RESUMO
Enoxaparin and daikenchuto are commonly administered to prevent venous thromboembolism and intestinal obstruction after gynecological malignancy surgery. However, the effects of their combined use on hepatic function are not well studied. This study aimed to clarify the effects of the coadministration of enoxaparin and daikenchuto on hepatic function. First, Japanese Adverse Drug Event Report (JADER) data were analyzed to identify signals of hepatic disorders. Second, a retrospective observational study of patients who underwent surgery for gynecological malignancies was conducted. This study defined hepatic disorders as an increase in aspartate aminotransferase (AST) or alanine aminotransaminase (ALT) levels above the reference values, using 1-h postoperative values as the baseline. The analysis of JADER data revealed an increased risk for hepatic disorders with the coadministration of enoxaparin and daikenchuto. An observational study also showed higher odds ratios (95% confidence intervals) for the occurrence of hepatic disorders in the coadministration group (4.27; 2.11-8.64) and enoxaparin alone group (2.48; 1.31-4.69) than in the daikenchuto alone group. The median increase in the ALT level was also higher in the coadministration group (34; 15-59) than in the enoxaparin alone (19; 6-38) and daikenchuto alone groups (8; 3-33). In conclusion, our study suggests that compared with the use of enoxaparin or daikenchuto alone, enoxaparin and daikenchuto coadministration increases the risk of hepatic disorders, with more significant increases in AST and ALT levels. Healthcare workers need to be aware of these potential side effects when combining these drugs after surgery for gynecological malignancies.
Assuntos
Neoplasias dos Genitais Femininos , Panax , Extratos Vegetais , Zanthoxylum , Zingiberaceae , Feminino , Humanos , Enoxaparina/efeitos adversos , Neoplasias dos Genitais Femininos/cirurgia , Neoplasias dos Genitais Femininos/tratamento farmacológico , Anticoagulantes/efeitos adversos , Complicações Pós-Operatórias/prevenção & controle , Complicações Pós-Operatórias/induzido quimicamente , Complicações Pós-Operatórias/tratamento farmacológicoRESUMO
DNA-protein crosslinks (DPCs) induced by aldehydes interfere with replication and transcription. Hereditary deficiencies in DPC repair and aldehyde clearance processes cause progeria, including Ruijs-Aalfs syndrome (RJALS) and AMeD syndrome (AMeDS) in humans. Although the elimination of DPC during replication has been well established, how cells overcome DPC lesions in transcription remains elusive. Here we show that endogenous aldehyde-induced DPC roadblocks are efficiently resolved by transcription-coupled repair (TCR). We develop a high-throughput sequencing technique to measure the genome-wide distribution of DPCs (DPC-seq). Using proteomics and DPC-seq, we demonstrate that the conventional TCR complex as well as VCP/p97 and the proteasome are required for the removal of formaldehyde-induced DPCs. TFIIS-dependent cleavage of RNAPII transcripts protects against transcription obstacles. Finally, a mouse model lacking both aldehyde clearance and TCR confirms endogenous DPC accumulation in actively transcribed regions. Collectively, our data provide evidence that transcription-coupled DPC repair (TC-DPCR) as well as aldehyde clearance are crucial for protecting against metabolic genotoxin, thus explaining the molecular pathogenesis of AMeDS and other disorders associated with defects in TCR, such as Cockayne syndrome.
Assuntos
Aldeídos , Reparo do DNA , Transcrição Gênica , Animais , Humanos , Aldeídos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Camundongos , DNA/metabolismo , DNA/genética , Dano ao DNA , Camundongos Knockout , Proteína com Valosina/metabolismo , Proteína com Valosina/genética , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Camundongos Endogâmicos C57BL , Formaldeído/toxicidade , Formaldeído/farmacologia , Reparo por ExcisãoRESUMO
CRISPR/Cas9-mediated gene editing has great potential utility for treating genetic diseases. However, its therapeutic applications are limited by unintended genomic alterations arising from DNA double-strand breaks and random integration of exogenous DNA. In this study, we propose NICER, a method for correcting heterozygous mutations that employs multiple nicks (MNs) induced by Cas9 nickase and a homologous chromosome as an endogenous repair template. Although a single nick near the mutation site rarely leads to successful gene correction, additional nicks on homologous chromosomes strongly enhance gene correction efficiency via interhomolog homologous recombination (IH-HR). This process partially depends on BRCA1 and BRCA2, suggesting the existence of several distinct pathways for MN-induced IH-HR. According to a genomic analysis, NICER rarely induces unintended genomic alterations. Furthermore, NICER restores the expression of disease-causing genes in cells derived from genetic diseases with compound heterozygous mutations. Overall, NICER provides a precise strategy for gene correction.
Assuntos
Antibacterianos , Recombinação Homóloga , Mutação , Quebras de DNA de Cadeia Dupla , Desoxirribonuclease IRESUMO
Xeroderma pigmentosum (XP) is a genodermatosis defined by cutaneous photosensitivity with an increased risk of skin tumors because of DNA repair deficiency. The worldwide prevalence of XP is ~1 to 4 in million, with higher incidence in some countries and regions including Japan (1 in 22,000) and North Africa due to founder mutations and a high degree of consanguinity. Among XP, the complementation group F (XP-F), is a rare form (1% of worldwide XP); however, this is underdiagnosed, because the ERCC4/XPF gene is essential for fetal development and most of previously reported ERCC4/XPF pathogenic variants are hypomorphs causing relatively mild phenotypes. From the largest Japanese XP cohort study, we report 17 XP-F cases bearing two pathogenic variants, both identified in deep intronic regions of the ERCC4/XPF gene. The first variant, located in intron 1, is a Japanese founder mutation, which additionally accounts for ~10% of the entire Japanese XP cases (MAF = 0.00196), causing an aberrant pre-mRNA splicing due to a miss-binding of U1snRNA. The second mutation located in intron eight induces an alternative polyadenylation. Both mutations cause a reduction of the ERCC4/XPF gene expression, resulting in XP clinical manifestations. Most cases developed early-onset skin cancers, indicating that these variants need critical attention. We further demonstrate that antisense oligonucleotides designed for the mutations can restore the XPF protein expression and DNA repair capacity in the patients' cells. Collectively, these pathogenic variants can be potential therapeutic targets for XP.
Assuntos
Dermatite , Xeroderma Pigmentoso , Humanos , Xeroderma Pigmentoso/genética , Xeroderma Pigmentoso/terapia , Xeroderma Pigmentoso/metabolismo , Reparo do DNA/genética , Íntrons/genética , Estudos de Coortes , Mutação , Dermatite/genéticaRESUMO
We present a mild case of Cockayne syndrome that was referred to us with an extreme sunburn at the age of 3. In early teens, although her cutaneous symptoms alleviated without any medications, she developed tremor and dysarthria. Neurological examination and brain imaging suggested demyelination disorders. The patient's cells indicated a reduced recovery of RNA synthesis, which was partially restored by the introduction of CSB (Cockayne Syndrome B)-cDNA. In addition, her cells indicated a substantially reduced level of CSB protein. Despite the insidious progression of neurological symptoms, she gave birth to a child. Such mild cases of Cockayne syndrome may be misdiagnosed.
Assuntos
Síndrome de Cockayne , Reparo do DNA , Humanos , Feminino , Criança , Adolescente , Síndrome de Cockayne/complicações , Síndrome de Cockayne/diagnóstico , Síndrome de Cockayne/genéticaRESUMO
Aicardi-Goutières syndrome (AGS) is a rare genetic disorder characterised by progressive encephalopathy, involving microcephaly, intracranial calcification, and cerebrospinal fluid lymphocytosis with increased interferon-α concentrations. The clinical features of AGS overlap with fetal cerebral anomalies caused by congenital infections, such as TORCH (toxoplasmosis, other, rubella, cytomegalovirus, and herpes), or with those of other genetic disorders showing neonatal microcephaly, including Cockayne syndrome (CS) with transcription-coupled DNA repair deficiency, and Seckel syndrome (SS) showing aberrant cell-cycle checkpoint signaling. Therefore, a differential diagnosis to confirm the genetic cause or a proof of infection should be considered. In this report, we describe an individual who showed primordial dwarfism and encephalopathy, and whose initial diagnosis was CS. First, we conducted conventional DNA repair proficiency tests for the patient derived fibroblast cells. Transcription-coupled nucleotide excision repair (TC-NER) activity, which is mostly compromised in CS cases, was slightly reduced in the patient's cells. However, unscheduled DNA synthesis (UDS) was significantly diminished. These cellular traits were inconsistent with the diagnosis of CS. We further performed whole exome sequencing for the case and identified a compound heterozygous loss-of-function variants in the SAMHD1 gene, mutations in which are known to cause AGS. As SAMHD1 encodes deoxyribonucleoside triphosphate triphosphohydrolase, we reasoned that the deoxyribonucleoside triphosphate (dNTP) pool size in the patient's cells was elevated, and the labeling efficiency of UDS-test was hindered due to the reduced concentration of phosphorylated ethynyl deoxyuridine (EdU), a nucleoside analogue used for the assay. In conclusion, UDS assay may be a useful diagnostic tool to distinguish between AGS with SAMHD1 mutations and other related diseases.
RESUMO
Transcription-blocking DNA lesions (TBLs) in genomic DNA are triggered by a wide variety of DNA-damaging agents. Such lesions cause stalling of elongating RNA polymerase II (RNA Pol II) enzymes and fully block transcription when unresolved. The toxic impact of DNA damage on transcription progression is commonly referred to as transcription stress. In response to RNA Pol II stalling, cells activate and employ transcription-coupled repair (TCR) machineries to repair cytotoxic TBLs and resume transcription. Increasing evidence indicates that the modification and processing of stalled RNA Pol II is an integral component of the cellular response to and the repair of TBLs. If TCR pathways fail, the prolonged stalling of RNA Pol II will impede global replication and transcription as well as block the access of other DNA repair pathways that may act upon the TBL. Consequently, such prolonged stalling will trigger profound genome instability and devastating clinical features. In this review, we will discuss the mechanisms by which various types of TBLs are repaired by distinct TCR pathways and how RNA Pol II processing is regulated during these processes. We will also discuss the clinical consequences of transcription stress and genotype-phenotype correlations of related TCR-deficiency disorders.
Assuntos
Dano ao DNA , Reparo do DNA , Instabilidade Genômica , RNA Polimerase II/metabolismo , Transcrição Gênica , Envelhecimento , Animais , DNA/metabolismo , Eucariotos/enzimologia , Eucariotos/genética , Eucariotos/metabolismo , HumanosRESUMO
Cells employ transcription-coupled repair (TCR) to eliminate transcription-blocking DNA lesions. DNA damage-induced binding of the TCR-specific repair factor CSB to RNA polymerase II (RNAPII) triggers RNAPII ubiquitylation of a single lysine (K1268) by the CRL4CSA ubiquitin ligase. How CRL4CSA is specifically directed towards K1268 is unknown. Here, we identify ELOF1 as the missing link that facilitates RNAPII ubiquitylation, a key signal for the assembly of downstream repair factors. This function requires its constitutive interaction with RNAPII close to K1268, revealing ELOF1 as a specificity factor that binds and positions CRL4CSA for optimal RNAPII ubiquitylation. Drug-genetic interaction screening also revealed a CSB-independent pathway in which ELOF1 prevents R-loops in active genes and protects cells against DNA replication stress. Our study offers key insights into the molecular mechanisms of TCR and provides a genetic framework of the interplay between transcriptional stress responses and DNA replication.
Assuntos
Dano ao DNA , Reparo do DNA , Fator 1 de Elongação de Peptídeos/metabolismo , RNA Polimerase II/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , DNA Helicases/genética , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Humanos , Fator 1 de Elongação de Peptídeos/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , RNA Polimerase II/genética , Elongação da Transcrição Genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/genéticaRESUMO
BACK GROUND: Krüppel-like transcription factor 5 (KLF5) is a transcription factor regulating cell proliferation, angiogenesis and differentiation. It has been recently reported that Am80, a synthetic retinoic acid receptor α-specific agonist, inhibits the expression of KLF5. In the present study, we have examined the expression of KLF5 in fibrotic peritoneum induced by chlorhexidine gluconate (CG) in mouse and evaluated that Am80, as an inhibitor of KLF5, can reduce peritoneal fibrosis. METHODS: Peritoneal fibrosis was induced by intraperitoneal injection of CG into peritoneal cavity of ICR mice. Am80 was administered orally for every day from the start of CG injection. Control mice received only a vehicle (0.5% carboxymethylcellulose solution). After 3 weeks of treatment, peritoneal equilibration test (PET) was performed and peritoneal tissues were examined by immunohistochemistry. RESULTS: The expression of KLF5 was less found in the peritoneal tissue of control mice, while KLF5 was expressed in the thickened submesothelial area of CG-injected mice receiving the vehicle. Am80 treatment reduced KLF5 expression and remarkably attenuated peritoneal thickening, accompanied with the reduction of type III collagen expression. The numbers of transforming growth factor ß-positive cells, α-smooth muscle actin-positive cells and infiltrating macrophages were significantly decreased in Am80-treated group. PET revealed the increased peritoneal permeability in CG mice, whereas Am80 administration significantly improved the peritoneal high permeability state. CONCLUSIONS: These results indicate the involvement of KLF5 in the progression of experimental peritoneal fibrosis and suggest that Am80 may be potentially useful for the prevention of peritoneal fibrosis through inhibition of KLF5 expression.
Assuntos
Fatores de Transcrição Kruppel-Like , Diálise Peritoneal , Fibrose Peritoneal , Animais , Fibrose , Fatores de Transcrição Kruppel-Like/antagonistas & inibidores , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Endogâmicos ICR , Fibrose Peritoneal/induzido quimicamente , Fibrose Peritoneal/prevenção & controle , Peritônio/patologiaRESUMO
Rs671 in the aldehyde dehydrogenase 2 gene (ALDH2) is the cause of Asian alcohol flushing response after drinking. ALDH2 detoxifies endogenous aldehydes, which are the major source of DNA damage repaired by the Fanconi anemia pathway. Here, we show that the rs671 defective allele in combination with mutations in the alcohol dehydrogenase 5 gene, which encodes formaldehyde dehydrogenase (ADH5FDH ), causes a previously unidentified disorder, AMeD (aplastic anemia, mental retardation, and dwarfism) syndrome. Cellular studies revealed that a decrease in the formaldehyde tolerance underlies a loss of differentiation and proliferation capacity of hematopoietic stem cells. Moreover, Adh5-/-Aldh2 E506K/E506K double-deficient mice recapitulated key clinical features of AMeDS, showing short life span, dwarfism, and hematopoietic failure. Collectively, our results suggest that the combined deficiency of formaldehyde clearance mechanisms leads to the complex clinical features due to overload of formaldehyde-induced DNA damage, thereby saturation of DNA repair processes.
RESUMO
Nucleotide excision repair (NER) removes helix-destabilizing adducts including ultraviolet (UV) lesions, cyclobutane pyrimidine dimers (CPDs), and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs). In comparison with CPDs, 6-4PPs have greater cytotoxicity and more strongly destabilizing properties of the DNA helix. It is generally believed that NER is the only DNA repair pathway that removes the UV lesions as evidenced by the previous data since no repair of UV lesions was detected in NER-deficient skin fibroblasts. Topoisomerase I (TOP1) constantly creates transient single-strand breaks (SSBs) releasing the torsional stress in genomic duplex DNA. Stalled TOP1-SSB complexes can form near DNA lesions including abasic sites and ribonucleotides embedded in chromosomal DNA. Here we show that base excision repair (BER) increases cellular tolerance to UV independently of NER in cancer cells. UV lesions irreversibly trap stable TOP1-SSB complexes near the UV damage in NER-deficient cells, and the resulting SSBs activate BER. Biochemical experiments show that 6-4PPs efficiently induce stable TOP1-SSB complexes, and the long-patch repair synthesis of BER removes 6-4PPs downstream of the SSB. Furthermore, NER-deficient cancer cell lines remove 6-4PPs within 24 h, but not CPDs, and the removal correlates with TOP1 expression. NER-deficient skin fibroblasts weakly express TOP1 and show no detectable repair of 6-4PPs. Remarkably, the ectopic expression of TOP1 in these fibroblasts led them to completely repair 6-4PPs within 24 h. In conclusion, we reveal a DNA repair pathway initiated by TOP1, which significantly contributes to cellular tolerance to UV-induced lesions particularly in malignant cancer cells overexpressing TOP1.
Assuntos
Quebras de DNA de Cadeia Simples/efeitos da radiação , Reparo do DNA , DNA Topoisomerases Tipo I/metabolismo , Raios Ultravioleta/efeitos adversos , Sistemas CRISPR-Cas/genética , DNA Polimerase beta/genética , DNA Polimerase beta/metabolismo , Fibroblastos , Técnicas de Inativação de Genes , Humanos , Células MCF-7 , Cultura Primária de Células , Pele/citologia , Pele/patologia , Pele/efeitos da radiação , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/genética , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/metabolismo , Xeroderma Pigmentoso/etiologia , Xeroderma Pigmentoso/patologia , Proteína de Xeroderma Pigmentoso Grupo A/genética , Proteína de Xeroderma Pigmentoso Grupo A/metabolismoRESUMO
Transcription-coupled nucleotide excision repair (TC-NER) is initiated by the stalling of elongating RNA polymerase II (RNAPIIo) at DNA lesions. The ubiquitination of RNAPIIo in response to DNA damage is an evolutionarily conserved event, but its function in mammals is unknown. Here, we identified a single DNA damage-induced ubiquitination site in RNAPII at RPB1-K1268, which regulates transcription recovery and DNA damage resistance. Mechanistically, RPB1-K1268 ubiquitination stimulates the association of the core-TFIIH complex with stalled RNAPIIo through a transfer mechanism that also involves UVSSA-K414 ubiquitination. We developed a strand-specific ChIP-seq method, which revealed RPB1-K1268 ubiquitination is important for repair and the resolution of transcriptional bottlenecks at DNA lesions. Finally, RPB1-K1268R knockin mice displayed a short life-span, premature aging, and neurodegeneration. Our results reveal RNAPII ubiquitination provides a two-tier protection mechanism by activating TC-NER and, in parallel, the processing of DNA damage-stalled RNAPIIo, which together prevent prolonged transcription arrest and protect against neurodegeneration.
Assuntos
Reparo do DNA/fisiologia , RNA Polimerase II/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , DNA/metabolismo , Dano ao DNA/fisiologia , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Feminino , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Polimerase II/genética , UbiquitinaçãoRESUMO
BACKGROUND: 3C/Ritscher-Schinzel syndrome is characterised by congenital cranio-cerebello-cardiac dysplasia, where CCDC22 and WASHC5 are accepted as the causative genes. In combination with the retromer or retriever complex, these genes play a role in endosomal membrane protein recycling. We aimed to identify the gene abnormality responsible for the pathogenicity in siblings with a 3C/Ritscher-Schinzel-like syndrome, displaying cranio-cerebello-cardiac dysplasia, coloboma, microphthalmia, chondrodysplasia punctata and complicated skeletal malformation. METHODS: Exome sequencing was performed to identify pathogenic variants. Cellular biological analyses and generation of knockout mice were carried out to elucidate the gene function and pathophysiological significance of the identified variants. RESULTS: We identified compound heterozygous pathogenic variants (c.1097dup; p.Cys366Trpfs*28 and c.2755G>A; p.Ala919Thr) in the VPS35L gene, which encodes a core protein of the retriever complex. The identified missense variant lacked the ability to form the retriever complex, and the frameshift variant induced non-sense-mediated mRNA decay, thereby confirming biallelic loss of function of VPS35L. In addition, VPS35L knockout cells showed decreased autophagic function in nutrient-rich and starvation conditions, as well as following treatment with Torin 1. We also generated Vps35l-/- mice and demonstrated that they were embryonic lethal at an early stage, between E7.5 and E10.5. CONCLUSIONS: Our results suggest that biallelic loss-of-function variants in VPS35L underlies 3C/Ritscher-Schinzel-like syndrome. Furthermore, VPS35L is necessary for autophagic function and essential for early embryonic development. The data presented here provide a new insight into the critical role of the retriever complex in fetal development.
Assuntos
Anormalidades Múltiplas/genética , Cerebelo/metabolismo , Anormalidades Craniofaciais/genética , Síndrome de Dandy-Walker/genética , Predisposição Genética para Doença , Comunicação Interatrial/genética , Proteínas de Transporte Vesicular/genética , Anormalidades Múltiplas/patologia , Animais , Cerebelo/patologia , Anormalidades Craniofaciais/patologia , Síndrome de Dandy-Walker/patologia , Feminino , Comunicação Interatrial/patologia , Humanos , Mutação com Perda de Função/genética , Camundongos , Camundongos Knockout , Mutação de Sentido Incorreto/genética , Naftiridinas/farmacologia , Fenótipo , Gravidez , Estabilidade de RNA/genéticaRESUMO
Background: Anaplastic thyroid carcinoma (ATC) is one of the most aggressive and refractory cancers, and a therapy with a new concept needs to be developed. Recently, research on cancer stem cells (CSCs) has progressed, and CSCs have been suggested to be responsible for metastasis, recurrence, and therapy resistance. In ATC-CSCs, aldehyde dehydrogenase (ALDH) activity is the most reliable marker to enrich CSCs. However, it is just a marker and is not involved in CSC properties. The present study therefore aimed to identify key signaling pathways specific for ATC-CSCs. Methods: A small interfering RNA library targeting 719 kinases was used in a sphere formation assay and cell survival assay using ATC cell lines to select target molecules specific for CSC properties. The functions of the selected candidates were confirmed by sphere formation, cell survival, soft agar, and nude mice xenograft assays using small compound inhibitors. Results: The study focused on PDGFR, JAK, and PIM, whose small interfering RNAs had a higher inhibitory effect on sphere formation, as well as a lower or no effect on regular cell growth in both FRO and KTC3 cells. Next, inhibitors of PDGFR, JAK, STAT3, PIM and NF-κB were used, and all of them successfully suppressed sphere formation in a dose-dependent manner but not regular cell growth, confirming the screening results. Inhibition of the JAK/STAT3 and NF-κB pathways also reduced anchorage-independent growth in soft agar and tumor growth in nude mice. Conclusions: These results suggest that JAK/STAT3 and NF-κB signals play important roles in ATC-CSCs. Targeting these signaling pathways may be a promising approach to treat ATC.
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Janus Quinases/fisiologia , NF-kappa B/fisiologia , Células-Tronco Neoplásicas/patologia , Fator de Transcrição STAT3/fisiologia , Carcinoma Anaplásico da Tireoide/patologia , Neoplasias da Glândula Tireoide/patologia , Aldeído Desidrogenase/metabolismo , Animais , Linhagem Celular Tumoral , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Receptores do Fator de Crescimento Derivado de Plaquetas/fisiologia , Transdução de Sinais/fisiologiaAssuntos
Mutação da Fase de Leitura , Glucosiltransferases/genética , Hiperpigmentação/genética , Dermatopatias Genéticas/genética , Dermatopatias Papuloescamosas/genética , Pigmentação da Pele/genética , Biópsia , Análise Mutacional de DNA , Feminino , Predisposição Genética para Doença , Humanos , Hiperpigmentação/diagnóstico , Hiperpigmentação/enzimologia , Japão , Pessoa de Meia-Idade , Fenótipo , Dermatopatias Genéticas/diagnóstico , Dermatopatias Genéticas/enzimologia , Dermatopatias Papuloescamosas/diagnóstico , Dermatopatias Papuloescamosas/enzimologiaRESUMO
This corrects the article DOI: 10.1038/ncomms16102.
RESUMO
The p300 and CBP histone acetyltransferases are recruited to DNA double-strand break (DSB) sites where they induce histone acetylation, thereby influencing the chromatin structure and DNA repair process. Whether p300/CBP at DSB sites also acetylate non-histone proteins, and how their acetylation affects DSB repair, remain unknown. Here we show that p300/CBP acetylate RAD52, a human homologous recombination (HR) DNA repair protein, at DSB sites. Using in vitro acetylated RAD52, we identified 13 potential acetylation sites in RAD52 by a mass spectrometry analysis. An immunofluorescence microscopy analysis revealed that RAD52 acetylation at DSBs sites is counteracted by SIRT2- and SIRT3-mediated deacetylation, and that non-acetylated RAD52 initially accumulates at DSB sites, but dissociates prematurely from them. In the absence of RAD52 acetylation, RAD51, which plays a central role in HR, also dissociates prematurely from DSB sites, and hence HR is impaired. Furthermore, inhibition of ataxia telangiectasia mutated (ATM) protein by siRNA or inhibitor treatment demonstrated that the acetylation of RAD52 at DSB sites is dependent on the ATM protein kinase activity, through the formation of RAD52, p300/CBP, SIRT2, and SIRT3 foci at DSB sites. Our findings clarify the importance of RAD52 acetylation in HR and its underlying mechanism.
Assuntos
Quebras de DNA de Cadeia Dupla , Histona Acetiltransferases/fisiologia , Histona Desacetilases/fisiologia , Recombinação Homóloga , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Acetilação , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Histona Acetiltransferases/genética , Histona Desacetilases/genética , Humanos , Microscopia de Fluorescência , Técnicas do Sistema de Duplo-HíbridoRESUMO
BACKGROUND: Cockayne syndrome (CS) is a rare, autosomal recessive multisystem disorder characterised by prenatal or postnatal growth failure, progressive neurological dysfunction, ocular and skeletal abnormalities and premature ageing. About half of the patients with symptoms diagnostic for CS show cutaneous photosensitivity and an abnormal cellular response to UV light due to mutations in either the ERCC8/CSA or ERCC6/CSB gene. Studies performed thus far have failed to delineate clear genotype-phenotype relationships. We have carried out a four-centre clinical, molecular and cellular analysis of 124 patients with CS. METHODS AND RESULTS: We assigned 39 patients to the ERCC8/CSA and 85 to the ERCC6/CSB genes. Most of the genetic variants were truncations. The missense variants were distributed non-randomly with concentrations in relatively short regions of the respective proteins. Our analyses revealed several hotspots and founder mutations in ERCC6/CSB. Although no unequivocal genotype-phenotype relationships could be made, patients were more likely to have severe clinical features if the mutation was downstream of the PiggyBac insertion in intron 5 of ERCC6/CSB than if it was upstream. Also a higher proportion of severely affected patients was found with mutations in ERCC6/CSB than in ERCC8/CSA. CONCLUSION: By identifying >70 novel homozygous or compound heterozygous genetic variants in 124 patients with CS with different disease severity and ethnic backgrounds, we considerably broaden the CSA and CSB mutation spectrum responsible for CS. Besides providing information relevant for diagnosis of and genetic counselling for this devastating disorder, this study improves the definition of the puzzling genotype-phenotype relationships in patients with CS.
Assuntos
Síndrome de Cockayne/genética , DNA Helicases/genética , Enzimas Reparadoras do DNA/genética , Transtornos de Fotossensibilidade/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , Fatores de Transcrição/genética , Adolescente , Adulto , Criança , Pré-Escolar , Síndrome de Cockayne/fisiopatologia , Estudos de Coortes , Feminino , Predisposição Genética para Doença , Humanos , Lactente , Íntrons/genética , Masculino , Mutação de Sentido Incorreto/genética , Transtornos de Fotossensibilidade/fisiopatologia , Gravidez , Raios Ultravioleta , Adulto JovemRESUMO
Autosomal recessive cerebellar ataxias (ARCAs) are clinically and genetically heterogeneous neurological disorders. Through whole-exome sequencing of Japanese ARCA patients, we identified three index patients from unrelated families who had biallelic mutations in ERCC4. ERCC4 mutations have been known to cause xeroderma pigmentosum complementation group F (XP-F), Cockayne syndrome, and Fanconi anemia phenotypes. All of the patients described here showed very slowly progressive cerebellar ataxia and cognitive decline with choreiform involuntary movement, with young adolescent or midlife onset. Brain MRI demonstrated atrophy that included the cerebellum and brainstem. Of note, cutaneous symptoms were very mild: there was normal to very mild pigmentation of exposed skin areas and/or an equivocal history of pathological sunburn. However, an unscheduled DNA synthesis assay of fibroblasts from the patient revealed impairment of nucleotide excision repair. A similar phenotype was very recently recognized through genetic analysis of Caucasian cerebellar ataxia patients. Our results confirm that biallelic ERCC4 mutations cause a cerebellar ataxia-dominant phenotype with mild cutaneous symptoms, possibly accounting for a high proportion of the genetic causes of ARCA in Japan, where XP-F is prevalent.