RESUMO
Minichromosome maintenance (MCM) proteins facilitate replication by licensing origins and unwinding the DNA double strand. Interestingly, the number of MCM hexamers greatly exceeds the number of firing origins suggesting additional roles of MCMs. Here we show a hitherto unanticipated function of MCM2 in cilia formation in human cells and zebrafish that is uncoupled from replication. Zebrafish depleted of MCM2 develop ciliopathy-phenotypes including microcephaly and aberrant heart looping due to malformed cilia. In non-cycling human fibroblasts, loss of MCM2 promotes transcription of a subset of genes, which cause cilia shortening and centriole overduplication. Chromatin immunoprecipitation experiments show that MCM2 binds to transcription start sites of cilia inhibiting genes. We propose that such binding may block RNA polymerase II-mediated transcription. Depletion of a second MCM (MCM7), which functions in complex with MCM2 during its canonical functions, reveals an overlapping cilia-deficiency phenotype likely unconnected to replication, although MCM7 appears to regulate a distinct subset of genes and pathways. Our data suggests that MCM2 and 7 exert a role in ciliogenesis in post-mitotic tissues.
Assuntos
Cílios/genética , DNA Helicases/genética , Componente 2 do Complexo de Manutenção de Minicromossomo/genética , Componente 7 do Complexo de Manutenção de Minicromossomo/genética , Transcrição Gênica , Animais , Cílios/patologia , Ciliopatias/genética , Ciliopatias/patologia , Humanos , Mitose/genética , Sítio de Iniciação de Transcrição , Peixe-Zebra/genéticaRESUMO
Chronic infection of hepatitis B virus (HBV) is associated with an increased incidence of hepatocellular carcinoma (HCC). HBV encodes an oncoprotein, hepatitis B x protein (HBx), that is crucial for viral replication and interferes with multiple cellular activities including gene expression, histone modifications, and genomic stability. To date, it remains unclear how disruption of these activities contributes to hepatocarcinogenesis. Here, we report that HBV exhibits antiresection activity by disrupting DNA end resection, thus impairing the initial steps of homologous recombination (HR). This antiresection activity occurs in primary human hepatocytes undergoing a natural viral infection-replication cycle as well as in cells with integrated HBV genomes. Among the seven HBV-encoded proteins, we identified HBx as the sole viral factor that inhibits resection. By disrupting an evolutionarily conserved Cullin4A-damage-specific DNA binding protein 1-RING type of E3 ligase, CRL4WDR70 , through its H-box, we show that HBx inhibits H2B monoubiquitylation at lysine 120 at double-strand breaks, thus reducing the efficiency of long-range resection. We further show that directly impairing H2B monoubiquitylation elicited tumorigenesis upon engraftment of deficient cells in athymic mice, confirming that the impairment of CRL4WDR70 function by HBx is sufficient to promote carcinogenesis. Finally, we demonstrate that lack of H2B monoubiquitylation is manifest in human HBV-associated HCC when compared with HBV-free HCC, implying corresponding defects of epigenetic regulation and end resection. Conclusion: The antiresection activity of HBx induces an HR defect and genomic instability and contributes to tumorigenesis of host hepatocytes.
Assuntos
Carcinoma Hepatocelular/virologia , Instabilidade Genômica/genética , Hepatite B/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/virologia , Transativadores/genética , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Células Cultivadas , Proteínas de Ligação a DNA/genética , Epigênese Genética , Hepatite B/patologia , Vírus da Hepatite B/genética , Hepatócitos/citologia , Hepatócitos/fisiologia , Humanos , Litostatina/genética , Neoplasias Hepáticas/patologia , Camundongos , Sensibilidade e Especificidade , Proteínas Virais Reguladoras e Acessórias , Replicação Viral/genéticaRESUMO
Protein phosphatase PP4C has been implicated in the DNA damage response (DDR), but its substrates in DDR remain largely unknown. We devised a novel proteomic strategy for systematic identification of proteins dephosphorylated by PP4C and identified KRAB-domain-associated protein 1 (KAP-1) as a substrate. Ionizing radiation leads to phosphorylation of KAP-1 at S824 (via ATM) and at S473 (via CHK2). A PP4C/R3ß complex interacts with KAP-1 and silencing this complex leads to persistence of phospho-S824 and phospho-S473. We identify a new role for KAP-1 in DDR by showing that phosphorylation of S473 impacts the G2/M checkpoint. Depletion of PP4R3ß or expression of the phosphomimetic KAP-1 S473 mutant (S473D) leads to a prolonged G2/M checkpoint. Phosphorylation of S824 is necessary for repair of heterochromatic DNA lesions and similar to cells expressing phosphomimetic KAP-1 S824 mutant (S824D), or PP4R3ß-silenced cells, display prolonged relaxation of chromatin with release of chromatin remodelling protein CHD3. Our results define a new role for PP4-mediated dephosphorylation in the DDR, including the regulation of a previously undescribed function of KAP-1 in checkpoint response.
Assuntos
Dano ao DNA , Fosfoproteínas Fosfatases/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Repressoras/metabolismo , Divisão Celular , DNA/efeitos da radiação , Fase G2 , Células HeLa , Humanos , Modelos Biológicos , Fosforilação , Radiação Ionizante , Proteína 28 com Motivo TripartidoRESUMO
Accumulation of DNA damage leading to adult stem cell exhaustion has been proposed to be a principal mechanism of ageing. Here we address this question by taking advantage of the highly specific role of DNA ligase IV in the repair of DNA double-strand breaks by non-homologous end-joining, and by the discovery of a unique mouse strain with a hypomorphic Lig4(Y288C) mutation. The Lig4(Y288C) mouse, identified by means of a mutagenesis screening programme, is a mouse model for human LIG4 syndrome, showing immunodeficiency and growth retardation. Diminished DNA double-strand break repair in the Lig4(Y288C) strain causes a progressive loss of haematopoietic stem cells and bone marrow cellularity during ageing, and severely impairs stem cell function in tissue culture and transplantation. The sensitivity of haematopoietic stem cells to non-homologous end-joining deficiency is therefore a key determinant of their ability to maintain themselves against physiological stress over time and to withstand culture and transplantation.
Assuntos
Envelhecimento/fisiologia , Reparo do DNA , Células-Tronco Hematopoéticas/citologia , Animais , Proliferação de Células , Senescência Celular/fisiologia , Quebras de DNA de Cadeia Dupla , Dano ao DNA , DNA Ligase Dependente de ATP , DNA Ligases/deficiência , DNA Ligases/genética , DNA Ligases/metabolismo , Células-Tronco Hematopoéticas/patologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Mutação de Sentido Incorreto/efeitos dos fármacos , Mutação de Sentido Incorreto/genética , SíndromeRESUMO
Purpose: The ongoing SARS-CoV-2 pandemic has resulted in over 6.3 million deaths and 560 million COVID-19 cases worldwide. Clinical management of hospitalized patients is complex due to the heterogeneous course of COVID-19. Low-dose radiation therapy is known to dampen localized chronic inflammation and has been suggested to be used to reduce lung inflammation in patients with COVID-19. However, it is unknown whether SARS-CoV-2 alters the radiation response and associated radiation exposure related risk. Methods and Materials: We generated gene expression profiles from circulating leukocytes of hospitalized patients with COVID-19 and healthy donors. Results: The p53 signaling pathway was found to be dysregulated, with mRNA levels of p53, ATM, and CHK2 being lower in patients with COVID-19. Several key p53 target genes involved in cell cycle arrest, apoptosis, and p53 feedback inhibition were upregulated in patients with COVID-19 while other p53 target genes were downregulated. This dysregulation has functional consequences as the transcription of p53-dependant genes (CCNG1, GADD45A, DDB2, SESN1, FDXR, APOBEC) was reduced 24 hours after x-ray exposure ex vivo to both low (100 mGy) or high (2 Gy) doses. Conclusions: SARS-CoV-2 infection affects a DNA damage response that may modify radiation-induced health risks in exposed patients with COVID-19.
RESUMO
Purpose: Humans are increasingly exposed to ionizing radiation (IR). Both low (<100 mGy) and high doses can cause stochastic effects, including cancer; whereas doses above 100 mGy are needed to promote tissue or cell damage. 10-15% of radiotherapy (RT) patients suffer adverse reactions, described as displaying radiosensitivity (RS). Sensitivity to IR's stochastic effects is termed radiosusceptibility (RSu). To optimize radiation protection we need to understand the range of individual variability and underlying mechanisms. We review the potential mechanisms contributing to RS/RSu focusing on RS following RT, the most tractable RS group.Conclusions: The IR-induced DNA damage response (DDR) has been well characterized. Patients with mutations in the DDR have been identified and display marked RS but they represent only a small percentage of the RT patients with adverse reactions. We review the impacting mechanisms and additional factors influencing RS/RSu. We discuss whether RS/RSu might be genetically determined. As a recommendation, we propose that a prospective study be established to assess RS following RT. The study should detail tumor site and encompass a well-defined grading system. Predictive assays should be independently validated. Detailed analysis of the inflammatory, stress and immune responses, mitochondrial function and life style factors should be included. Existing cohorts should also be optimally exploited.
Assuntos
Neoplasias Induzidas por Radiação/diagnóstico , Radiação Ionizante , Transporte Ativo do Núcleo Celular , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Carbono/metabolismo , Ciclo Celular , Dano ao DNA , Relação Dose-Resposta à Radiação , Humanos , Neoplasias/radioterapia , Estresse Oxidativo , Oxigênio/metabolismo , Lesões por Radiação , Proteção Radiológica , Tolerância a Radiação , Radioterapia , Processos EstocásticosRESUMO
Radiosusceptibility is the sensitivity of a biological organism to ionising radiation (IR)-induced carcinogenesis, an outcome of IR exposure relevant following low doses. The tissue response is strongly influenced by the DNA damage response (DDR) activated in stem and progenitor cells. We previously reported that in vivo exposure to 2 Gy X-rays activates apoptosis, proliferation arrest and premature differentiation in neural progenitor cells (transit amplifying cells and neuroblasts) but not in neural stem cells (NSCs) of the largest neurogenic region of the adult brain, the subventricular zone (SVZ). These responses promote adult quiescent NSC (qNSC) activation after 2 Gy. In contrast, neonatal (P5) SVZ neural progenitors continue proliferating and do not activate qNSCs. Significantly, the human and mouse neonatal brain is radiosusceptible. Here, we examine the response of stem and progenitor cells in the SVZ to low IR doses (50-500 mGy). We observe a linear dose-response for apoptosis but, in contrast, proliferation arrest and neuroblast differentiation require a threshold dose of 200 or 500 mGy, respectively. Importantly, qNSCs were not activated at doses below 500 mGy. Thus, full DDR activation in the neural stem cell compartment in vivo necessitates a threshold dose, which can be considered of significance when evaluating IR-induced cancer risk and dose extrapolation.
Assuntos
Células-Tronco Adultas/citologia , Células-Tronco Adultas/efeitos da radiação , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos da radiação , Animais , Proliferação de Células/efeitos da radiação , Relação Dose-Resposta à Radiação , CamundongosRESUMO
Meier-Gorlin syndrome (MGS) is a rare, congenital primordial microcephalic dwarfism disorder. MGS is caused by genetic variants of components of the origin recognition complex (ORC) consisting of ORC1-6 and the pre-replication complex, which together enable origin firing and hence genome replication. In addition, ORC1 has previously been shown to play a role in ciliogenesis. Here, we extend this work and investigate the function of ORC1 and two other members of the complex on cilia at an organismal level. Knockdown experiments in zebrafish confirmed the impact of ORC1 on cilia. ORC1-deficiency confers defects anticipated to arise from impaired cilia function such as formation of oedema, kidney cysts, curved bodies and left-right asymmetry defects. We found ORC1 furthermore required for cilium formation in zebrafish and demonstrate that ciliopathy phenotypes in ORC1-depleted zebrafish could not be rescued by reconstitution with ORC1 bearing a genetic variant previously identified in MGS patients. Loss-of-function of Orc4 and Orc6, respectively, conferred similar ciliopathy phenotypes and cilium shortening in zebrafish, suggesting that several, if not all, components of the ORC regulate ciliogenesis downstream to or in addition to their canonical function in replication initiation. This study presents the first in vivo evidence of an influence of the MGS genes of the ORC family on cilia, and consolidates the possibility that cilia dysfunction could contribute to the clinical manifestation of ORC-deficient MGS.
Assuntos
Cílios/metabolismo , Embrião não Mamífero/metabolismo , Complexo de Reconhecimento de Origem/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Ciliopatias/genética , Organogênese , FenótipoRESUMO
Double-strand breaks (DSBs) arise endogenously during normal cellular processes and exogenously by genotoxic agents such as ionizing radiation (IR). DSBs are one of the most severe types of DNA damage, which if left unrepaired are lethal to the cell. Several different DNA repair pathways combat DSBs, with nonhomologous end-joining (NHEJ) being one of the most important in mammalian cells. Competent NHEJ catalyses repair of DSBs by joining together and ligating two free DNA ends of little homology (microhomology) or DNA ends of no homology. The core components of mammalian NHEJ are the catalytic subunit of DNA protein kinase (DNA-PK(cs)), Ku subunits Ku70 and Ku80, Artemis, XRCC4 and DNA ligase IV. DNA-PK is a nuclear serine/threonine protein kinase that comprises a catalytic subunit (DNA-PK(cs)), with the Ku subunits acting as the regulatory element. It has been proposed that DNA-PK is a molecular sensor for DNA damage that enhances the signal via phosphorylation of many downstream targets. The crucial role of DNA-PK in the repair of DSBs is highlighted by the hypersensitivity of DNA-PK(-/-) mice to IR and the high levels of unrepaired DSBs after genotoxic insult. Recently, DNA-PK has emerged as a suitable genetic target for molecular therapeutics such as siRNA, antisense and novel inhibitory small molecules. This review encompasses the recent literature regarding the role of DNA-PK in the protection of genomic stability and focuses on how this knowledge has aided the development of specific DNA-PK inhibitors, via both small molecule and directed molecular targeting techniques. This review promotes the inhibition of DNA-PK as a valid approach to enhance the tumor-cell-killing effects of treatments such as IR.
Assuntos
Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/farmacologia , Terapia Genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/farmacologia , DNA , Proteína Quinase Ativada por DNA , Humanos , Neoplasias/genética , Proteínas Nucleares , Interferência de RNARESUMO
53BP1 plays multiple roles in mammalian DNA damage repair, mediating pathway choice and facilitating DNA double-strand break repair in heterochromatin. Although it possesses a C-terminal BRCT2 domain, commonly involved in phospho-peptide binding in other proteins, initial recruitment of 53BP1 to sites of DNA damage depends on interaction with histone post-translational modifications--H4K20me2 and H2AK13/K15ub--downstream of the early γH2AX phosphorylation mark of DNA damage. We now show that, contrary to current models, the 53BP1-BRCT2 domain binds γH2AX directly, providing a third post-translational mark regulating 53BP1 function. We find that the interaction of 53BP1 with γH2AX is required for sustaining the 53BP1-dependent focal concentration of activated ATM that facilitates repair of DNA double-strand breaks in heterochromatin in G1.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Reparo do DNA/fisiologia , Heterocromatina/metabolismo , Histonas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Animais , Proteínas Cromossômicas não Histona/metabolismo , Cristalografia por Raios X , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/metabolismo , Imunofluorescência , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Processamento de Proteína Pós-Traducional , Estrutura Quaternária de Proteína , RNA Interferente Pequeno , Transfecção , Proteína 1 de Ligação à Proteína Supressora de Tumor p53RESUMO
A DNA double-strand break (DSB) has long been recognized as a severe cellular lesion, potentially representing an initiating event for carcinogenesis or cell death. The evolution of DSB repair pathways as well as additional processes, such as cell cycle checkpoint arrest, to minimize the cellular impact of DSB formation was, therefore, not surprising. However, the depth and complexity of the DNA damage responses being revealed by current studies were unexpected. Perhaps the most surprising finding to emerge is the dramatic changes to chromatin architecture that arise in the DSB vicinity. In this review, we overview the cellular response to DSBs focusing on DNA repair pathways and the interface between them. We consider additional events which impact upon these DSB repair pathways, including regulated arrest of cell cycle progression and chromatin architecture alterations. Finally, we discuss the impact of defects in these processes to human disease.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Transdução de Sinais , Animais , Reparo do DNA por Junção de Extremidades , Doença/genética , Humanos , Recombinação Genética , Reparo de DNA por RecombinaçãoRESUMO
Previous studies have shown that dormant licensed replication origins can be exploited to enhance recovery from replication stress. Since tumor cells express high levels of origin-licensing proteins, we examined whether depletion of such factors might specifically sensitize tumor versus nontumor cells. Consistent with previous findings, we observed that three tumor-derived cell lines overexpress ORC1, a licensing component, compared with four nontumor cell lines and that a greater level of ORC1 was required to maintain viability in the tumor cells. We determined siRNA-mediated knockdown conditions for each line that maximally reduced ORC1 but did not impact upon viability, which we considered would optimally deplete dormant origins. ORC1 depletion hypersensitized the tumor-derived cells to hydroxyurea and H202 but did not affect the sensitivity of the nontumor lines. Similar results were observed following depletion of ORC6 or CDC6. Furthermore, codepletion of p53 and ORC1 modestly impaired viability of 1BR3hTERT nontumor fibroblasts and more dramatically caused hypersensitivity to hydroxyurea. Finally, overexpression of the c-Myc oncogene combined with ORC1 depletion in nontumor BJhTERT cells diminished viability. Collectively, these findings suggest that tumor cells may have a reliance on origin-licensing capacity, suggesting that licensing factors could represent a target for drug-based cancer therapy.
Assuntos
Neoplasias Ósseas/genética , Proteínas de Ciclo Celular/genética , Dano ao DNA , Replicação do DNA/fisiologia , Osteossarcoma/genética , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Proteínas de Ciclo Celular/metabolismo , Processos de Crescimento Celular/fisiologia , Técnicas de Silenciamento de Genes , Humanos , Osteossarcoma/metabolismo , Osteossarcoma/patologia , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , TransfecçãoRESUMO
DNA double strand breaks (DSBs) induced by ionizing radiation (IR) are deleterious damages. Two major pathways repair DSBs in human cells, DNA non-homologous end-joining (NHEJ) and homologous recombination (HR). It has been suggested that the balance between the two repair pathways varies depending on the chromatin structure surrounding the damage site and/or the complexity of damage at the DNA break ends. Heavy ion radiation is known to induce complex-type DSBs, and the efficiency of NHEJ in repairing these DSBs was shown to be diminished. Taking advantage of the ability of high linear energy transfer (LET) radiation to produce complex DSBs effectively, we investigated how the complexity of DSB end structure influences DNA damage responses. An early step in HR is the generation of 3'-single strand DNA (SSD) via a process of DNA end resection that requires CtIP. To assess this process, we analyzed the level of phosphorylated CtIP, as well as RPA phosphorylation and focus formation, which occur on the exposed SSD. We show that complex DSBs efficiently activate DNA end resection. After heavy ion beam irradiation, resection signals appear both in the vicinity of heterochromatic areas, which is also observed after X-irradiation, and additionally in euchromatic areas. Consequently, ~85% of complex DSBs are subjected to resection in heavy ion particle tracks. Furthermore, around 20-40% of G1 cells exhibit resection signals. Taken together, our observations reveal that the complexity of DSB ends is a critical factor regulating the choice of DSB repair pathway and drastically alters the balance toward resection-mediated rejoining. As demonstrated here, studies on DNA damage responses induced by heavy ion radiation provide an important tool to shed light on mechanisms regulating DNA end resection.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades/genética , Reparo de DNA por Recombinação , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Transporte/metabolismo , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Ciclo Celular/efeitos da radiação , Linhagem Celular , DNA de Cadeia Simples/metabolismo , Endodesoxirribonucleases , Células HeLa , Heterocromatina/genética , Heterocromatina/metabolismo , Humanos , Transferência Linear de Energia , Camundongos , Morfolinas/farmacologia , Proteínas Nucleares/metabolismo , Fosforilação , Pironas/farmacologia , Radiação Ionizante , Proteína de Replicação A/metabolismoRESUMO
KAP-1 poses a substantial barrier to DNA double-strand break (DSB) repair within heterochromatin that is alleviated by ATM-dependent KAP-1 phosphorylation (pKAP-1). Here we address the mechanistic consequences of pKAP-1 that promote heterochromatic DSB repair and chromatin relaxation. KAP-1 function involves autoSUMOylation and recruitment of nucleosome deacetylation, methylation and remodeling activities. Although heterochromatin acetylation or methylation changes were not detected, radiation-induced pKAP-1 dispersed the nucleosome remodeler CHD3 from DSBs and triggered concomitant chromatin relaxation; pKAP-1 loss reversed these effects. Depletion or inactivation of CHD3, or ablation of its interaction with KAP-1(SUMO1), bypassed pKAP-1's role in repair. Though KAP-1 SUMOylation was unaffected after irradiation, CHD3 dissociated from KAP-1(SUMO1) in a pKAP-1-dependent manner. We demonstrate that KAP-1(Ser824) phosphorylation generates a motif that directly perturbs interactions between CHD3's SUMO-interacting motif and SUMO1, dispersing CHD3 from heterochromatin DSBs and enabling repair.
Assuntos
Quebras de DNA de Cadeia Dupla , DNA Helicases/fisiologia , Reparo do DNA , Proteínas Nucleares/fisiologia , Nucleossomos/metabolismo , Proteínas Repressoras/fisiologia , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/fisiologia , DNA Helicases/química , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/fisiologia , Células HEK293 , Células HeLa , Heterocromatina/metabolismo , Histonas/metabolismo , Humanos , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase , Camundongos , Morfolinas/farmacologia , Células NIH 3T3 , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Nucleossomos/efeitos dos fármacos , Fosforilação , Proteínas Serina-Treonina Quinases/fisiologia , Pironas/farmacologia , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Proteína SUMO-1/química , Proteína SUMO-1/metabolismo , Proteína SUMO-1/fisiologia , Sumoilação , Proteína 28 com Motivo Tripartido , Proteínas Supressoras de Tumor/fisiologiaRESUMO
DNA double-strand breaks (DSBs) trigger ATM (ataxia telangiectasia mutated) signalling and elicit genomic rearrangements and chromosomal fragmentation if misrepaired or unrepaired. Although most DSB repair is ATM-independent, approximately 15% of ionizing radiation (IR)-induced breaks persist in the absence of ATM-signalling. 53BP1 (p53-binding protein 1) facilitates ATM-dependent DSB repair but is largely dispensable for ATM activation or checkpoint arrest. ATM promotes DSB repair within heterochromatin by phosphorylating KAP-1 (KRAB-associated protein 1, also known as TIF1beta, TRIM28 or KRIP-1; ref. 2). Here, we show that the ATM signalling mediator proteins MDC1, RNF8, RNF168 and 53BP1 are also required for heterochromatic DSB repair. Although KAP-1 phosphorylation is critical for 53BP1-mediated repair, overall phosphorylated KAP-1 (pKAP-1) levels are only modestly affected by 53BP1 loss. pKAP-1 is transiently pan-nuclear but also forms foci overlapping with gammaH2AX in heterochromatin. Cells that do not form 53BP1 foci, including human RIDDLE (radiosensitivity, immunodeficiency, dysmorphic features and learning difficulties) syndrome cells, fail to form pKAP-1 foci. 53BP1 amplifies Mre11-NBS1 accumulation at late-repairing DSBs, concentrating active ATM and leading to robust, localized pKAP-1. We propose that ionizing-radiation induced foci (IRIF) spatially concentrate ATM activity to promote localized alterations in regions of chromatin otherwise inhibitory to repair.
Assuntos
Reparo do DNA/fisiologia , Heterocromatina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas Repressoras/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Células Cultivadas , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Reparo do DNA/genética , Enzimas Reparadoras do DNA , Proteínas de Ligação a DNA/metabolismo , Imunofluorescência , Heterocromatina/efeitos da radiação , Humanos , Immunoblotting , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteína Homóloga a MRE11 , Camundongos , Células NIH 3T3 , Proteínas Nucleares/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Radiação Ionizante , Transativadores/metabolismo , Proteína 28 com Motivo Tripartido , Proteínas Supressoras de Tumor/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53 , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Hypomorphic mutations in DNA ligase IV (LIG4) cause a human syndrome of immunodeficiency, radiosensitivity, and growth retardation due to defective DNA repair by the nonhomologous end-joining (NHEJ) pathway. Lig4-null mice are embryonic lethal, and better mouse models are needed to study human LigIV syndrome. We recently identified a viable mouse strain with a Y288C hypomorphic mutation in the Lig4 gene. Lig4Y288C mice exhibit a greater than 10-fold reduction of LigIV activity in vivo and recapitulate the immunodeficiency and growth retardation seen in human patients. Here, we have demonstrated that the Lig4Y288C mutation leads to multiple defects in lymphocyte development and function, including impaired V(D)J recombination, peripheral lymphocyte survival and proliferation, and B cell class switch recombination. We also highlight a high incidence of thymic tumors in the Lig4Y288C mice, suggesting that wild-type LigIV protects against malignant transformation. These findings provide explanations for the complex lymphoid phenotype of human LigIV syndrome.
Assuntos
Diferenciação Celular/imunologia , DNA Ligases/metabolismo , Switching de Imunoglobulina/imunologia , Isotipos de Imunoglobulinas/imunologia , Linfócitos/enzimologia , Linfócitos/imunologia , Neoplasias do Timo/enzimologia , Animais , Formação de Anticorpos/imunologia , Sobrevivência Celular , DNA Ligases/deficiência , DNA Ligases/genética , Modelos Animais de Doenças , Linfócitos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação/genética , Síndrome , Neoplasias do Timo/genética , Neoplasias do Timo/imunologia , Neoplasias do Timo/patologiaRESUMO
Chromosomal instability plays a pivotal role in multistep carcinogenesis by facilitating the acquisition of the multiple genetic alterations necessary for malignant transformation. In order to study the role of abnormal DNA repair in malignant melanoma, we measured the ability of cell lines from malignant melanoma and that of primary melanocytes to process 4 different kinds of DNA damage (pyrimidine dimers, oxidative DNA lesions, replication errors, and DNA double-strand breaks) using 4 different plasmid assays. Based on the number of chromosomes, the DNA index, and the rates of spontaneous micronuclei, the chromosomal stability in primary melanocytes and the melanoma line LIBR was characterized as being high, intermediate in M1, and low in MeWo. Repair of UVB photoproducts, of oxidative DNA damage, and of replication errors was not impaired in any melanoma line. Using linearized shuttle vector plasmid pZ189, LIBR cells and primary melanocytes exhibited a high efficiency of joining overlapping DNA ends, reflecting proficient repair of DNA double-strand breaks. Joining efficiency was reduced slightly in M1 and 2.9-fold in MeWo. This indicates that in the melanoma cell lines studied here, an increase in chromosomal instability is accompanied by a pronounced impairment in the ability to join DNA ends. Although a small sample was studied, this raises the possibility that defects in DNA end joining may also contribute to genetic instability and chromosome aberrations in melanoma.