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1.
Cell ; 184(4): 1081-1097.e19, 2021 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-33606978

RESUMO

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.


Assuntos
Dano ao DNA , Edição de Genes , Testes Genéticos , Sequência de Aminoácidos , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Sequência de Bases , Sistemas CRISPR-Cas/genética , Camptotecina/farmacologia , Linhagem Celular , Dano ao DNA/genética , Reparo do DNA/genética , Feminino , Humanos , Mutação/genética , Fenótipo , Ligação Proteica , Domínios Proteicos , RNA Guia de Cinetoplastídeos/genética , Inibidores da Topoisomerase/farmacologia , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/química , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Ubiquitina Tiolesterase/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
2.
Cell ; 184(24): 5970-5984.e18, 2021 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-34793701

RESUMO

Numerous DNA double-strand breaks (DSBs) arise during meiosis to initiate homologous recombination. These DSBs are usually repaired faithfully, but here, we uncover a distinct type of mutational event in which deletions form via joining of ends from two closely spaced DSBs (double cuts) within a single hotspot or at adjacent hotspots on the same or different chromatids. Deletions occur in normal meiosis but are much more frequent when DSB formation is dysregulated in the absence of the ATM kinase. Events between chromosome homologs point to multi-chromatid damage and aborted gap repair. Some deletions contain DNA from other hotspots, indicating that double cutting at distant sites creates substrates for insertional mutagenesis. End joining at double cuts can also yield tandem duplications or extrachromosomal circles. Our findings highlight the importance of DSB regulation and reveal a previously hidden potential for meiotic mutagenesis that is likely to affect human health and genome evolution.


Assuntos
Deleção de Genes , Duplicação Gênica , Células Germinativas/metabolismo , Recombinação Genética/genética , Animais , Proteínas Mutadas de Ataxia Telangiectasia/deficiência , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Sequência de Bases , Cromátides/metabolismo , Cromossomos de Mamíferos/genética , Cruzamentos Genéticos , Quebras de DNA de Cadeia Dupla , DNA Circular/genética , Feminino , Genoma , Haplótipos/genética , Recombinação Homóloga/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Mutagênese Insercional/genética , Mutação/genética
3.
Annu Rev Biochem ; 84: 711-38, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25580527

RESUMO

The ataxia-telangiectasia mutated (ATM) protein kinase is a master regulator of the DNA damage response, and it coordinates checkpoint activation, DNA repair, and metabolic changes in eukaryotic cells in response to DNA double-strand breaks and oxidative stress. Loss of ATM activity in humans results in the pleiotropic neurodegeneration disorder ataxia-telangiectasia. ATM exists in an inactive state in resting cells but can be activated by the Mre11-Rad50-Nbs1 (MRN) complex and other factors at sites of DNA breaks. In addition, oxidation of ATM activates the kinase independently of the MRN complex. This review discusses these mechanisms of activation, as well as the posttranslational modifications that affect this process and the cellular factors that affect the efficiency and specificity of ATM activation and substrate phosphorylation. I highlight functional similarities between the activation mechanisms of ATM, phosphatidylinositol 3-kinases (PI3Ks), and the other PI3K-like kinases, as well as recent structural insights into their regulation.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Animais , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática , Humanos , Estresse Oxidativo , Fosfatidilinositol 3-Quinase/metabolismo , Processamento de Proteína Pós-Traducional
4.
Mol Cell ; 82(1): 159-176.e12, 2022 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-34847357

RESUMO

The MYCN oncoprotein drives the development of numerous neuroendocrine and pediatric tumors. Here we show that MYCN interacts with the nuclear RNA exosome, a 3'-5' exoribonuclease complex, and recruits the exosome to its target genes. In the absence of the exosome, MYCN-directed elongation by RNA polymerase II (RNAPII) is slow and non-productive on a large group of cell-cycle-regulated genes. During the S phase of MYCN-driven tumor cells, the exosome is required to prevent the accumulation of stalled replication forks and of double-strand breaks close to the transcription start sites. Upon depletion of the exosome, activation of ATM causes recruitment of BRCA1, which stabilizes nuclear mRNA decapping complexes, leading to MYCN-dependent transcription termination. Disruption of mRNA decapping in turn activates ATR, indicating transcription-replication conflicts. We propose that exosome recruitment by MYCN maintains productive transcription elongation during S phase and prevents transcription-replication conflicts to maintain the rapid proliferation of neuroendocrine tumor cells.


Assuntos
Núcleo Celular/enzimologia , Proliferação de Células , Replicação do DNA , Exossomos/enzimologia , Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/enzimologia , RNA Polimerase II/metabolismo , Transcrição Gênica , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/genética , Quebras de DNA de Cadeia Dupla , Exorribonucleases/genética , Exorribonucleases/metabolismo , Exossomos/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Masculino , Camundongos , Proteína Proto-Oncogênica N-Myc/genética , Células NIH 3T3 , Neuroblastoma/genética , Neuroblastoma/patologia , Regiões Promotoras Genéticas , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , RNA Polimerase II/genética , Terminação da Transcrição Genética
5.
Genes Dev ; 36(5-6): 278-293, 2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-35318271

RESUMO

DNA repair and DNA damage signaling pathways are critical for the maintenance of genomic stability. Defects of DNA repair and damage signaling contribute to tumorigenesis, but also render cancer cells vulnerable to DNA damage and reliant on remaining repair and signaling activities. Here, we review the major classes of DNA repair and damage signaling defects in cancer, the genomic instability that they give rise to, and therapeutic strategies to exploit the resulting vulnerabilities. Furthermore, we discuss the impacts of DNA repair defects on both targeted therapy and immunotherapy, and highlight emerging principles for targeting DNA repair defects in cancer therapy.


Assuntos
Reparo do DNA , Neoplasias , Dano ao DNA/genética , Reparo do DNA/genética , Instabilidade Genômica/genética , Humanos , Imunoterapia , Neoplasias/tratamento farmacológico , Neoplasias/terapia
6.
Mol Cell ; 81(7): 1515-1533.e5, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33571423

RESUMO

Loss of the ataxia-telangiectasia mutated (ATM) kinase causes cerebellum-specific neurodegeneration in humans. We previously demonstrated that deficiency in ATM activation via oxidative stress generates insoluble protein aggregates in human cells, reminiscent of protein dysfunction in common neurodegenerative disorders. Here, we show that this process is driven by poly-ADP-ribose polymerases (PARPs) and that the insoluble protein species arise from intrinsically disordered proteins associating with PAR-associated genomic sites in ATM-deficient cells. The lesions implicated in this process are single-strand DNA breaks dependent on reactive oxygen species, transcription, and R-loops. Human cells expressing Mre11 A-T-like disorder mutants also show PARP-dependent aggregation identical to ATM deficiency. Lastly, analysis of A-T patient cerebellum samples shows widespread protein aggregation as well as loss of proteins known to be critical in human spinocerebellar ataxias that is not observed in neocortex tissues. These results provide a hypothesis accounting for loss of protein integrity and cerebellum function in A-T.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/deficiência , Quebras de DNA de Cadeia Simples , Proteína Homóloga a MRE11/deficiência , Neocórtex/metabolismo , Poli ADP Ribosilação , Proteostase , Ataxias Espinocerebelares/metabolismo , Adulto , Linhagem Celular Tumoral , Feminino , Humanos , Masculino , Neocórtex/patologia , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/patologia
7.
Mol Cell ; 81(5): 1084-1099.e6, 2021 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-33450211

RESUMO

Cells have evolved an elaborate DNA repair network to ensure complete and accurate DNA replication. Defects in these repair machineries can fuel genome instability and drive carcinogenesis while creating vulnerabilities that may be exploited in therapy. Here, we use nascent chromatin capture (NCC) proteomics to characterize the repair of replication-associated DNA double-strand breaks (DSBs) triggered by topoisomerase 1 (TOP1) inhibitors. We reveal profound changes in the fork proteome, including the chromatin environment and nuclear membrane interactions, and identify three classes of repair factors according to their enrichment at broken and/or stalled forks. ATM inhibition dramatically rewired the broken fork proteome, revealing that ataxia telangiectasia mutated (ATM) signalling stimulates DNA end resection, recruits PLK1, and concomitantly suppresses the canonical DSB ubiquitination response by preventing accumulation of RNF168 and BRCA1-A. This work and collection of replication fork proteomes provide a new framework to understand how cells orchestrate homologous recombination repair of replication-associated DSBs.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas de Ciclo Celular/genética , Replicação do DNA , DNA Topoisomerases Tipo I/genética , DNA/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Reparo de DNA por Recombinação , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Camptotecina/farmacologia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Cromatina/química , Cromatina/metabolismo , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , DNA Topoisomerases Tipo I/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Regulação da Expressão Gênica , Células HeLa , Humanos , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Proteômica/métodos , Proteínas Proto-Oncogênicas/metabolismo , Piridinas/farmacologia , Quinolinas/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Inibidores da Topoisomerase I/farmacologia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/efeitos dos fármacos , Quinase 1 Polo-Like
8.
Mol Cell ; 79(3): 425-442.e7, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32615088

RESUMO

Double-strand breaks (DSBs) are the most deleterious DNA lesions, which, if left unrepaired, may lead to genome instability or cell death. Here, we report that, in response to DSBs, the RNA methyltransferase METTL3 is activated by ATM-mediated phosphorylation at S43. Phosphorylated METTL3 is then localized to DNA damage sites, where it methylates the N6 position of adenosine (m6A) in DNA damage-associated RNAs, which recruits the m6A reader protein YTHDC1 for protection. In this way, the METTL3-m6A-YTHDC1 axis modulates accumulation of DNA-RNA hybrids at DSBs sites, which then recruit RAD51 and BRCA1 for homologous recombination (HR)-mediated repair. METTL3-deficient cells display defective HR, accumulation of unrepaired DSBs, and genome instability. Accordingly, depletion of METTL3 significantly enhances the sensitivity of cancer cells and murine xenografts to DNA damage-based therapy. These findings uncover the function of METTL3 and YTHDC1 in HR-mediated DSB repair, which may have implications for cancer therapy.


Assuntos
Adenosina/análogos & derivados , Neoplasias de Cabeça e Pescoço/genética , Metiltransferases/genética , Proteínas do Tecido Nervoso/genética , Fatores de Processamento de RNA/genética , Reparo de DNA por Recombinação/efeitos dos fármacos , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Adenosina/metabolismo , Animais , Antibióticos Antineoplásicos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Bleomicina/farmacologia , Linhagem Celular Tumoral , DNA/genética , DNA/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Células HEK293 , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/mortalidade , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Metiltransferases/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas do Tecido Nervoso/metabolismo , Hibridização de Ácido Nucleico , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoblastos/patologia , Fosforilação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Fatores de Processamento de RNA/metabolismo , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Ribonuclease H/genética , Ribonuclease H/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/mortalidade , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Análise de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Mol Cell ; 79(1): 115-126.e6, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32497497

RESUMO

Extension of telomeres is a critical step in the immortalization of cancer cells. This complex reaction requires proper spatiotemporal coordination of telomerase and telomeres and remains poorly understood at the cellular level. To understand how cancer cells execute this process, we combine CRISPR genome editing and MS2 RNA tagging to image single molecules of telomerase RNA (hTR). Real-time dynamics and photoactivation experiments of hTR in Cajal bodies (CBs) reveal that hTERT controls the exit of hTR from CBs. Single-molecule tracking of hTR at telomeres shows that TPP1-mediated recruitment results in short telomere-telomerase scanning interactions, and then base pairing between hTR and telomere ssDNA promotes long interactions required for stable telomerase retention. Interestingly, POT1 OB-fold mutations that result in abnormally long telomeres in cancers act by enhancing this retention step. In summary, single-molecule imaging unveils the life cycle of telomerase RNA and provides a framework to reveal how cancer-associated mutations mechanistically drive defects in telomere homeostasis.


Assuntos
Corpos Enovelados/metabolismo , DNA de Cadeia Simples/metabolismo , RNA/metabolismo , Imagem Individual de Molécula/métodos , Telomerase/metabolismo , Homeostase do Telômero , Telômero/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA de Cadeia Simples/genética , Edição de Genes , Células HeLa , Humanos , Mutação , RNA/genética , Complexo Shelterina , Telomerase/genética , Telômero/genética , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo
10.
Genes Dev ; 34(11-12): 806-818, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32354835

RESUMO

Exonucleolytic resection, critical to repair double-strand breaks (DSBs) by recombination, is not well understood, particularly in mammalian meiosis. Here, we define structures of resected DSBs in mouse spermatocytes genome-wide at nucleotide resolution. Resection tracts averaged 1100 nt, but with substantial fine-scale heterogeneity at individual hot spots. Surprisingly, EXO1 is not the major 5' → 3' exonuclease, but the DSB-responsive kinase ATM proved a key regulator of both initiation and extension of resection. In wild type, apparent intermolecular recombination intermediates clustered near to but offset from DSB positions, consistent with joint molecules with incompletely invaded 3' ends. Finally, we provide evidence for PRDM9-dependent chromatin remodeling leading to increased accessibility at recombination sites. Our findings give insight into the mechanisms of DSB processing and repair in meiotic chromatin.


Assuntos
Reparo do DNA/fisiologia , Meiose , Animais , Cromatina/química , Cromatina/metabolismo , DNA/química , Quebras de DNA de Cadeia Dupla , Histona-Lisina N-Metiltransferase/metabolismo , Camundongos , Estrutura Molecular , Recombinação Genética
11.
Genes Dev ; 34(11-12): 731-732, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32482713

RESUMO

The exchange of genetic information between parental chromosomes in meiosis is an integral process for the creation of gametes. To generate a crossover, hundreds of DNA double-strand breaks (DSBs) are introduced in the genome of each meiotic cell by the SPO11 protein. The nucleolytic resection of DSB-adjacent DNA is a key step in meiotic DSB repair, but this process has remained understudied. In this issue of Genes & Development, Yamada and colleagues (pp. 806-818) capture some of the first details of resection and DSB repair intermediates in mouse meiosis using a method that maps blunt-ended DNA after ssDNA digestion. This yields some of the first genome-wide insights into DSB resection and repair in a mammalian genome and offers a tantalizing glimpse of how to quantitatively dissect this difficult to study, yet integral, nuclear process.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA/fisiologia , Meiose , Animais , Cromatina/química , Cromatina/metabolismo , DNA/química , Meiose/genética , Estrutura Molecular , Recombinação Genética
12.
EMBO J ; 42(6): e112094, 2023 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-36727301

RESUMO

DNA-PKcs is a key regulator of DNA double-strand break repair. Apart from its canonical role in the DNA damage response, DNA-PKcs is involved in the cellular response to oxidative stress (OS), but its exact role remains unclear. Here, we report that DNA-PKcs-deficient human cells display depolarized mitochondria membrane potential (MMP) and reoriented metabolism, supporting a role for DNA-PKcs in oxidative phosphorylation (OXPHOS). DNA-PKcs directly interacts with mitochondria proteins ANT2 and VDAC2, and formation of the DNA-PKcs/ANT2/VDAC2 (DAV) complex supports optimal exchange of ADP and ATP across mitochondrial membranes to energize the cell via OXPHOS and to maintain MMP. Moreover, we demonstrate that the DAV complex temporarily dissociates in response to oxidative stress to attenuate ADP-ATP exchange, a rate-limiting step for OXPHOS. Finally, we found that dissociation of the DAV complex is mediated by phosphorylation of DNA-PKcs at its Thr2609 cluster by ATM kinase. Based on these findings, we propose that the coordination between the DAV complex and ATM serves as a novel oxidative stress checkpoint to decrease ROS production from mitochondrial OXPHOS and to hasten cellular recovery from OS.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ligação a DNA , Estresse Oxidativo , Humanos , Trifosfato de Adenosina/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Mitocôndrias/metabolismo , Fosforilação
13.
EMBO J ; 42(15): e112684, 2023 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-37303233

RESUMO

Upon DNA damage, cells activate the DNA damage response (DDR) to coordinate proliferation and DNA repair. Dietary, metabolic, and environmental inputs are emerging as modulators of how DNA surveillance and repair take place. Lipids hold potential to convey these cues, although little is known about how. We observed that lipid droplet (LD) number specifically increased in response to DNA breaks. Using Saccharomyces cerevisiae and cultured human cells, we show that the selective storage of sterols into these LD concomitantly stabilizes phosphatidylinositol-4-phosphate (PI(4)P) at the Golgi, where it binds the DDR kinase ATM. In turn, this titration attenuates the initial nuclear ATM-driven response to DNA breaks, thus allowing processive repair. Furthermore, manipulating this loop impacts the kinetics of DNA damage signaling and repair in a predictable manner. Thus, our findings have major implications for tackling genetic instability pathologies through dietary and pharmacological interventions.


Assuntos
Proteínas Serina-Treonina Quinases , Proteínas de Saccharomyces cerevisiae , Humanos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Esteróis/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Dano ao DNA , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo
14.
Am J Hum Genet ; 2024 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-39317201

RESUMO

The ClinGen Hereditary Breast, Ovarian, and Pancreatic Cancer (HBOP) Variant Curation Expert Panel (VCEP) is composed of internationally recognized experts in clinical genetics, molecular biology, and variant interpretation. This VCEP made specifications for the American College of Medical Genetics and Association for Molecular Pathology (ACMG/AMP) guidelines for the ataxia telangiectasia mutated (ATM) gene according to the ClinGen protocol. These gene-specific rules for ATM were modified from the ACMG/AMP guidelines and were tested against 33 ATM variants of various types and classifications in a pilot curation phase. The pilot revealed a majority agreement between the HBOP VCEP classifications and the ClinVar-deposited classifications. Six pilot variants had conflicting interpretations in ClinVar, and re-evaluation with the VCEP's ATM-specific rules resulted in four that were classified as benign, one as likely pathogenic, and one as a variant of uncertain significance (VUS) by the VCEP, improving the certainty of interpretations in the public domain. Overall, 28 of the 33 pilot variants were not VUS, leading to an 85% classification rate. The ClinGen-approved, modified rules demonstrated value for improved interpretation of variants in ATM.

15.
Mol Cell ; 75(4): 669-682.e5, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31302002

RESUMO

Phosphorylated IKKα(p45) is a nuclear active form of the IKKα kinase that is induced by the MAP kinases BRAF and TAK1 and promotes tumor growth independent of canonical NF-κB signaling. Insights into the sources of IKKα(p45) activation and its downstream substrates in the nucleus remain to be defined. Here, we discover that IKKα(p45) is rapidly activated by DNA damage independent of ATM-ATR, but dependent on BRAF-TAK1-p38-MAPK, and is required for robust ATM activation and efficient DNA repair. Abolishing BRAF or IKKα activity attenuates ATM, Chk1, MDC1, Kap1, and 53BP1 phosphorylation, compromises 53BP1 and RIF1 co-recruitment to sites of DNA lesions, and inhibits 53BP1-dependent fusion of dysfunctional telomeres. Furthermore, IKKα or BRAF inhibition synergistically enhances the therapeutic potential of 5-FU and irinotecan to eradicate chemotherapy-resistant metastatic human tumors in vivo. Our results implicate BRAF and IKKα kinases in the DDR and reveal a combination strategy for cancer treatment.


Assuntos
Dano ao DNA , Resistencia a Medicamentos Antineoplásicos , Fluoruracila/farmacologia , Quinase I-kappa B/metabolismo , Irinotecano/farmacologia , Sistema de Sinalização das MAP Quinases , Proteínas de Neoplasias , Neoplasias , Animais , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Células HCT116 , Humanos , Quinase I-kappa B/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Células MCF-7 , Camundongos , Camundongos Nus , Metástase Neoplásica , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Telômero/genética , Telômero/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Hum Mol Genet ; 33(18): 1605-1617, 2024 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-38888340

RESUMO

The MRE11/RAD50/NBS1 (MRN) complex plays critical roles in cellular responses to DNA double-strand breaks. MRN is involved in end binding and processing, and it also induces cell cycle checkpoints by activating the ataxia-telangiectasia mutated (ATM) protein kinase. Hypomorphic pathogenic variants in the MRE11, RAD50, or NBS1 genes cause autosomal recessive genome instability syndromes featuring variable degrees of dwarfism, neurological defects, anemia, and cancer predisposition. Disease-associated MRN alleles include missense and nonsense variants, and many cause reduced protein levels of the entire MRN complex. However, the dramatic variability in the disease manifestation of MRN pathogenic variants is not understood. We sought to determine if low protein levels are a significant contributor to disease sequelae and therefore generated a transgenic murine model expressing MRE11 at low levels. These mice display dramatic phenotypes including small body size, severe anemia, and impaired DNA repair. We demonstrate that, distinct from ataxia telangiectasia-like disorder caused by MRE11 pathogenic missense or nonsense variants, mice and cultured cells expressing low MRE11 levels do not display the anticipated defects in ATM activation. Our findings indicate that ATM signaling can be supported by very low levels of the MRN complex and imply that defective ATM activation results from perturbation of MRN function caused by specific hypomorphic disease mutations. These distinct phenotypic outcomes underline the importance of understanding the impact of specific pathogenic MRE11 variants, which may help direct appropriate early surveillance for patients with these complicated disorders in a clinical setting.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia , Ataxia Telangiectasia , Reparo do DNA , Proteínas de Ligação a DNA , Proteína Homóloga a MRE11 , Camundongos Transgênicos , Fenótipo , Animais , Proteína Homóloga a MRE11/genética , Proteína Homóloga a MRE11/metabolismo , Camundongos , Ataxia Telangiectasia/genética , Ataxia Telangiectasia/metabolismo , Ataxia Telangiectasia/patologia , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Humanos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Reparo do DNA/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Modelos Animais de Doenças , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Quebras de DNA de Cadeia Dupla
17.
Immunity ; 47(3): 421-434.e3, 2017 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-28930658

RESUMO

Environmental insults are often detected by multiple sensors that activate diverse signaling pathways and transcriptional regulators, leading to a tailored transcriptional output. To understand how a tailored response is coordinated, we examined the inflammatory response elicited in mouse macrophages by ionizing radiation (IR). RNA-sequencing studies revealed that most radiation-induced genes were strongly dependent on only one of a small number of sensors and signaling pathways, notably the DNA damage-induced kinase ATM, which regulated many IR-response genes, including interferon response genes, via an atypical IRF1-dependent, STING-independent mechanism. Moreover, small, defined sets of genes activated by p53 and NRF2 accounted for the selective response to radiation in comparison to a microbial inducer of inflammation. Our findings reveal that genes comprising an environmental response are activated by defined sensing mechanisms with a high degree of selectivity, and they identify distinct components of the radiation response that might be susceptible to therapeutic perturbation.


Assuntos
Regulação da Expressão Gênica/efeitos da radiação , Inflamação/genética , Inflamação/metabolismo , Radiação Ionizante , Transdução de Sinais , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Análise por Conglomerados , Proteína Quinase Ativada por DNA/metabolismo , Relação Dose-Resposta à Radiação , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Humanos , Interferons/metabolismo , Interferons/farmacologia , Macrófagos/metabolismo , Macrófagos/efeitos da radiação , Proteínas de Membrana/metabolismo , Camundongos , Fator 88 de Diferenciação Mieloide/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptor de Interferon alfa e beta/genética , Receptor de Interferon alfa e beta/metabolismo , Transcrição Gênica/efeitos da radiação , Ativação Transcricional , Regulador Transcricional ERG/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
18.
EMBO Rep ; 25(9): 3970-3989, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39090319

RESUMO

The tandem Tudor-like domain-containing protein Spindlin1 (SPIN1) is a transcriptional coactivator with critical functions in embryonic development and emerging roles in cancer. However, the involvement of SPIN1 in DNA damage repair has remained unclear. Our study shows that SPIN1 is recruited to DNA lesions through its N-terminal disordered region that binds to Poly-ADP-ribose (PAR), and facilitates homologous recombination (HR)-mediated DNA damage repair. SPIN1 promotes H3K9me3 accumulation at DNA damage sites and enhances the interaction between H3K9me3 and Tip60, thereby promoting the activation of ATM and HR repair. We also show that SPIN1 increases chemoresistance. These findings reveal a novel role for SPIN1 in the activation of H3K9me3-dependent DNA repair pathways, and suggest that SPIN1 may contribute to cancer chemoresistance by modulating the efficiency of double-strand break (DSB) repair.


Assuntos
Proteínas de Ciclo Celular , Resistencia a Medicamentos Antineoplásicos , Histonas , Lisina Acetiltransferase 5 , Fosfoproteínas , Ligação Proteica , Humanos , Resistencia a Medicamentos Antineoplásicos/genética , Histonas/metabolismo , Lisina Acetiltransferase 5/metabolismo , Lisina Acetiltransferase 5/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Quebras de DNA de Cadeia Dupla , Reparo de DNA por Recombinação , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Poli Adenosina Difosfato Ribose/metabolismo , Linhagem Celular Tumoral , Dano ao DNA , Reparo do DNA , Proteínas Associadas aos Microtúbulos
19.
EMBO Rep ; 25(3): 1469-1489, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38366255

RESUMO

Tumor acidosis is associated with increased invasiveness and drug resistance. Here, we take an unbiased approach to identify vulnerabilities of acid-exposed cancer cells by combining pH-dependent flow cytometry cell sorting from 3D colorectal tumor spheroids and transcriptomic profiling. Besides metabolic rewiring, we identify an increase in tetraploid cell frequency and DNA damage response as consistent hallmarks of acid-exposed cancer cells, supported by the activation of ATM and ATR signaling pathways. We find that regardless of the cell replication error status, both ATM and ATR inhibitors exert preferential growth inhibitory effects on acid-exposed cancer cells. The efficacy of a combination of these drugs with 5-FU is further documented in 3D spheroids as well as in patient-derived colorectal tumor organoids. These data position tumor acidosis as a revelator of the therapeutic potential of DNA repair blockers and as an attractive clinical biomarker to predict the response to a combination with chemotherapy.


Assuntos
Neoplasias Colorretais , Tetraploidia , Humanos , Proteínas Mutadas de Ataxia Telangiectasia/genética , Transdução de Sinais , Dano ao DNA , Reparo do DNA , Inibidores de Proteínas Quinases/farmacologia
20.
Mol Cell ; 71(6): 897-910.e8, 2018 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-30122534

RESUMO

Chromatin ubiquitination by the ubiquitin ligase RNF168 is critical to regulate the DNA damage response (DDR). DDR deficiencies lead to cancer-prone syndromes, but whether this reflects DNA repair defects is still elusive. We identified key factors of the RNF168 pathway as essential mediators of efficient DNA replication in unperturbed S phase. We found that loss of RNF168 leads to reduced replication fork progression and to reversed fork accumulation, particularly evident at repetitive sequences stalling replication. Slow fork progression depends on MRE11-dependent degradation of reversed forks, implicating RNF168 in reversed fork protection and restart. Consistent with regular nucleosomal organization of reversed forks, the replication function of RNF168 requires H2A ubiquitination. As this novel function is shared with the key DDR players ATM, γH2A.X, RNF8, and 53BP1, we propose that double-stranded ends at reversed forks engage classical DDR factors, suggesting an alternative function of this pathway in preventing genome instability and human disease.


Assuntos
Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , Histonas/metabolismo , Linhagem Celular , Quebras de DNA de Cadeia Dupla , Replicação do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Humanos , Fase S/fisiologia , Transdução de Sinais , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/fisiologia
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