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1.
Cell ; 184(4): 1081-1097.e19, 2021 02 18.
Artículo en Inglés | MEDLINE | ID: mdl-33606978

RESUMEN

Mutations in DNA damage response (DDR) genes endanger genome integrity and predispose to cancer and genetic disorders. Here, using CRISPR-dependent cytosine base editing screens, we identify > 2,000 sgRNAs that generate nucleotide variants in 86 DDR genes, resulting in altered cellular fitness upon DNA damage. Among those variants, we discover loss- and gain-of-function mutants in the Tudor domain of the DDR regulator 53BP1 that define a non-canonical surface required for binding the deubiquitinase USP28. Moreover, we characterize variants of the TRAIP ubiquitin ligase that define a domain, whose loss renders cells resistant to topoisomerase I inhibition. Finally, we identify mutations in the ATM kinase with opposing genome stability phenotypes and loss-of-function mutations in the CHK2 kinase previously categorized as variants of uncertain significance for breast cancer. We anticipate that this resource will enable the discovery of additional DDR gene functions and expedite studies of DDR variants in human disease.


Asunto(s)
Daño del ADN , Edición Génica , Pruebas Genéticas , Secuencia de Aminoácidos , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Secuencia de Bases , Sistemas CRISPR-Cas/genética , Camptotecina/farmacología , Línea Celular , Daño del ADN/genética , Reparación del ADN/genética , Femenino , Humanos , Mutación/genética , Fenotipo , Unión Proteica , Dominios Proteicos , ARN Guía de Kinetoplastida/genética , Inhibidores de Topoisomerasa/farmacología , Proteína 1 de Unión al Supresor Tumoral P53/química , Proteína 1 de Unión al Supresor Tumoral P53/genética , Ubiquitina Tiolesterasa/metabolismo , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo
2.
Cell ; 173(4): 972-988.e23, 2018 05 03.
Artículo en Inglés | MEDLINE | ID: mdl-29656893

RESUMEN

Repair of damaged DNA is essential for maintaining genome integrity and for preventing genome-instability-associated diseases, such as cancer. By combining proximity labeling with quantitative mass spectrometry, we generated high-resolution interaction neighborhood maps of the endogenously expressed DNA repair factors 53BP1, BRCA1, and MDC1. Our spatially resolved interaction maps reveal rich network intricacies, identify shared and bait-specific interaction modules, and implicate previously concealed regulators in this process. We identified a novel vertebrate-specific protein complex, shieldin, comprising REV7 plus three previously uncharacterized proteins, RINN1 (CTC-534A2.2), RINN2 (FAM35A), and RINN3 (C20ORF196). Recruitment of shieldin to DSBs, via the ATM-RNF8-RNF168-53BP1-RIF1 axis, promotes NHEJ-dependent repair of intrachromosomal breaks, immunoglobulin class-switch recombination (CSR), and fusion of unprotected telomeres. Shieldin functions as a downstream effector of 53BP1-RIF1 in restraining DNA end resection and in sensitizing BRCA1-deficient cells to PARP inhibitors. These findings have implications for understanding cancer-associated PARPi resistance and the evolution of antibody CSR in higher vertebrates.


Asunto(s)
Reparación del ADN por Unión de Extremidades/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Proteínas Adaptadoras Transductoras de Señales , Proteína BRCA1/antagonistas & inhibidores , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteínas de Ciclo Celular , Línea Celular Tumoral , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Humanos , Cambio de Clase de Inmunoglobulina/efectos de los fármacos , Proteínas Mad2/antagonistas & inhibidores , Proteínas Mad2/genética , Proteínas Mad2/metabolismo , Mutagénesis Sitio-Dirigida , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteínas de Unión a Telómeros/antagonistas & inhibidores , Proteínas de Unión a Telómeros/genética , Proteínas de Unión a Telómeros/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/antagonistas & inhibidores , Proteína 1 de Unión al Supresor Tumoral P53/genética , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo
3.
Nat Rev Mol Cell Biol ; 21(5): 284-299, 2020 05.
Artículo en Inglés | MEDLINE | ID: mdl-32094664

RESUMEN

The tumour suppressor breast cancer type 1 susceptibility protein (BRCA1) promotes DNA double-strand break (DSB) repair by homologous recombination and protects DNA replication forks from attrition. BRCA1 partners with BRCA1-associated RING domain protein 1 (BARD1) and other tumour suppressor proteins to mediate the initial nucleolytic resection of DNA lesions and the recruitment and regulation of the recombinase RAD51. The discovery of the opposing functions of BRCA1 and the p53-binding protein 1 (53BP1)-associated complex in DNA resection sheds light on how BRCA1 influences the choice of homologous recombination over non-homologous end joining and potentially other mutagenic pathways of DSB repair. Understanding the functional crosstalk between BRCA1-BARD1 and its cofactors and antagonists will illuminate the molecular basis of cancers that arise from a deficiency or misregulation of chromosome damage repair and replication fork maintenance. Such knowledge will also be valuable for understanding acquired tumour resistance to poly(ADP-ribose) polymerase (PARP) inhibitors and other therapeutics and for the development of new treatments. In this Review, we discuss recent advances in elucidating the mechanisms by which BRCA1-BARD1 functions in DNA repair, replication fork maintenance and tumour suppression, and its therapeutic relevance.


Asunto(s)
Proteína BRCA1/genética , Neoplasias de la Mama/genética , Proteínas Supresoras de Tumor/genética , Proteína 1 de Unión al Supresor Tumoral P53/genética , Ubiquitina-Proteína Ligasas/genética , Neoplasias de la Mama/patología , Reparación del ADN por Unión de Extremidades/genética , Replicación del ADN/genética , Femenino , Humanos , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Poli(ADP-Ribosa) Polimerasas/genética , Unión Proteica/genética , Reparación del ADN por Recombinación/genética
4.
Mol Cell ; 84(5): 839-853.e12, 2024 Mar 07.
Artículo en Inglés | MEDLINE | ID: mdl-38242129

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
5.
Mol Cell ; 83(7): 1043-1060.e10, 2023 04 06.
Artículo en Inglés | MEDLINE | ID: mdl-36854302

RESUMEN

Repair of DNA double-strand breaks (DSBs) elicits three-dimensional (3D) chromatin topological changes. A recent finding reveals that 53BP1 assembles into a 3D chromatin topology pattern around DSBs. How this formation of a higher-order structure is configured and regulated remains enigmatic. Here, we report that SLFN5 is a critical factor for 53BP1 topological arrangement at DSBs. Using super-resolution imaging, we find that SLFN5 binds to 53BP1 chromatin domains to assemble a higher-order microdomain architecture by driving damaged chromatin dynamics at both DSBs and deprotected telomeres. Mechanistically, we propose that 53BP1 topology is shaped by two processes: (1) chromatin mobility driven by the SLFN5-LINC-microtubule axis and (2) the assembly of 53BP1 oligomers mediated by SLFN5. In mammals, SLFN5 deficiency disrupts the DSB repair topology and impairs non-homologous end joining, telomere fusions, class switch recombination, and sensitivity to poly (ADP-ribose) polymerase inhibitor. We establish a molecular mechanism that shapes higher-order chromatin topologies to safeguard genomic stability.


Asunto(s)
Cromatina , Reparación del ADN , Animales , Cromatina/genética , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Mamíferos/metabolismo , Proteínas de Unión a Telómeros/genética , Proteína 1 de Unión al Supresor Tumoral P53/genética , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Proteínas de Ciclo Celular/metabolismo
6.
Mol Cell ; 82(10): 1924-1939.e10, 2022 05 19.
Artículo en Inglés | MEDLINE | ID: mdl-35439434

RESUMEN

The 53BP1-RIF1-shieldin pathway maintains genome stability by suppressing nucleolytic degradation of DNA ends at double-strand breaks (DSBs). Although RIF1 interacts with damaged chromatin via phospho-53BP1 and facilitates recruitment of the shieldin complex to DSBs, it is unclear whether other regulatory cues contribute to this response. Here, we implicate methylation of histone H3 at lysine 4 by SETD1A-BOD1L in the recruitment of RIF1 to DSBs. Compromising SETD1A or BOD1L expression or deregulating H3K4 methylation allows uncontrolled resection of DNA ends, impairs end-joining of dysfunctional telomeres, and abrogates class switch recombination. Moreover, defects in RIF1 localization to DSBs are evident in patient cells bearing loss-of-function mutations in SETD1A. Loss of SETD1A-dependent RIF1 recruitment in BRCA1-deficient cells restores homologous recombination and leads to resistance to poly(ADP-ribose)polymerase inhibition, reinforcing the clinical relevance of these observations. Mechanistically, RIF1 binds directly to methylated H3K4, facilitating its recruitment to, or stabilization at, DSBs.


Asunto(s)
Roturas del ADN de Doble Cadena , Proteínas de Unión a Telómeros , Proteína BRCA1/genética , ADN/metabolismo , Reparación del ADN por Unión de Extremidades , Reparación del ADN , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Metilación , Proteínas de Unión a Telómeros/genética , Proteínas de Unión a Telómeros/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/genética , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo
7.
Mol Cell ; 82(7): 1359-1371.e9, 2022 04 07.
Artículo en Inglés | MEDLINE | ID: mdl-35216668

RESUMEN

The chromatin-binding protein 53BP1 promotes DNA repair by orchestrating the recruitment of downstream effectors including PTIP, RIF1, and shieldin to DNA double-strand break sites. While we know how PTIP recognizes 53BP1, the molecular details of RIF1 recruitment to DNA-damage sites remains undefined. Here, we report that RIF1 is a phosphopeptide-binding protein that directly interacts with three phosphorylated 53BP1 epitopes. The RIF1-binding sites on 53BP1 share an essential LxL motif followed by two closely apposed phosphorylated residues. Simultaneous mutation of these sites on 53BP1 abrogates RIF1 accumulation into ionizing-radiation-induced foci, but surprisingly, only fully compromises 53BP1-dependent DNA repair when an alternative mode of shieldin recruitment to DNA-damage sites is also disabled. Intriguingly, this alternative mode of recruitment still depends on RIF1 but does not require its interaction with 53BP1. RIF1 therefore employs phosphopeptide recognition to promote DNA repair but also modifies shieldin action independently of 53BP1 binding.


Asunto(s)
Fosfopéptidos , Proteínas de Unión a Telómeros , Proteína BRCA1/genética , Proteínas Portadoras/metabolismo , ADN/metabolismo , Reparación del ADN por Unión de Extremidades , Reparación del ADN , Fosfopéptidos/genética , Fosfopéptidos/metabolismo , Proteínas de Unión a Telómeros/genética , Proteínas de Unión a Telómeros/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/genética , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo
8.
Mol Cell ; 81(12): 2596-2610.e7, 2021 06 17.
Artículo en Inglés | MEDLINE | ID: mdl-33961796

RESUMEN

p53-binding protein 1 (53BP1) regulates both the DNA damage response and p53 signaling. Although 53BP1's function is well established in DNA double-strand break repair, how its role in p53 signaling is modulated remains poorly understood. Here, we identify the scaffolding protein AHNAK as a G1 phase-enriched interactor of 53BP1. We demonstrate that AHNAK binds to the 53BP1 oligomerization domain and controls its multimerization potential. Loss of AHNAK results in hyper-accumulation of 53BP1 on chromatin and enhanced phase separation, culminating in an elevated p53 response, compromising cell survival in cancer cells but leading to senescence in non-transformed cells. Cancer transcriptome analyses indicate that AHNAK-53BP1 cooperation contributes to the suppression of p53 target gene networks in tumors and that loss of AHNAK sensitizes cells to combinatorial cancer treatments. These findings highlight AHNAK as a rheostat of 53BP1 function, which surveys cell proliferation by preventing an excessive p53 response.


Asunto(s)
Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Línea Celular Tumoral , Cromatina/metabolismo , ADN/genética , Roturas del ADN de Doble Cadena , Reparación del ADN , Fase G1/fisiología , Histonas/metabolismo , Humanos , Células MCF-7 , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiología , Transducción de Señal/fisiología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/genética , Proteína 1 de Unión al Supresor Tumoral P53/fisiología
9.
Mol Cell ; 81(22): 4692-4708.e9, 2021 11 18.
Artículo en Inglés | MEDLINE | ID: mdl-34555355

RESUMEN

Inhibitors of poly(ADP-ribose) (PAR) polymerase (PARPi) have entered the clinic for the treatment of homologous recombination (HR)-deficient cancers. Despite the success of this approach, preclinical and clinical research with PARPi has revealed multiple resistance mechanisms, highlighting the need for identification of novel functional biomarkers and combination treatment strategies. Functional genetic screens performed in cells and organoids that acquired resistance to PARPi by loss of 53BP1 identified loss of LIG3 as an enhancer of PARPi toxicity in BRCA1-deficient cells. Enhancement of PARPi toxicity by LIG3 depletion is dependent on BRCA1 deficiency but independent of the loss of 53BP1 pathway. Mechanistically, we show that LIG3 loss promotes formation of MRE11-mediated post-replicative ssDNA gaps in BRCA1-deficient and BRCA1/53BP1 double-deficient cells exposed to PARPi, leading to an accumulation of chromosomal abnormalities. LIG3 depletion also enhances efficacy of PARPi against BRCA1-deficient mammary tumors in mice, suggesting LIG3 as a potential therapeutic target.


Asunto(s)
Proteína BRCA1/genética , ADN Ligasa (ATP)/genética , ADN de Cadena Simple , Proteína Homóloga de MRE11/genética , Neoplasias Ováricas/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Proteínas de Unión a Poli-ADP-Ribosa/genética , Neoplasias de la Mama Triple Negativas/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/genética , Animales , Biopsia , Sistemas CRISPR-Cas , Línea Celular , Núcleo Celular/metabolismo , Proliferación Celular , Aberraciones Cromosómicas , Daño del ADN , ADN Ligasa (ATP)/metabolismo , Femenino , Humanos , Lentivirus/genética , Neoplasias Mamarias Animales , Ratones , Mutación , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , ARN Interferente Pequeño/metabolismo , Transgenes
10.
Mol Cell ; 81(15): 3128-3144.e7, 2021 08 05.
Artículo en Inglés | MEDLINE | ID: mdl-34216544

RESUMEN

Mutations in BRCA1 or BRCA2 (BRCA) is synthetic lethal with poly(ADP-ribose) polymerase inhibitors (PARPi). Lethality is thought to derive from DNA double-stranded breaks (DSBs) necessitating BRCA function in homologous recombination (HR) and/or fork protection (FP). Here, we report instead that toxicity derives from replication gaps. BRCA1- or FANCJ-deficient cells, with common repair defects but distinct PARPi responses, reveal gaps as a distinguishing factor. We further uncouple HR, FP, and fork speed from PARPi response. Instead, gaps characterize BRCA-deficient cells, are diminished upon resistance, restored upon resensitization, and, when exposed, augment PARPi toxicity. Unchallenged BRCA1-deficient cells have elevated poly(ADP-ribose) and chromatin-associated PARP1, but aberrantly low XRCC1 consistent with defects in backup Okazaki fragment processing (OFP). 53BP1 loss resuscitates OFP by restoring XRCC1-LIG3 that suppresses the sensitivity of BRCA1-deficient cells to drugs targeting OFP or generating gaps. We highlight gaps as a determinant of PARPi toxicity changing the paradigm for synthetic lethal interactions.


Asunto(s)
Proteína BRCA1/genética , Replicación del ADN/efectos de los fármacos , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Animales , Línea Celular , Cisplatino/farmacología , ADN/genética , ADN/metabolismo , ADN de Cadena Simple/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Recombinación Homóloga/efectos de los fármacos , Humanos , Ratones Endogámicos NOD , ARN Helicasas/genética , Recombinasa Rad51/genética , Proteína de Replicación A/genética , Proteína 1 de Unión al Supresor Tumoral P53/genética
11.
Genes Dev ; 35(19-20): 1356-1367, 2021 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-34503990

RESUMEN

Double-strand break (DSB) repair choice is greatly influenced by the initial processing of DNA ends. 53BP1 limits the formation of recombinogenic single-strand DNA (ssDNA) in BRCA1-deficient cells, leading to defects in homologous recombination (HR). However, the exact mechanisms by which 53BP1 inhibits DSB resection remain unclear. Previous studies have identified two potential pathways: protection against DNA2/EXO1 exonucleases presumably through the Shieldin (SHLD) complex binding to ssDNA, and localized DNA synthesis through the CTC1-STN1-TEN1 (CST) and DNA polymerase α (Polα) to counteract resection. Using a combinatorial approach of END-seq, SAR-seq, and RPA ChIP-seq, we directly assessed the extent of resection, DNA synthesis, and ssDNA, respectively, at restriction enzyme-induced DSBs. We show that, in the presence of 53BP1, Polα-dependent DNA synthesis reduces the fraction of resected DSBs and the resection lengths in G0/G1, supporting a previous model that fill-in synthesis can limit the extent of resection. However, in the absence of 53BP1, Polα activity is sustained on ssDNA yet does not substantially counter resection. In contrast, EXO1 nuclease activity is essential for hyperresection in the absence of 53BP1. Thus, Polα-mediated fill-in partially limits resection in the presence of 53BP1 but cannot counter extensive hyperresection due to the loss of 53BP1 exonuclease blockade. These data provide the first nucleotide mapping of DNA synthesis at resected DSBs and provide insight into the relationship between fill-in polymerases and resection exonucleases.


Asunto(s)
Roturas del ADN de Doble Cadena , Replicación del ADN , Reparación del ADN/genética , Replicación del ADN/genética , ADN de Cadena Simple/genética , Recombinación Homóloga/genética , Proteína 1 de Unión al Supresor Tumoral P53/genética , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo
12.
Mol Cell ; 78(5): 951-959.e6, 2020 06 04.
Artículo en Inglés | MEDLINE | ID: mdl-32359443

RESUMEN

BRCA1 promotes the DNA end resection and RAD51 loading steps of homologous recombination (HR). Whether these functions can be uncoupled, and whether mutant proteins retaining partial activity can complement one another, is unclear and could affect the severity of BRCA1-associated Fanconi anemia (FA). Here we generated a Brca1CC mouse with a coiled-coil (CC) domain deletion. Brca1CC/CC mice are born at low frequencies, and post-natal mice have FA-like abnormalities, including bone marrow failure. Intercrossing with Brca1Δ11, which is homozygous lethal, generated Brca1CC/Δ11 mice at Mendelian frequencies that were indistinguishable from Brca1+/+ mice. Brca1CC and Brca1Δ11 proteins were individually responsible for counteracting 53BP1-RIF1-Shieldin activity and promoting RAD51 loading, respectively. Thus, Brca1CC and Brca1Δ11 alleles represent separation-of-function mutations that combine to provide a level of HR sufficient for normal development and hematopoiesis. Because BRCA1 activities can be genetically separated, compound heterozygosity for functional complementary mutations may protect individuals from FA.


Asunto(s)
Proteína BRCA1/genética , Recombinación Homóloga/genética , Proteína 1 de Unión al Supresor Tumoral P53/genética , Animales , Proteína BRCA1/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN , Exones , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo
13.
Mol Cell ; 77(1): 26-38.e7, 2020 01 02.
Artículo en Inglés | MEDLINE | ID: mdl-31653568

RESUMEN

53BP1 activity drives genome instability and lethality in BRCA1-deficient mice by inhibiting homologous recombination (HR). The anti-recombinogenic functions of 53BP1 require phosphorylation-dependent interactions with PTIP and RIF1/shieldin effector complexes. While RIF1/shieldin blocks 5'-3' nucleolytic processing of DNA ends, it remains unclear how PTIP antagonizes HR. Here, we show that mutation of the PTIP interaction site in 53BP1 (S25A) allows sufficient DNA2-dependent end resection to rescue the lethality of BRCA1Δ11 mice, despite increasing RIF1 "end-blocking" at DNA damage sites. However, double-mutant cells fail to complete HR, as excessive shieldin activity also inhibits RNF168-mediated loading of PALB2/RAD51. As a result, BRCA1Δ1153BP1S25A mice exhibit hallmark features of HR insufficiency, including premature aging and hypersensitivity to PARPi. Disruption of shieldin or forced targeting of PALB2 to ssDNA in BRCA1D1153BP1S25A cells restores RNF168 recruitment, RAD51 nucleofilament formation, and PARPi resistance. Our study therefore reveals a critical function of shieldin post-resection that limits the loading of RAD51.


Asunto(s)
Recombinación Homóloga/genética , Proteína 1 de Unión al Supresor Tumoral P53/genética , Envejecimiento/efectos de los fármacos , Envejecimiento/genética , Animales , Proteína BRCA1/genética , Roturas del ADN de Doble Cadena/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Daño del ADN/genética , Inestabilidad Genómica/efectos de los fármacos , Inestabilidad Genómica/genética , Recombinación Homóloga/efectos de los fármacos , Ratones , Mutación/efectos de los fármacos , Mutación/genética , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Recombinasa Rad51/genética , Ubiquitina-Proteína Ligasas/genética
14.
Genes Dev ; 34(1-2): 7-23, 2020 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-31896689

RESUMEN

53BP1 is an enigmatic DNA damage response factor that gained prominence because it determines the efficacy of PARP1 inhibitory drugs (PARPi) in BRCA1-deficient cancers. Recent studies have elevated 53BP1 from its modest status of (yet another) DNA damage factor to master regulator of double-strand break (DSB) repair pathway choice. Our review of the literature suggests an alternative view. We propose that 53BP1 has evolved to avoid mutagenic repair outcomes and does so by controlling the processing of DNA ends and the dynamics of DSBs. The consequences of 53BP1 deficiency, such as diminished PARPi efficacy in BRCA1-deficient cells and altered repair of damaged telomeres, can be explained from this viewpoint. We further propose that some of the fidelity functions of 53BP1 coevolved with class switch recombination (CSR) in the immune system. We speculate that, rather than being deterministic in DSB repair pathway choice, 53BP1 functions as a DSB escort that guards against illegitimate and potentially tumorigenic recombination.


Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN/genética , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Evolución Molecular , Humanos , Cambio de Clase de Inmunoglobulina/genética , Telómero/genética , Proteína 1 de Unión al Supresor Tumoral P53/deficiencia , Proteína 1 de Unión al Supresor Tumoral P53/genética
15.
PLoS Biol ; 22(9): e3002760, 2024 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-39226322

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
16.
Mol Cell ; 73(6): 1267-1281.e7, 2019 03 21.
Artículo en Inglés | MEDLINE | ID: mdl-30704900

RESUMEN

BRCA1 functions at two distinct steps during homologous recombination (HR). Initially, it promotes DNA end resection, and subsequently it recruits the PALB2 and BRCA2 mediator complex, which stabilizes RAD51-DNA nucleoprotein filaments. Loss of 53BP1 rescues the HR defect in BRCA1-deficient cells by increasing resection, suggesting that BRCA1's downstream role in RAD51 loading is dispensable when 53BP1 is absent. Here we show that the E3 ubiquitin ligase RNF168, in addition to its canonical role in inhibiting end resection, acts in a redundant manner with BRCA1 to load PALB2 onto damaged DNA. Loss of RNF168 negates the synthetic rescue of BRCA1 deficiency by 53BP1 deletion, and it predisposes BRCA1 heterozygous mice to cancer. BRCA1+/-RNF168-/- cells lack RAD51 foci and are hypersensitive to PARP inhibitor, whereas forced targeting of PALB2 to DNA breaks in mutant cells circumvents BRCA1 haploinsufficiency. Inhibiting the chromatin ubiquitin pathway may, therefore, be a synthetic lethality strategy for BRCA1-deficient cancers.


Asunto(s)
Proteína BRCA1/genética , Cromatina/enzimología , Fibroblastos/enzimología , Haploinsuficiencia , Neoplasias/enzimología , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Animales , Proteína BRCA2/genética , Línea Celular Tumoral , Cromatina/genética , Daño del ADN , Proteína del Grupo de Complementación N de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación N de la Anemia de Fanconi/metabolismo , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/patología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , Reparación del ADN por Recombinación , Proteína 1 de Unión al Supresor Tumoral P53/genética , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Ubiquitina-Proteína Ligasas/deficiencia , Ubiquitina-Proteína Ligasas/genética
17.
Proc Natl Acad Sci U S A ; 121(28): e2322972121, 2024 Jul 09.
Artículo en Inglés | MEDLINE | ID: mdl-38968116

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
18.
Mol Cell ; 72(2): 250-262.e6, 2018 10 18.
Artículo en Inglés | MEDLINE | ID: mdl-30270107

RESUMEN

Double-strand breaks (DSBs) are extremely detrimental DNA lesions that can lead to cancer-driving mutations and translocations. Non-homologous end joining (NHEJ) and homologous recombination (HR) represent the two main repair pathways operating in the context of chromatin to ensure genome stability. Despite extensive efforts, our knowledge of DSB-induced chromatin still remains fragmented. Here, we describe the distribution of 20 chromatin features at multiple DSBs spread throughout the human genome using ChIP-seq. We provide the most comprehensive picture of the chromatin landscape set up at DSBs and identify NHEJ- and HR-specific chromatin events. This study revealed the existence of a DSB-induced monoubiquitination-to-acetylation switch on histone H2B lysine 120, likely mediated by the SAGA complex, as well as higher-order signaling at HR-repaired DSBs whereby histone H1 is evicted while ubiquitin and 53BP1 accumulate over the entire γH2AX domains.


Asunto(s)
Cromatina/genética , Reparación del ADN/genética , Histonas/genética , Línea Celular Tumoral , Roturas del ADN de Doble Cadena , Inestabilidad Genómica/genética , Recombinación Homóloga/genética , Humanos , Células K562 , Proteína 1 de Unión al Supresor Tumoral P53/genética
19.
Nucleic Acids Res ; 52(13): 7687-7703, 2024 Jul 22.
Artículo en Inglés | MEDLINE | ID: mdl-38884202

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
20.
J Biol Chem ; 300(8): 107545, 2024 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-38992439

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
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