RESUMO
The mammalian SUMO-targeted E3 ubiquitin ligase Rnf4 has been reported to act as a regulator of DNA repair, but the importance of RNF4 as a tumor suppressor has not been tested. Using a conditional-knockout mouse model, we deleted Rnf4 in the B cell lineage to test the importance of RNF4 for growth of somatic cells. Although Rnf4-conditional-knockout B cells exhibited substantial genomic instability, Rnf4 deletion caused no increase in tumor susceptibility. In contrast, Rnf4 deletion extended the healthy lifespan of mice expressing an oncogenic c-myc transgene. Rnf4 activity is essential for normal DNA replication, and in its absence, there was a failure in ATR-CHK1 signaling of replication stress. Factors that normally mediate replication fork stability, including members of the Fanconi anemia gene family and the helicases PIF1 and RECQL5, showed reduced accumulation at replication forks in the absence of RNF4. RNF4 deficiency also resulted in an accumulation of hyper-SUMOylated proteins in chromatin, including members of the SMC5/6 complex, which contributes to replication failure by a mechanism dependent on RAD51. These findings indicate that RNF4, which shows increased expression in multiple human tumor types, is a potential target for anticancer therapy, especially in tumors expressing c-myc.
Assuntos
Replicação do DNA , Proteínas Proto-Oncogênicas c-myc , Animais , Humanos , Camundongos , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Linfócitos B/metabolismo , Linfócitos B/patologia , Carcinogênese/genética , Carcinogênese/metabolismo , Quinase 1 do Ponto de Checagem/metabolismo , Quinase 1 do Ponto de Checagem/genética , Instabilidade Genômica , Camundongos Knockout , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transdução de Sinais , Sumoilação , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
BRCA1 maintains genome integrity and suppresses tumorigenesis by promoting homologous recombination (HR)-mediated repair of DNA double-strand breaks (DSB) and DNA damage-induced cell-cycle checkpoints. Phosphorylation of BRCA1 by ATM, ATR, CHK2, CDK, and PLK1 kinases has been reported to regulate its functions. Here we show that ATR and ATM-mediated phosphorylation of BRCA1 on T1394, a highly conserved but functionally uncharacterized site, is a key modification for its function in the DNA damage response (DDR). Following DNA damage, T1394 phosphorylation ensured faithful repair of DSBs by promoting HR and preventing single-strand annealing, a deletion-generating repair process. BRCA1 T1394 phosphorylation further safeguarded chromosomal integrity by maintaining the G2-M checkpoint. Moreover, multiple patient-derived BRCA1 variants of unknown significance were shown to affect T1394 phosphorylation. These results establish an important regulatory mechanism of BRCA1 function in the DDR and may have implications in the development or prognosis of BRCA1-associated cancers. SIGNIFICANCE: This study identifies a BRCA1 phosphorylation event critical for its DNA repair function and reveals the functional defects of several BRCA1 variants of unknown significance.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Reparo de DNA por Recombinação , Sequência de Aminoácidos , Aminoácidos/química , Aminoácidos/metabolismo , Animais , Proteína BRCA1/química , Proteína BRCA1/genética , Linhagem Celular Tumoral , Dano ao DNA , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Camundongos , Camundongos Transgênicos , Modelos Biológicos , FosforilaçãoRESUMO
Mutations in the BRCA1 gene cause an extremely high lifetime risk of breast and ovarian cancer, but the exact mechanism by which the BRCA1 protein acts to prevent cancer onset remains unclear. In this edition of Cancer Research, Park and colleagues describe a new mouse model featuring a single amino acid substitution in the coiled-coil motif of BRCA1. This change prevents BRCA1 from interacting with PALB2 (partner and localizer of BRCA2), causing rapid cancer onset and a loss of blood cells similar to Fanconi anemia.See related article by Park et al., p. 4172.
Assuntos
Proteína BRCA1 , Anemia de Fanconi , Animais , Proteína BRCA1/genética , Proteína BRCA2/genética , Anemia de Fanconi/genética , Proteína do Grupo de Complementação N da Anemia de Fanconi/genética , Feminino , Genes BRCA1 , Genes BRCA2 , Humanos , Camundongos , Proteínas Supressoras de Tumor/genéticaRESUMO
'BRCAness' is a term used to describe cancer cells that behave similarly to tumors with BRCA1 or BRCA2 mutations. The BRCAness phenotype is associated with hypersensitivity to chemotherapy agents including PARP inhibitors, which are a promising class of recently-licensed anti-cancer treatments. This hypersensitivity arises because of a deficiency in the homologous recombination (HR) pathway for DNA double-strand break repair. To gain further insight into how genetic modifiers of HR contribute to the BRCAness phenotype, we created a new mouse model of BRCAness by generating mice that are deficient in BLM helicase and the Exo1 exonuclease, which are involved in the early stages of HR. We find that cells lacking BLM and Exo1 exhibit a BRCAness phenotype, with diminished HR, and hypersensitivity to PARP inhibitors. We further tested how 53BP1, an important regulator of HR, affects repair efficiency in our BRCAness model. We find that deletion of 53BP1 can relieve several of the repair deficiencies observed in cells lacking BLM and Exo1, just as it does in cells lacking BRCA1. These results substantiate the importance of BRCAness as a concept for classification of cancer cases, and further clarify the role of 53BP1 in regulation of DNA repair pathway choice in mammalian cells.
Assuntos
Enzimas Reparadoras do DNA/genética , Reparo do DNA/efeitos dos fármacos , Exodesoxirribonucleases/genética , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , RecQ Helicases/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Linfócitos B/patologia , Linfócitos B/efeitos da radiação , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , DNA/genética , DNA/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Enzimas Reparadoras do DNA/deficiência , Exodesoxirribonucleases/deficiência , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos da radiação , Raios gama , Deleção de Genes , Expressão Gênica , Instabilidade Genômica , Humanos , Camundongos , Camundongos Knockout , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Cultura Primária de Células , RecQ Helicases/deficiência , Troca de Cromátide Irmã , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/deficiênciaRESUMO
DNA double-strand breaks (DSBs) arise regularly in cells and when left unrepaired cause senescence or cell death. Homologous recombination (HR) and nonhomologous end-joining (NHEJ) are the two major DNA-repair pathways. Whereas HR allows faithful DSB repair and healthy cell growth, NHEJ has higher potential to contribute to mutations and malignancy. Many regulatory mechanisms influence which of these two pathways is used in DSB repair. These mechanisms depend on the cell cycle, post-translational modifications, and chromatin effects. Here, we summarize current research into these mechanisms, with a focus on mammalian cells, and also discuss repair by "alternative end-joining" and single-strand annealing.
Assuntos
Ciclo Celular , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Recombinação Homóloga , Transdução de Sinais , Animais , HumanosRESUMO
Complete replication of the genome is an essential prerequisite for normal cell division, but a variety of factors can block the replisome, triggering replication stress and potentially causing mutation or cell death. The cellular response to replication stress involves recruitment of proteins to stabilize the replication fork and transmit a stress signal to pause the cell cycle and allow fork restart. We find that the ubiquitously expressed DNA damage response factor 53BP1 is required for the normal response to replication stress. Using primary, ex vivo B cells, we showed that a population of 53BP1-/- cells in early S phase is hypersensitive to short-term exposure to three different agents that induce replication stress. 53BP1 localizes to a subset of replication forks following induced replication stress, and an absence of 53BP1 leads to defective ATR-Chk1-p53 signaling and caspase 3-mediated cell death. Nascent replicated DNA additionally undergoes degradation in 53BP1-/- cells. These results show that 53BP1 plays an important role in protecting replication forks during the cellular response to replication stress, in addition to the previously characterized role of 53BP1 in DNA double-strand break repair.
Assuntos
Quinase 1 do Ponto de Checagem/genética , Replicação do DNA/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Linfócitos B/fisiologia , Caspase 3/genética , Proteínas de Ciclo Celular/genética , Morte Celular/genética , Divisão Celular/genética , Células Cultivadas , DNA/genética , Dano ao DNA/genética , Reparo do DNA/genética , Células HEK293 , Humanos , Camundongos , Fase S/genética , Transdução de Sinais/genéticaRESUMO
The BLM gene product, BLM, is a RECQ helicase that is involved in DNA replication and repair of DNA double-strand breaks by the homologous recombination (HR) pathway. During HR, BLM has both pro- and anti-recombinogenic activities, either of which may contribute to maintenance of genomic integrity. We find that in cells expressing a mutant version of BRCA1, an essential HR factor, ablation of BLM rescues genomic integrity and cell survival in the presence of DNA double-strand breaks. Improved genomic integrity in these cells is linked to a substantial increase in the stability of RAD51 at DNA double-strand break sites and in the overall efficiency of HR. Ablation of BLM also rescues RAD51 foci and HR in cells lacking BRCA2 or XRCC2. These results indicate that the anti-recombinase activity of BLM is of general importance for normal retention of RAD51 at DNA break sites and regulation of HR.
Assuntos
Quebras de DNA de Cadeia Dupla , Linfócitos/enzimologia , Neoplasias/enzimologia , Rad51 Recombinase/metabolismo , RecQ Helicases/metabolismo , Reparo de DNA por Recombinação , Animais , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/deficiência , Proteína BRCA2/genética , Linhagem Celular Tumoral , Sobrevivência Celular , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Instabilidade Genômica , Genótipo , Humanos , Linfócitos/patologia , Camundongos Knockout , Mutação , Neoplasias/genética , Neoplasias/patologia , Fenótipo , Estabilidade Proteica , Interferência de RNA , Rad51 Recombinase/genética , RecQ Helicases/deficiência , RecQ Helicases/genética , Transfecção , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/deficiência , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genéticaRESUMO
BRCA1 mutations strongly predispose affected individuals to breast and ovarian cancer, but the mechanism by which BRCA1 acts as a tumor suppressor is not fully understood. Homozygous deletion of exon 2 of the mouse Brca1 gene normally causes embryonic lethality, but we show that exon 2-deleted alleles of Brca1 are expressed as a mutant isoform that lacks the N-terminal RING domain. This "RING-less" BRCA1 protein is stable and efficiently recruited to the sites of DNA damage. Surprisingly, robust RAD51 foci form in cells expressing RING-less BRCA1 in response to DNA damage, but the cells nonetheless display the substantial genomic instability. Genomic instability can be rescued by the deletion of Trp53bp1, which encodes the DNA damage response factor 53BP1, and mice expressing RING-less BRCA1 do not show an increased susceptibility to tumors in the absence of 53BP1. Genomic instability in cells expressing RING-less BRCA1 correlates with the loss of BARD1 and a defect in restart of replication forks after hydroxyurea treatment, suggesting a role of BRCA1-BARD1 in genomic integrity that is independent of RAD51 loading.
Assuntos
Instabilidade Genômica , Proteínas Supressoras de Tumor/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Animais , Proteína BRCA1 , Sequência de Bases , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA , Éxons/genética , Feminino , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA , Deleção de Sequência , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/deficiência , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genéticaRESUMO
Telomerase is a unique ribonucleoprotein enzyme that is required for continued cell proliferation. To generate catalytically active telomerase, human telomerase reverse transcriptase (hTERT) must translocate to the nucleus and assemble with the RNA component of telomerase. The molecular chaperones heat shock protein 90 (Hsp90) and p23 maintain hTERT in a conformation that enables nuclear translocation. However, the regulatory role of chaperones in nuclear transport of hTERT remains unclear. In this work, we demonstrate that immunophilin FK506-binding protein (FKBP)52 linked the hTERT-Hsp90 complex to the dynein-dynactin motor, thereby promoting the transport of hTERT to the nucleus along microtubules. FKBP52 interacted with the hTERT-Hsp90 complex through binding of the tetratricopeptide repeat domain to Hsp90 and binding of the dynamitin (Dyt) component of the dynein-associated dynactin complex to the peptidyl prolyl isomerase domain. The depletion of FKBP52 inhibited nuclear transport of hTERT, resulting in cytoplasmic accumulation. Cytoplasmic hTERT was rapidly degraded through ubiquitin (Ub)-dependent proteolysis, thereby abrogating telomerase activity. In addition, overexpression of dynamitin, which is known to dissociate the dynein-dynactin motor from its cargoes, reduced telomerase activity. Collectively, these results provide a molecular mechanism by which FKBP52 modulates telomerase activity by promoting dynein-dynactin-dependent nuclear import of hTERT.
Assuntos
Citoplasma/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Ligação a Tacrolimo/metabolismo , Telomerase/metabolismo , Transporte Ativo do Núcleo Celular/genética , Transporte Ativo do Núcleo Celular/fisiologia , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proteínas de Choque Térmico HSP90/genética , Humanos , Immunoblotting , Imunoprecipitação , Prostaglandina-E Sintases/genética , Prostaglandina-E Sintases/metabolismo , Interferência de RNA , Proteínas de Ligação a Tacrolimo/genética , Telomerase/genética , Técnicas do Sistema de Duplo-Híbrido , Ubiquitinação/genética , Ubiquitinação/fisiologiaRESUMO
Sustaining genomic integrity is essential for preventing onset of cancers. Therefore, human cells evolve to have refined biological pathways to defend genetic materials from various genomic insults. DNA damage response and DNA repair pathways essential for genome maintenance are accomplished by cooperative executions of multiple factors including breast cancer type 1 susceptibility protein (BRCA1). BRCA1 is initially identified as an altered gene in the hereditary breast cancer patients. Since then, tremendous efforts to understand the functions of BRAC1 reveal that BRCA1 is found in distinct complexes, including BRCA1-A, BRCA1-B, BRCA1-C, and the BRCA1/PALB2/BRCA2 complex, and plays diverse roles in a context-dependent manner. Among the complexes, BRCA1-A is critical for BRCA1 recruitment to the sites of DNA damage. Factors comprising the BRCA1-A include RAP80, CCDC98/Abraxas, BRCC36, BRCC45, BARD1, BRCA1, and MERIT40, a RAP80-associated factor. In this review, we summarize recent findings of the factors that form the BRCA1-A complex.
Assuntos
Proteína BRCA1/metabolismo , Dano ao DNA , Proteína BRCA1/química , Proteína BRCA1/genética , HumanosRESUMO
The human telomeric protein TRF2 protects chromosome ends by facilitating their organization into the protective capping structure. Here we show that the stability of TRF2 is regulated via modification by the small ubiquitin-like modifiers (SUMO). TRF2 specifically interacts with and is sumoylated by PIAS1 in mammalian cells. The proteasome inhibitor stabilizes SUMO-conjugated TRF2 without affecting the level of unmodified TRF2, suggesting that SUMO conjugation is required for proteasomal degradation of TRF2. We also show that RNF4, a mammalian SUMO-targeted ubiquitin ligase, interacts with TRF2 in a SUMO-dependent manner and preferentially targets SUMO-conjugated TRF2 for ubiquitination. Collectively, our data demonstrate that the PIAS1-mediated sumoylation status of TRF2 serves as a molecular switch that controls the level of TRF2 at telomeres.
Assuntos
Proteínas Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Inibidoras de STAT Ativados/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo , Fatores de Transcrição/metabolismo , Linhagem Celular , Células HEK293 , Humanos , Células MCF-7 , Inibidores de Proteassoma/farmacologia , Sumoilação , Telômero/metabolismo , UbiquitinaçãoRESUMO
Continued cell proliferation requires telomerase to maintain functional telomeres that are essential for chromosome integrity. Although the core enzyme includes a telomerase reverse transcriptase (TERT) and a telomerase RNA component (TERC), a number of auxiliary proteins have been identified to regulate telomerase assembly, localization, and enzymatic activity. Here we describe the characterization of the AAA-ATPase NVL2 as a novel hTERT-interacting protein. NVL2 interacts and co-localizes with hTERT in the nucleolus. NLV2 is also found in association with catalytically competent telomerase in cell lysates through an interaction with hTERT. Depletion of endogenous NVL2 by small interfering RNA led to a decrease in hTERT without affecting the steady-state levels of hTERT mRNA, thereby reducing telomerase activity, suggesting that NVL2 is an essential component of the telomerase holoenzyme. We also found that ATP-binding activity of NVL2 is required for hTERT binding as well as telomerase assembly. Our findings suggest that NVL2, in addition to its role in ribosome biosynthesis, is essential for telomerase biogenesis and provides an alternative approach for inhibiting telomerase activity in cancer.