA1/genética , Transducción de Señal , Linfocitos T CD8-positivos/inmunología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Ratones Endogámicos C57BL , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Ratones Noqueados , Carcinoma Ductal Pancreático/inmunología , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Inmunidad Innata

RESUMEN

DNA double-strand breaks (DSBs) elicit an elaborate response to signal damage and trigger repair via two major pathways: nonhomologous end-joining (NHEJ), which functions throughout the interphase, and homologous recombination (HR), restricted to S/G2 phases. The DNA damage response relies, on post-translational modifications of nuclear factors to coordinate the mending of breaks. Ubiquitylation of histones and chromatin-associated factors regulates DSB repair and numerous E3 ubiquitin ligases are involved in this process. Despite significant progress, our understanding of ubiquitin-mediated DNA damage response regulation remains incomplete. Here, we have performed a localization screen to identify RING/U-box E3 ligases involved in genome maintenance. Our approach uncovered 7 novel E3 ligases that are recruited to microirradiation stripes, suggesting potential roles in DNA damage signaling and repair. Among these factors, the DELTEX family E3 ligase DTX2 is rapidly mobilized to lesions in a poly ADP-ribosylation-dependent manner. DTX2 is recruited and retained at DSBs via its WWE and DELTEX conserved C-terminal domains. In cells, both domains are required for optimal binding to mono and poly ADP-ribosylated proteins with WWEs playing a prominent role in this process. Supporting its involvement in DSB repair, DTX2 depletion decreases HR efficiency and moderately enhances NHEJ. Furthermore, DTX2 depletion impeded BRCA1 foci formation and increased 53BP1 accumulation at DSBs, suggesting a fine-tuning role for this E3 ligase in repair pathway choice. Finally, DTX2 depletion sensitized cancer cells to X-rays and PARP inhibition and these susceptibilities could be rescued by DTX2 reexpression. Altogether, our work identifies DTX2 as a novel ADP-ribosylation-dependent regulator of HR-mediated DSB repair.

Asunto(s)

Roturas del ADN de Doble Cadena , Ubiquitina-Proteína Ligasas , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Humanos , ADP-Ribosilación , Reparación del ADN , Reparación del ADN por Unión de Extremidades , Proteína BRCA1/metabolismo , Proteína BRCA1/genética , Ubiquitinación , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/genética

RESUMEN

G0-G1 phase alternative end joining (A-EJ) is a recently defined mutagenic pathway characterized by resected deletion and translocation joints that are predominantly direct and are distinguished from A-EJ in cycling cells that rely much more on microhomology-mediated end joining (MMEJ). Using chemical and genetic approaches, we systematically evaluate potential A-EJ factors and DNA damage response (DDR) genes to support this mechanism by mapping the repair fates of RAG1/2-initiated double-strand breaks in the context of Igκ locus V-J recombination and chromosome translocation. Our findings highlight a polymerase theta-independent Parp1-XRCC1/LigIII axis as central A-EJ components, supported by 53BP1 in the context of an Ataxia-telangiectasia mutated (ATM)-activated DDR. Mechanistically, we demonstrate varied changes in short-range resection, MMEJ, and translocation, imposed by compromising specific DDR activities, which include polymerase alpha, Ataxia-telangiectasia and Rad3-related (ATR), DNA2, and Mre11. This study advances our understanding of DNA damage repair within the 53BP1 regulatory domain and the RAG1/2 postcleavage complex.

Asunto(s)

Proteínas de la Ataxia Telangiectasia Mutada , Reparación del ADN por Unión de Extremidades , Proteína 1 de Unión al Supresor Tumoral P53 , Recombinación V(D)J , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/genética , Animales , Humanos , Roturas del ADN de Doble Cadena , Ratones , Daño del ADN , Translocación Genética

RESUMEN

Mitotic errors generate micronuclei entrapping mis-segregated chromosomes, which are susceptible to catastrophic fragmentation through chromothripsis. The reassembly of fragmented chromosomes by error-prone DNA double-strand break (DSB) repair generates diverse genomic rearrangements associated with human diseases. How specific repair pathways recognize and process these lesions remains poorly understood. Here we use CRISPR/Cas9 to systematically inactivate distinct DSB repair pathways and interrogate the rearrangement landscape of fragmented chromosomes. Deletion of canonical non-homologous end joining (NHEJ) components substantially reduces complex rearrangements and shifts the rearrangement landscape toward simple alterations without the characteristic patterns of chromothripsis. Following reincorporation into the nucleus, fragmented chromosomes localize within sub-nuclear micronuclei bodies (MN bodies) and undergo ligation by NHEJ within a single cell cycle. In the absence of NHEJ, chromosome fragments are rarely engaged by alternative end-joining or recombination-based mechanisms, resulting in delayed repair kinetics, persistent 53BP1-labeled MN bodies, and cell cycle arrest. Thus, we provide evidence supporting NHEJ as the exclusive DSB repair pathway generating complex rearrangements from mitotic errors.

Asunto(s)

Sistemas CRISPR-Cas , Cromotripsis , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Mitosis , Mitosis/genética , Humanos , Reordenamiento Génico , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/genética , Micronúcleos con Defecto Cromosómico

RESUMEN

Poly(ADP-ribose) polymerase inhibitors (PARPis) exhibit remarkable anticancer activity in tumors with homologous recombination (HR) gene mutations. However, the role of other DNA repair proteins in PARPi-induced lethality remains elusive. Here, we reveal that FANCM promotes PARPi resistance independent of the core Fanconi anemia (FA) complex. FANCM-depleted cells retain HR proficiency, acting independently of BRCA1 in response to PARPis. FANCM depletion leads to increased DNA damage in the second S phase after PARPi exposure, driven by elevated single-strand DNA (ssDNA) gap formation behind replication forks in the first S phase. These gaps arise from both 53BP1- and primase and DNA directed polymerase (PRIMPOL)-dependent mechanisms. Notably, FANCM-depleted cells also exhibit reduced resection of collapsed forks, while 53BP1 deletion restores resection and mitigates PARPi sensitivity. Our results suggest that FANCM counteracts 53BP1 to repair PARPi-induced DNA damage. Furthermore, FANCM depletion leads to increased chromatin bridges and micronuclei formation after PARPi treatment, elucidating the mechanism underlying extensive cell death in FANCM-depleted cells.

Asunto(s)

ADN de Cadena Simple , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Proteína 1 de Unión al Supresor Tumoral P53 , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Humanos , ADN de Cadena Simple/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/genética , ADN Helicasas/metabolismo , ADN Helicasas/genética , Daño del ADN , Reparación del ADN/efectos de los fármacos , Recombinación Homóloga/efectos de los fármacos , Proteína BRCA1/metabolismo , Proteína BRCA1/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Línea Celular Tumoral

RESUMEN

Rapid accumulation of repair factors at DNA double-strand breaks (DSBs) is essential for DSB repair. Several factors involved in DSB repair have been found undergoing liquid-liquid phase separation (LLPS) at DSB sites to facilitate DNA repair. RNF168, a RING-type E3 ubiquitin ligase, catalyzes H2A.X ubiquitination for recruiting DNA repair factors. Yet, whether RNF168 undergoes LLPS at DSB sites remains unclear. Here, we identified K63-linked polyubiquitin-triggered RNF168 condensation which further promoted RNF168-mediated DSB repair. RNF168 formed liquid-like condensates upon irradiation in the nucleus while purified RNF168 protein also condensed in vitro. An intrinsically disordered region containing amino acids 460-550 was identified as the essential domain for RNF168 condensation. Interestingly, LLPS of RNF168 was significantly enhanced by K63-linked polyubiquitin chains, and LLPS largely enhanced the RNF168-mediated H2A.X ubiquitination, suggesting a positive feedback loop to facilitate RNF168 rapid accumulation and its catalytic activity. Functionally, LLPS deficiency of RNF168 resulted in delayed recruitment of 53BP1 and BRCA1 and subsequent impairment in DSB repair. Taken together, our finding demonstrates the pivotal effect of LLPS in RNF168-mediated DSB repair.

Asunto(s)

Reparación del ADN , Ubiquitina-Proteína Ligasas , Humanos , Roturas del ADN de Doble Cadena , Histonas/metabolismo , Histonas/genética , Poliubiquitina/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación

RESUMEN

RAD18 is an E3 ubiquitin ligase that prevents replication fork collapse by promoting DNA translesion synthesis and template switching. Besides this classical role, RAD18 has been implicated in homologous recombination; however, this function is incompletely understood. Here, we show that RAD18 is recruited to DNA lesions by monoubiquitination of histone H2A at K15 and counteracts accumulation of 53BP1. Super-resolution microscopy revealed that RAD18 localizes to the proximity of DNA double strand breaks and limits the distribution of 53BP1 to the peripheral chromatin nanodomains. Whereas auto-ubiquitination of RAD18 mediated by RAD6 inhibits its recruitment to DNA breaks, interaction with SLF1 promotes RAD18 accumulation at DNA breaks in the post-replicative chromatin by recognition of histone H4K20me0. Surprisingly, suppression of 53BP1 function by RAD18 is not involved in homologous recombination and rather leads to reduction of non-homologous end joining. Instead, we provide evidence that RAD18 promotes HR repair by recruiting the SMC5/6 complex to DNA breaks. Finally, we identified several new loss-of-function mutations in RAD18 in cancer patients suggesting that RAD18 could be involved in cancer development.

Asunto(s)

Cromatina , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN , Histonas , Proteína 1 de Unión al Supresor Tumoral P53 , Ubiquitina-Proteína Ligasas , Ubiquitinación , Humanos , Cromatina/metabolismo , Cromatina/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/genética , Histonas/metabolismo , Recombinación Homóloga/genética , Reparación del ADN por Recombinación , Replicación del ADN , Reparación del ADN , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Reparación del ADN por Unión de Extremidades

RESUMEN

Maintenance of genome integrity requires tight control of DNA damage response (DDR) signalling and repair, with phosphorylation and ubiquitination representing key elements. How these events are coordinated to achieve productive DNA repair remains elusive. Here we identify the ubiquitin-conjugating enzyme UBE2D3 as a regulator of ATM kinase-induced DDR that promotes non-homologous end-joining (NHEJ) at telomeres. UBE2D3 contributes to DDR-induced chromatin ubiquitination and recruitment of the NHEJ-promoting factor 53BP1, both mediated by RNF168 upon ATM activation. Additionally, UBE2D3 promotes NHEJ by limiting RNF168 accumulation and facilitating ATM-mediated phosphorylation of KAP1-S824. Mechanistically, defective KAP1-S824 phosphorylation and telomeric NHEJ upon UBE2D3-deficiency are linked to RNF168 hyperaccumulation and aberrant PP2A phosphatase activity. Together, our results identify UBE2D3 as a multi-level regulator of NHEJ that orchestrates ATM and RNF168 activities. Moreover, they reveal a negative regulatory circuit in the DDR that is constrained by UBE2D3 and consists of RNF168- and phosphatase-mediated restriction of KAP1 phosphorylation.

Asunto(s)

Proteínas de la Ataxia Telangiectasia Mutada , Reparación del ADN por Unión de Extremidades , Transducción de Señal , Proteína 28 que Contiene Motivos Tripartito , Proteína 1 de Unión al Supresor Tumoral P53 , Enzimas Ubiquitina-Conjugadoras , Ubiquitina-Proteína Ligasas , Ubiquitinación , Enzimas Ubiquitina-Conjugadoras/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/genética , Humanos , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Fosforilación , Proteína 28 que Contiene Motivos Tripartito/metabolismo , Proteína 28 que Contiene Motivos Tripartito/genética , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/genética , Células HEK293 , Telómero/metabolismo , Daño del ADN , Cromatina/metabolismo , Animales

RESUMEN

The master DNA damage repair histone protein, H2AX, is essential for orchestrating the recruitment of downstream mediator and effector proteins at damaged chromatin. The phosphorylation of H2AX at S139, γH2AX, is well-studied for its DNA repair function. However, the extended C-terminal tail is not characterized. Here, we define the minimal motif on H2AX for the canonical function in activating the MDC1-RNF8-RNF168 phosphorylation-ubiquitination pathway that is important for recruiting repair proteins, such as 53BP1 and BRCA1. Interestingly, H2AX recruits 53BP1 independently from the MDC1-RNF8-RNF168 pathway through its evolved C-terminal linker region with S139 phosphorylation. Mechanistically, 53BP1 recruitment to damaged chromatin is mediated by the interaction between the H2AX C-terminal tail and the 53BP1 Oligomerization-Tudor domains. Moreover, γH2AX-linker mediated 53BP1 recruitment leads to camptothecin resistance in H2AX knockout cells. Overall, our study uncovers an evolved mechanism within the H2AX C-terminal tail for regulating DNA repair proteins at damaged chromatin.

Asunto(s)

Cromatina , Daño del ADN , Histonas , Proteína 1 de Unión al Supresor Tumoral P53 , Ubiquitinación , Humanos , Proteínas Adaptadoras Transductoras de Señales , Proteína BRCA1/metabolismo , Proteína BRCA1/genética , Camptotecina/farmacología , Proteínas de Ciclo Celular , Cromatina/metabolismo , Reparación del ADN , Células HEK293 , Histonas/metabolismo , Histonas/genética , Fosforilación , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética

RESUMEN

Histone H2AX plays a key role in DNA damage signalling in the surrounding regions of DNA double-strand breaks (DSBs). In response to DNA damage, H2AX becomes phosphorylated on serine residue 139 (known as γH2AX), resulting in the recruitment of the DNA repair effectors 53BP1 and BRCA1. Here, by studying resistance to poly(ADP-ribose) polymerase (PARP) inhibitors in BRCA1/2-deficient mammary tumours, we identify a function for γH2AX in orchestrating drug-induced replication fork degradation. Mechanistically, γH2AX-driven replication fork degradation is elicited by suppressing CtIP-mediated fork protection. As a result, H2AX loss restores replication fork stability and increases chemoresistance in BRCA1/2-deficient tumour cells without restoring homology-directed DNA repair, as highlighted by the lack of DNA damage-induced RAD51 foci. Furthermore, in the attempt to discover acquired genetic vulnerabilities, we find that ATM but not ATR inhibition overcomes PARP inhibitor (PARPi) resistance in H2AX-deficient tumours by interfering with CtIP-mediated fork protection. In summary, our results demonstrate a role for H2AX in replication fork biology in BRCA-deficient tumours and establish a function of H2AX separable from its classical role in DNA damage signalling and DSB repair.

Asunto(s)

Proteína BRCA1 , Proteína BRCA2 , Replicación del ADN , Resistencia a Antineoplásicos , Histonas , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Animales , Femenino , Humanos , Ratones , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteína BRCA1/metabolismo , Proteína BRCA1/deficiencia , Proteína BRCA1/genética , Proteína BRCA2/metabolismo , Proteína BRCA2/genética , Proteína BRCA2/deficiencia , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Neoplasias de la Mama/tratamiento farmacológico , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Línea Celular Tumoral , Roturas del ADN de Doble Cadena , Daño del ADN , Reparación del ADN , Replicación del ADN/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Histonas/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Recombinasa Rad51/metabolismo , Recombinasa Rad51/genética , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/genética , Ratones Desnudos

RESUMEN

Mitotic duration is tightly constrained, and extended mitosis is characteristic of problematic cells prone to chromosome missegregation and genomic instability. We show here that mitotic extension leads to the formation of p53-binding protein 1 (53BP1)-ubiquitin-specific protease 28 (USP28)-p53 protein complexes that are transmitted to, and stably retained by, daughter cells. Complexes assembled through a Polo-like kinase 1-dependent mechanism during extended mitosis and elicited a p53 response in G1 that prevented the proliferation of the progeny of cells that experienced an approximately threefold extended mitosis or successive less extended mitoses. The ability to monitor mitotic extension was lost in p53-mutant cancers and some p53-wild-type (p53-WT) cancers, consistent with classification of TP53BP1 and USP28 as tumor suppressors. Cancers retaining the ability to monitor mitotic extension exhibited sensitivity to antimitotic agents.

Asunto(s)

Proliferación Celular , Mitosis , Neoplasias , Proteína 1 de Unión al Supresor Tumoral P53 , Ubiquitina Tiolesterasa , Humanos , Proliferación Celular/genética , Inestabilidad Genómica , Mitosis/efectos de los fármacos , Mitosis/genética , Neoplasias/genética , Neoplasias/patología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina Tiolesterasa/genética , Ubiquitina Tiolesterasa/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/genética , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Línea Celular Tumoral , Quinasa Tipo Polo 1/metabolismo , Antimitóticos/farmacología , Resistencia a Antineoplásicos

RESUMEN

Reprogramming to pluripotency is associated with DNA damage and requires the functions of the BRCA1 tumor suppressor. Here, we leverage separation-of-function mutations in BRCA1/2 as well as the physical and/or genetic interactions between BRCA1 and its associated repair proteins to ascertain the relevance of homology-directed repair (HDR), stalled fork protection (SFP), and replication gap suppression (RGS) in somatic cell reprogramming. Surprisingly, loss of SFP and RGS is inconsequential for the transition to pluripotency. In contrast, cells deficient in HDR, but proficient in SFP and RGS, reprogram with reduced efficiency. Conversely, the restoration of HDR function through inactivation of 53bp1 rescues reprogramming in Brca1-deficient cells, and 53bp1 loss leads to elevated HDR and enhanced reprogramming in mouse and human cells. These results demonstrate that somatic cell reprogramming is especially dependent on repair of replication-associated double-strand breaks (DSBs) by the HDR activity of BRCA1 and BRCA2 and can be improved in the absence of 53BP1.

Asunto(s)

Proteína BRCA1 , Reprogramación Celular , Roturas del ADN de Doble Cadena , Reparación del ADN , Proteína 1 de Unión al Supresor Tumoral P53 , Animales , Humanos , Ratones , Proteína BRCA1/metabolismo , Proteína BRCA1/genética , Replicación del ADN , Reparación del ADN por Recombinación , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/genética

RESUMEN

RNF168 plays a central role in the DNA damage response (DDR) by ubiquitylating histone H2A at K13 and K15. These modifications direct BRCA1-BARD1 and 53BP1 foci formation in chromatin, essential for cell-cycle-dependent DNA double-strand break (DSB) repair pathway selection. The mechanism by which RNF168 catalyzes the targeted accumulation of H2A ubiquitin conjugates to form repair foci around DSBs remains unclear. Here, using cryoelectron microscopy (cryo-EM), nuclear magnetic resonance (NMR) spectroscopy, and functional assays, we provide a molecular description of the reaction cycle and dynamics of RNF168 as it modifies the nucleosome and recognizes its ubiquitylation products. We demonstrate an interaction of a canonical ubiquitin-binding domain within full-length RNF168, which not only engages ubiquitin but also the nucleosome surface, clarifying how such site-specific ubiquitin recognition propels a signal amplification loop. Beyond offering mechanistic insights into a key DDR protein, our study aids in understanding site specificity in both generating and interpreting chromatin ubiquitylation.

Asunto(s)

Nucleosomas , Ubiquitina-Proteína Ligasas , Nucleosomas/genética , Microscopía por Crioelectrón , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Histonas/metabolismo , Cromatina/genética , Reparación del ADN , Ubiquitina/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/genética , Daño del ADN

RESUMEN

Methyl-lysine reader p53 binding protein 1 (53BP1) is a central mediator of DNA break repair and is associated with various human diseases, including cancer. Thus, high-quality 53BP1 chemical probes can aid in further understanding the role of 53BP1 in genome repair pathways. Herein, we utilized focused DNA-encoded library screening to identify the novel hit compound UNC8531, which binds the 53BP1 tandem Tudor domain (TTD) with an IC50 of 0.47 ± 0.09 µM in a TR-FRET assay and Kd values of 0.85 ± 0.17 and 0.79 ± 0.52 µM in ITC and SPR, respectively. UNC8531 was cocrystallized with the 53BP1 TTD to guide further optimization efforts, leading to UNC9512. NanoBRET and 53BP1-dependent foci formation experiments confirmed cellular target engagement. These results show that UNC9512 is a best-in-class small molecule 53BP1 antagonist that can aid further studies investigating the role of 53BP1 in DNA repair, gene editing, and oncogenesis.

Asunto(s)

Reparación del ADN , Péptidos y Proteínas de Señalización Intracelular , Humanos , ADN , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/química , Proteína 1 de Unión al Supresor Tumoral P53/genética , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Dominio Tudor

RESUMEN

Repair of DSB induced by IR is primarily carried out by Non-Homologous End Joining (NHEJ), a pathway in which 53BP1 plays a key role. We have discovered that the EMT-inducing transcriptional repressor ZEB1 (i) interacts with 53BP1 and that this interaction occurs rapidly and is significantly amplified following exposure of cells to IR; (ii) is required for the localization of 53BP1 to a subset of double-stranded breaks, and for physiological DSB repair; (iii) co-localizes with 53BP1 at IR-induced foci (IRIF); (iv) promotes NHEJ and inhibits Homologous Recombination (HR); (v) depletion increases resection at DSBs and (vi) confers PARP inhibitor (PARPi) sensitivity on BRCA1-deficient cells. Lastly, ZEB1's effects on repair pathway choice, resection, and PARPi sensitivity all rely on its homeodomain. In contrast to the well-characterized therapeutic resistance of high ZEB1-expressing cancer cells, the novel ZEB1-53BP1-shieldin resection axis described here exposes a therapeutic vulnerability: ZEB1 levels in BRCA1-deficient tumors may serve as a predictive biomarker of response to PARPis.

Asunto(s)

Reparación del ADN por Unión de Extremidades , Homeobox 1 de Unión a la E-Box con Dedos de Zinc , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/genética , Proteína 1 de Unión al Supresor Tumoral P53/genética , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Línea Celular Tumoral , Humanos , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismo

RESUMEN

53BP1 is a chromatin-binding protein that promotes DNA double-strand break repair through the recruitment of downstream effectors including RIF1, shieldin, and CST. The structural basis of the protein-protein interactions within the 53BP1-RIF1-shieldin-CST pathway that are essential for its DNA repair activity is largely unknown. Here, we used AlphaFold2-Multimer (AF2) to predict all possible pairwise combinations of proteins within this pathway and provide structural models of seven previously characterized interactions. This analysis also predicted an entirely novel binding interface between the HEAT-repeat domain of RIF1 and the eIF4E-like domain of SHLD3. Extensive interrogation of this interface through both in vitro pulldown analysis and cellular assays supports the AF2-predicted model and demonstrates that RIF1-SHLD3 binding is essential for shieldin recruitment to sites of DNA damage, and for its role in antibody class switch recombination and PARP inhibitor sensitivity. Direct physical interaction between RIF1 and SHLD3 is therefore essential for 53BP1-RIF1-shieldin-CST pathway activity.

Asunto(s)

Proteínas de Unión al ADN , Furilfuramida , Proteína 1 de Unión al Supresor Tumoral P53/genética , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Proteínas de Unión al ADN/metabolismo , Reparación del ADN , ADN/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Proteínas de Unión a Telómeros/genética , Proteínas de Unión a Telómeros/metabolismo