RESUMEN
BACKGROUND: The mode of action of narrowband ultraviolet B (NB-UVB) therapy in clearing psoriasis is incompletely understood, and in vivo studies at the molecular level in patients undergoing NB-UVB therapy are limited. We previously demonstrated increased expression and activity of double-stranded RNA (dsRNA) receptors in psoriasis lesions, and suggested that this enhanced innate signalling contributed to the maintenance of psoriatic inflammation. OBJECTIVES: We investigated whether NB-UVB affects dsRNA receptor expression and function in vivo as well as in vitro. METHODS: Skin samples of patients with psoriasis undergoing NB-UVB treatment were analysed for epidermal messenger RNA (mRNA) expression of the various dsRNA receptors by microarray and quantitative reverse transcription-polymerase chain reaction. Primary human keratinocytes were irradiated with NB-UVB and stimulated with interferon (IFN)-α or IFN-γ, critical cytokines in psoriasis. The dsRNA analogue polyriboinosinic-polyribocytidylic acid was used to assess the functional responsiveness of the cells to dsRNA. RESULTS: NB-UVB therapy of patients with psoriasis resulted in a significantly reduced mRNA expression of the activating dsRNA receptors MDA5 (IFIH1) and RIG-I (DDX58). On the other hand, expression of LGP2 (DHX58), toll-like receptor 3 (TLR3) and PKR (EIF2AK2) was not affected. In vitro, NB-UVB irradiation completely blocked the upregulation of four of the dsRNA receptors in primary human keratinocytes stimulated with IFN-α or IFN-γ, resulting in an attenuated inflammatory response to dsRNA. CONCLUSIONS: Our results show that NB-UVB irradiation inhibits the local innate inflammatory response to dsRNA, and suggest a novel mechanism of action of NB-UVB phototherapy in psoriasis.
Asunto(s)
Queratinocitos , Psoriasis , ARN Bicatenario/efectos de la radiación , Receptores de Reconocimiento de Patrones/metabolismo , Terapia Ultravioleta , Adulto , Anciano , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/metabolismo , Femenino , Humanos , Helicasa Inducida por Interferón IFIH1 , Interferones/farmacología , Queratinocitos/metabolismo , Queratinocitos/efectos de la radiación , Masculino , Análisis por Micromatrices , Persona de Mediana Edad , Psoriasis/metabolismo , Psoriasis/radioterapia , ARN Helicasas/metabolismo , ARN Bicatenario/metabolismo , ARN Mensajero/metabolismo , ARN Mensajero/efectos de la radiación , Receptores Inmunológicos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Piel/metabolismo , Piel/efectos de la radiación , Receptor Toll-Like 3/metabolismo , Terapia Ultravioleta/métodosRESUMEN
Cerebro-oculo-facio-skeletal (COFS) syndrome is an autosomal recessive disorder characterized by microcephaly, congenital cataracts, facial dysmorphism, neurogenic arthrogryposis, growth failure and severe psychomotor retardation. We report a large consanguineous pedigree from northern Finland with six individuals belonging into four different sibships and affected with typical COFS syndrome phenotype. Two deceased patients have been published previously in 1982 as the first cases exhibiting cerebral calcifications typical for this disorder. Two living and one of the deceased patients were all shown to possess a novel homozygous mutation in the ERCC6 [Cockayne syndrome B (CSB)] gene, thereby confirming the diagnosis on molecular genetic level even for the earlier published cases. Genealogical investigation showed a common ancestor living in a northeastern village in Finland in the 18th century for all six patients implying a founder effect.
Asunto(s)
Anomalías Múltiples/genética , ADN Helicasas/genética , Enzimas Reparadoras del ADN/genética , Mutación , Secuencia de Bases , Catarata/genética , Preescolar , Síndrome de Cockayne/genética , Consanguinidad , Análisis Mutacional de ADN , Finlandia , Humanos , Masculino , Microcefalia/genética , Datos de Secuencia Molecular , Fenotipo , Proteínas de Unión a Poli-ADP-Ribosa , SíndromeRESUMEN
Defects in the xeroderma pigmentosum type B (XPB) gene (ERCC3), a DNA helicase involved in nucleotide excision repair (NER) and an essential subunit of the basal transcription factor, TFIIH, have been described in only three families. We report three new XPB families: one has two sisters with relatively mild xeroderma pigmentosum (XP) symptoms not previously associated with XPB mutations and two have severe XP/Cockayne syndrome (CS) complex symptoms. All XP-B cells had reduced NER and post-ultraviolet (UV) cell viability. Surprisingly, cells from the milder XP sisters had the same missense mutation (c.296T>C, p.F99S) that was previously reported in two mild XP/CS complex brothers. These cells had higher levels of XPB protein than the severely affected XP/CS complex patients. An XPB expression vector with the p.F99S mutation partially complemented the NER defect in XP-B cells. The three severely affected XP/CS complex families all have the same splice acceptor site mutation (c.2218-6C>A, p.Q739insX42) in one allele. This resulted in alteration of 41 amino acids at the C terminus, producing partial NER complementation. This limited number of mutations probably reflects the very restricted range of alterations of this vital protein that are compatible with life. We found new mutations in the second allele yielding markedly truncated proteins in all five XP or XP/CS complex families: c.1273C>T, p.R425X; c.471+1G>A, p.K157insTSDSX; c.807-808delTT, p.F270X; c.1421-1422insA, p.D474EfsX475; and c.1633C>T, p.Q545X. The remarkable phenotypic heterogeneity of XPB is associated with partially active missense mutations in milder patients while severe XP/CS complex patients have nonsense mutations in both alleles with low levels of altered XPB proteins.
Asunto(s)
Síndrome de Cockayne/complicaciones , ADN Helicasas/genética , Proteínas de Unión al ADN/genética , Xerodermia Pigmentosa/complicaciones , Xerodermia Pigmentosa/genética , Anomalías Múltiples/genética , Adulto , Empalme Alternativo , Carcinoma Basocelular/genética , Supervivencia Celular/efectos de la radiación , ADN Helicasas/metabolismo , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Enfermedades Desmielinizantes/genética , Neoplasias del Ojo/genética , Femenino , Expresión Génica , Genoma Humano , Heterocigoto , Humanos , Masculino , Melanoma/genética , Persona de Mediana Edad , Mutación , Núcleo Familiar , Fenotipo , Trastornos por Fotosensibilidad/genética , Polimorfismo Genético , ARN/biosíntesis , Rayos Ultravioleta/efectos adversosRESUMEN
Interstrand cross-links (ICLs) are an extremely toxic class of DNA damage incurred during normal metabolism or cancer chemotherapy. ICLs covalently tether both strands of duplex DNA, preventing the strand unwinding that is essential for polymerase access. The mechanism of ICL repair in mammalian cells is poorly understood. However, genetic data implicate the Ercc1-Xpf endonuclease and proteins required for homologous recombination-mediated double-strand break (DSB) repair. To examine the role of Ercc1-Xpf in ICL repair, we monitored the phosphorylation of histone variant H2AX (gamma-H2AX). The phosphoprotein accumulates at DSBs, forming foci that can be detected by immunostaining. Treatment of wild-type cells with mitomycin C (MMC) induced gamma-H2AX foci and increased the amount of DSBs detected by pulsed-field gel electrophoresis. Surprisingly, gamma-H2AX foci were also induced in Ercc1(-/-) cells by MMC treatment. Thus, DSBs occur after cross-link damage via an Ercc1-independent mechanism. Instead, ICL-induced DSB formation required cell cycle progression into S phase, suggesting that DSBs are an intermediate of ICL repair that form during DNA replication. In Ercc1(-/-) cells, MMC-induced gamma-H2AX foci persisted at least 48 h longer than in wild-type cells, demonstrating that Ercc1 is required for the resolution of cross-link-induced DSBs. MMC triggered sister chromatid exchanges in wild-type cells but chromatid fusions in Ercc1(-/-) and Xpf mutant cells, indicating that in their absence, repair of DSBs is prevented. Collectively, these data support a role for Ercc1-Xpf in processing ICL-induced DSBs so that these cytotoxic intermediates can be repaired by homologous recombination.
Asunto(s)
Daño del ADN , Reparación del ADN , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Animales , Ciclo Celular , Línea Celular , Aberraciones Cromosómicas , ADN/química , ADN/metabolismo , Daño del ADN/efectos de los fármacos , Daño del ADN/efectos de la radiación , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/fisiología , Endonucleasas/deficiencia , Endonucleasas/fisiología , Rayos gamma , Histonas/análisis , Inmunohistoquímica , Ratones , Ratones Noqueados , Mitomicina/farmacología , Conformación de Ácido Nucleico , Rayos UltravioletaRESUMEN
Cerebro-oculo-facio-skeletal (COFS) syndrome is a recessively inherited rapidly progressive neurologic disorder leading to brain atrophy, with calcifications, cataracts, microcornea, optic atrophy, progressive joint contractures, and growth failure. Cockayne syndrome (CS) is a recessively inherited neurodegenerative disorder characterized by low to normal birth weight, growth failure, brain dysmyelination with calcium deposits, cutaneous photosensitivity, pigmentary retinopathy and/or cataracts, and sensorineural hearing loss. Cultured CS cells are hypersensitive to UV radiation, because of impaired nucleotide-excision repair (NER) of UV-induced damage in actively transcribed DNA, whereas global genome NER is unaffected. The abnormalities in CS are caused by mutated CSA or CSB genes. Another class of patients with CS symptoms have mutations in the XPB, XPD, or XPG genes, which result in UV hypersensitivity as well as defective global NER; such patients may concurrently have clinical features of another NER syndrome, xeroderma pigmentosum (XP). Clinically observed similarities between COFS syndrome and CS have been followed by discoveries of cases of COFS syndrome that are associated with mutations in the XPG and CSB genes. Here we report the first involvement of the XPD gene in a new case of UV-sensitive COFS syndrome, with heterozygous substitutions-a R616W null mutation (previously seen in patients in XP complementation group D) and a unique D681N mutation-demonstrating that a third gene can be involved in COFS syndrome. We propose that COFS syndrome be included within the already known spectrum of NER disorders: XP, CS, and trichothiodystrophy. We predict that future patients with COFS syndrome will be found to have mutations in the CSA or XPB genes, and we document successful use of DNA repair for prenatal diagnosis in triplet and singleton pregnancies at risk for COFS syndrome. This result strongly underlines the need for screening of patients with COFS syndrome, for either UV sensitivity or DNA-repair abnormalities.
Asunto(s)
Anomalías Múltiples/genética , ADN Helicasas , Reparación del ADN/genética , Proteínas de Unión al ADN , Enfermedades Fetales/genética , Mutación Missense/genética , Diagnóstico Prenatal , Proteínas/genética , Factores de Transcripción , Trillizos/genética , Anomalías Múltiples/diagnóstico , Anomalías Múltiples/fisiopatología , Secuencia de Aminoácidos , Disparidad de Par Base/genética , Secuencia de Bases , Preescolar , Síndrome de Cockayne/genética , Síndrome de Cockayne/fisiopatología , Análisis Mutacional de ADN , Replicación del ADN/genética , Replicación del ADN/efectos de la radiación , Femenino , Enfermedades Fetales/diagnóstico , Enfermedades Fetales/fisiopatología , Humanos , Lactante , Recién Nacido , Judíos/genética , Masculino , Datos de Secuencia Molecular , Embarazo , Proteínas/metabolismo , Síndrome , Rayos Ultravioleta/efectos adversos , Xerodermia Pigmentosa/genética , Xerodermia Pigmentosa/fisiopatología , Proteína de la Xerodermia Pigmentosa del Grupo DRESUMEN
Rombo syndrome is a rare entity characterized by the presence of atrophoderma vermiculatum of the face, multiple milia, telangiectases, acral erythema and a propensity to develop basal cell carcinomas. We describe a patient whose clinical and histopathological abnormalities are consistent with this diagnosis.
Asunto(s)
Eritema/diagnóstico , Dermatosis Facial/diagnóstico , Adulto , Eritema/patología , Dermatosis Facial/patología , Humanos , Masculino , Fenotipo , SíndromeRESUMEN
The xeroderma pigmentosum group D (XPD) helicase subunit of TFIIH functions in DNA repair and transcription initiation. Different mutations in XPD give rise to three ultraviolet-sensitive syndromes: the skin cancer-prone disorder xeroderma pigmentosum (XP), in which repair of ultraviolet damage is affected; and the severe neurodevelopmental conditions Cockayne syndrome (CS) and trichothiodystrophy (TTD). In the latter two, the basal transcription function of TFIIH is also presumed to be affected. Here we report four unusual TTD patients with fever-dependent reversible deterioration of TTD features such as brittle hair. Cells from these patients show an in vivo temperature-sensitive defect of transcription and DNA repair due to thermo-instability of TFIIH. Our findings reveal the clinical consequences of impaired basal transcription and mutations in very fundamental processes in humans, which previously were only known in lower organisms.
Asunto(s)
ADN Helicasas , Reparación del ADN/genética , Proteínas de Unión al ADN , Enfermedades del Cabello/genética , Mutación , Proteínas/genética , Factores de Transcripción , Secuencia de Bases , Células Cultivadas , ADN Complementario/genética , Femenino , Fiebre/patología , Cabello/metabolismo , Cabello/patología , Enfermedades del Cabello/metabolismo , Enfermedades del Cabello/patología , Humanos , Lactante , Síndrome , Temperatura , Proteína de la Xerodermia Pigmentosa del Grupo DRESUMEN
We describe a premature, small for gestational age infant girl with micropthalmia, bilateral congenital cataracts, hearing impairment, progressive somatic and neurodevelopmental arrest, and infantile spasms. She presented a massive photosensitive reaction with erythema and blistering after minimal sun exposure, which slowly gave place to small skin cancers. Her skin fibroblasts were 10-fold more sensitive than normal to UV exposure due to a severe deficiency in nucleotide excision repair. By complementation analysis, the patient XPCS4RO was assigned to the very rare xeroderma pigmentosum (XP) group G (XP-G). One allele of her XPG gene contained a 526C-->T transition that changed Gln-176 to a premature UAG stop codon. Only a minor fraction of XPG mRNA was encoded by this allele. The second, more significantly expressed XPG allele contained a 215C-->A transversion. This changed the highly conserved Pro-72 to a histidine, a substitution that would be expected to seriously impair the 3' endonuclease function of XPG in nucleotide excision repair. In cases suspected of having XP and/or early-onset Cockayne syndrome, extensive DNA repair studies should be performed to reach a correct diagnosis, thereby allowing reliable genetic counseling and prenatal diagnosis.
Asunto(s)
Síndrome de Cockayne , Proteínas de Unión al ADN/genética , Xerodermia Pigmentosa , Alelos , Síndrome de Cockayne/genética , Endonucleasas , Femenino , Humanos , Lactante , Mutación , Proteínas Nucleares , Factores de Transcripción , Xerodermia Pigmentosa/genéticaRESUMEN
Nijmegen breakage syndrome (NBS) and ataxia telangiectasia (AT) are rare autosomal recessive hereditary disorders characterized by radiosensitivity, chromosomal instability, immunodeficiency and proneness to cancer. Although the clinical features of both syndromes are quite distinct, the cellular characteristics are very similar. Cells from both NBS and AT patients are hypersensitive to ionizing radiation (IR), show elevated levels of chromosomal aberrations and display radioresistant DNA synthesis (RDS). The proteins defective in NBS and AT, NBS1 and ATM, respectively, are involved in the same pathway, but their exact relationship is not yet fully understood. Stumm et al. (Am. J. Hum. Genet. 60 (1997) 1246) have reported that hybrids of AT and NBS lymphoblasts were not complemented for chromosomal aberrations. In contrast, we found that X-ray-induced cell killing as well as chromosomal aberrations were complemented in proliferating NBS-1LBI/AT5BIVA hybrids, comparable to that in NBS-1LBI cells after transfer of a single human chromosome 8 providing the NBS1 gene. RDS observed in AT5BIVA cells was reduced in these hybrids to the level of that seen in immortal NBS-1LBI cells. However, the level of DNA synthesis, following ionizing radiation, in SV40 transformed wild-type cell lines was the same as in NBS-1LBI cells. Only primary wild-type cells showed stronger inhibition of DNA synthesis. In summary, these results clearly indicate that RDS cannot be used as an endpoint in functional complementation studies with immortal NBS-1LBI cells, whereas the cytogenetic assay is suitable for complementation studies with immortal AT and NBS cells.
Asunto(s)
Anomalías Múltiples/genética , Ataxia Telangiectasia/genética , Aberraciones Cromosómicas , Tolerancia a Radiación/genética , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular , Muerte Celular , Replicación del ADN/efectos de la radiación , Proteínas de Unión al ADN , Relación Dosis-Respuesta en la Radiación , Prueba de Complementación Genética , Predisposición Genética a la Enfermedad , Humanos , Células Híbridas , Ratones , Proteínas Nucleares , Proteínas Serina-Treonina Quinasas , Síndrome , Proteínas Supresoras de Tumor , Rayos XRESUMEN
We have assessed the ability of xeroderma pigmentosum and normal keratinocytes grown out from skin biopsies to undergo apoptosis after irradiation with ultraviolet B. Keratinocytes have been studied from xeroderma pigmentosum complementation groups A (three biopsies), C (three biopsies), D (one biopsy), xeroderma pigmentosum variant (two biopsies), and Cockayne syndrome (one biopsy). The three xeroderma pigmentosum group A and the xeroderma pigmentosum group D samples were at least six times more sensitive than normal cells to ultraviolet B-induced apoptosis. The xeroderma pigmentosum variant samples showed intermediate susceptibility. Xeroderma pigmentosum group C samples proved heterogeneous: one showed high sensitivity to apoptosis, whereas two showed near normal susceptibility. The Cockayne syndrome sample showed the high susceptibility of xeroderma pigmentosum groups A and D only at a higher fluence. These results suggest that the relationships between repair deficiency, apoptosis, and susceptibility to skin cancer are not straightforward. Ultraviolet B-induced skin cancer is also thought to be due in part to ultraviolet B-induced impairment of immune responses. The release of the inflammatory cytokines interleukin-6 and tumor necrosis factor-alpha from cultured xeroderma pigmentosum keratinocytes tended to occur at lower fluences than in normals, but was less extensive, and was more readily inhibited at higher fluences of ultraviolet B.
Asunto(s)
Queratinocitos/citología , Rayos Ultravioleta , Xerodermia Pigmentosa/patología , Apoptosis/efectos de la radiación , Células Cultivadas , Citocinas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Fibroblastos/efectos de la radiación , Humanos , Etiquetado Corte-Fin in Situ , Recién Nacido , Interleucina-6/metabolismo , Queratinocitos/efectos de la radiación , Masculino , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
We show that hypomorphic mutations in hMRE11, but not in ATM, are present in certain individuals with an ataxia-telangiectasia-like disorder (ATLD). The cellular features resulting from these hMRE11 mutations are similar to those seen in A-T as well as NBS and include hypersensitivity to ionizing radiation, radioresistant DNA synthesis, and abrogation of ATM-dependent events, such as the activation of Jun kinase following exposure to gamma irradiation. Although the mutant hMre11 proteins retain some ability to interact with hRad50 and Nbs1, formation of ionizing radiation-induced hMre11 and Nbs1 foci was absent in hMRE11 mutant cells. These data demonstrate that ATM and the hMre11/hRad50/Nbs1 protein complex act in the same DNA damage response pathway and link hMre11 to the complex pathology of A-T.
Asunto(s)
Ataxia Telangiectasia/genética , Enzimas Reparadoras del ADN , Reparación del ADN/genética , Proteínas de Unión al ADN/genética , Proteínas Nucleares , Ácido Anhídrido Hidrolasas , Ataxia Telangiectasia/metabolismo , Ataxia Telangiectasia/patología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Daño del ADN/genética , Análisis Mutacional de ADN , Proteínas de Unión al ADN/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patología , Fibroblastos/efectos de la radiación , Rayos gamma , Humanos , Proteína Homóloga de MRE11 , Mutación Missense/genéticaAsunto(s)
Replicación del ADN/efectos de la radiación , Animales , Radioisótopos de Carbono , Técnicas de Cultivo de Célula/métodos , Células Cultivadas , Enfermedades Genéticas Congénitas/genética , Humanos , Mamíferos , Radiación Ionizante , Técnica de Dilución de Radioisótopos , Timidina/metabolismo , Tritio , Rayos XRESUMEN
Nijmegen Breakage Syndrome (NBS) is a very rare autosomal recessive chromosomal instability disorder characterized by microcephaly, growth retardation, immunodeficiency and a high incidence of malignancies. Cells from NBS patients are hypersensitive to ionizing radiation (IR) and display radioresistant DNA synthesis (RDS). NBS is caused by mutations in the NBS1 gene on chromosome 8q21 encoding a protein called nibrin. This protein is a component of the hMre11/hRad50 protein complex, suggesting a defect in DNA double-strand break (DSB) repair and/or cell cycle checkpoint function in NBS cells. We established SV40 transformed, immortal NBS fibroblasts, from primary cells derived from a Polish patient, carrying the common founder mutation 657del5. Immortalized NBS cells, like primary cells, are X-ray sensitive (2-fold) and display RDS following IR. They show an increased sensitivity to bleomycin (3.5-fold), etoposide (2.5-fold), camptothecin (3-fold) and mitomycin C (1.5-fold), but normal sensitivity towards UV-C. Despite the clear hypersensitivity towards DSB-inducing agents, the overall rates of DSB-rejoining in NBS cells as measured by pulsed field gel electrophoresis were found to be very similar to those of wild type cells. This indicates that the X-ray sensitivity of NBS cells is not directly caused by an overt defect in DSB repair.
Asunto(s)
Anomalías Múltiples/genética , Transformación Celular Viral , Rotura Cromosómica , Fibroblastos/virología , Anomalías Múltiples/patología , Antineoplásicos/farmacología , Bleomicina/farmacología , Camptotecina/farmacología , Línea Celular , Línea Celular Transformada , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Preescolar , ADN/efectos de los fármacos , ADN/genética , ADN/efectos de la radiación , Daño del ADN , Reparación del ADN , Etopósido/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/efectos de la radiación , Células HeLa , Humanos , Mitomicina/farmacología , Mutación , Síndrome , Rayos XAsunto(s)
Reparación del ADN , Endonucleasas , Cromatina/metabolismo , Proteínas de Unión al ADN/genética , Humanos , Modelos Genéticos , Proteínas Nucleares , Proteínas/genética , Proteína de Replicación A , Factor de Transcripción TFIIH , Factores de Transcripción/genética , Factores de Transcripción TFII , Rayos Ultravioleta/efectos adversos , Xerodermia Pigmentosa/genética , Proteína de la Xerodermia Pigmentosa del Grupo ARESUMEN
Germline mutations in the ATM gene are responsible for the autosomal recessive disorder ataxia-telangiectasia (A-T). In our study, we have determined the ATM mutation spectrum in 19 classical A-T patients, including some immigrant populations, as well as 12 of Dutch ethnic origin. Both the protein truncation test (PTT) and the restriction endonuclease fingerprinting (REF) method were used and compared for their detection efficiency, identifying 76% and 60% of the mutations, respectively. Most patients were found to be compound heterozygote. Seventeen mutations were distinct, of which 10 were not reported previously. Mutations are small deletions or point mutations frequently affecting splice sites. Moreover, a 16.7-kb genomic deletion of the 3' end of the gene, most likely a result of recombination between two LINE elements, was identified. The most frequently found mutation, identified in three unrelated Turkish A-T individuals, was previously described to be a Turkish A-T founder mutation. The presence of a founder mutation among relatively small ethnic population groups in Western Europe could indicate a high carrier frequency in such communities. In patients of Dutch ethnic origin, however, no significant founder effect could be identified. The observed genetic heterogeneity including the relative high percentage of splice-site mutations had no reflection on the phenotype. All patients manifested classical A-T and increased cellular radioresistant DNA synthesis.
Asunto(s)
Ataxia Telangiectasia/genética , Mutación de Línea Germinal , Proteínas Serina-Treonina Quinasas , Proteínas/genética , Ataxia Telangiectasia/etnología , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular , Células Cultivadas , Análisis Mutacional de ADN , Proteínas de Unión al ADN , Efecto Fundador , Humanos , Países Bajos , Proteínas Supresoras de TumorRESUMEN
ERCC1-XPF is a heterodimeric protein complexinvolved in nucleotide excision repair and recombinational processes. Like its homologous complex in Saccharomyces cerevisiae , Rad10-Rad1, it acts as a structure-specific DNA endonuclease, cleaving at duplex-single-stranded DNA junctions. In repair, ERCC1-XPF and Rad10-Rad1 make an incision on the the 5'-side of the lesion. No humans with a defect in the ERCC1 subunit of this protein complex have been identified and ERCC1-deficient mice suffer from severe developmental problems and signs of premature aging on top of a repair-deficient phenotype. Xeroderma pigmentosum group F patients carry mutations in the XPF subunit and generally show the clinical symptoms of mild DNA repair deficiency. All XP-F patients examined demonstrate reduced levels of XPF and ERCC1 protein, suggesting that proper complex formation is required for stability of the two proteins. To better understand the molecular and clinical consequences of mutations in the ERCC1-XPF complex, we decided to map the interaction domains between the two subunits. The XPF-binding domain comprises C-terminal residues 224-297 of ERCC1. Intriguingly, this domain resides outside the region of homology with its yeast Rad10 counterpart. The ERCC1-binding domain in XPF maps to C-terminal residues 814-905. ERCC1-XPF complex formation is established by a direct interaction between these two binding domains. A mutation from an XP-F patient that alters the ERCC1-binding domain in XPF indeed affects complex formation with ERCC1.
Asunto(s)
Reparación del ADN , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Proteínas/química , Proteínas/metabolismo , Proteínas de Saccharomyces cerevisiae , Animales , Enzimas Reparadoras del ADN , Proteínas de Unión al ADN/genética , Endonucleasas/metabolismo , Proteínas Fúngicas/metabolismo , Humanos , Sustancias Macromoleculares , Mamíferos , Ratones , Ratones Noqueados , Reacción en Cadena de la Polimerasa , Biosíntesis de Proteínas , Proteínas/genética , Eliminación de Secuencia , Endonucleasas Específicas del ADN y ARN con un Solo Filamento , Transcripción GenéticaRESUMEN
The human single-stranded DNA-binding replication A protein (RPA) is involved in various DNA-processing events. By comparing the affinity of hRPA for artificial DNA hairpin structures with 3'- or 5'-protruding single-stranded arms, we found that hRPA binds ssDNA with a defined polarity; a strong ssDNA interaction domain of hRPA is positioned at the 5' side of its binding region, a weak ssDNA-binding domain resides at the 3' side. Polarity appears crucial for positioning of the excision repair nucleases XPG and ERCC1-XPF on the DNA. With the 3'-oriented side of hRPA facing a duplex ssDNA junction, hRPA interacts with and stimulates ERCC1-XPF, whereas the 5'-oriented side of hRPA at a DNA junction allows stable binding of XPG to hRPA. Our data pinpoint hRPA to the undamaged strand during nucleotide excision repair. Polarity of hRPA on ssDNA is likely to contribute to the directionality of other hRPA-dependent processes as well.
Asunto(s)
Reparación del ADN , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Endonucleasas/metabolismo , Proteínas/metabolismo , Animales , Células Cultivadas , ADN de Cadena Simple/metabolismo , Humanos , Insectos , Proteínas Nucleares , Unión Proteica , Proteína de Replicación A , Especificidad por Sustrato , Factores de TranscripciónRESUMEN
Xeroderma pigmentosum (XP) and Cockayne syndrome (CS) are human hereditary disorders characterized at the cellular level by an inability to repair certain types of DNA damage. Usually, XP and CS are clinically and genetically distinct. However, in rare cases, CS patients have been shown to have mutations in genes that were previously linked to the development of XP. The linkage between XP and CS has been difficult to study because few permanent cell lines have been established from XP/CS patients. To generate permanent cell lines, primary fibroblast cultures from two patients, displaying characteristics associated with CS and belonging to XP complementation group G, were transformed with anorigin-of-replication-deficient simian virus 40 (SV40). The new cell lines, summation operatorXPCS1LVo- and summation operatorXPCS1ROo-,were characterized phenotypically and genotypically to verify that properties of the primary cells are preserved after transformation. The cell lines exhibited rapid growth in culture and were shown, by immunostaining, to express the SV40 T antigen. The summation operatorXPCS1LVo- and summation operatorXPCS1ROo- cell lines were hypersensitive to UV light and had an impaired ability to reactivate a UV-irradiated reporter gene. Using polymerase chain reaction (PCR) amplification and restriction enzyme cleavage, the summation operatorXPCS1ROo- cells were shown to retain the homozygous T deletion at XPG position 2972. This mutation also characterizes the parental primary cells and was evident in the XPG RNA. Finally, to characterize the XPG DNA repair deficiency in these cell lines, an episomal expression vector containing wild-type XPG cDNA was used to correct UV-induced damage in a beta-galactosidase reporter gene.
Asunto(s)
Línea Celular Transformada/metabolismo , Síndrome de Cockayne/genética , Reparación del ADN/genética , Proteínas de Unión al ADN/genética , Fibroblastos/metabolismo , Xerodermia Pigmentosa/genética , Línea Celular Transformada/efectos de la radiación , Transformación Celular Viral , Síndrome de Cockayne/complicaciones , Proteínas de Unión al ADN/metabolismo , Endonucleasas , Fibroblastos/efectos de la radiación , Genotipo , Humanos , Proteínas Nucleares , Fenotipo , Virus 40 de los Simios , Factores de Transcripción , Rayos Ultravioleta , Xerodermia Pigmentosa/complicacionesRESUMEN
Host-cell reactivation (HCR) of UV-C-irradiated herpes simplex virus type 1 (HSV-1) has been determined in skin fibroblasts from the following hereditary cancer-prone syndromes: aniridia (AN), dysplastic nevus syndrome (DNS), Von Hippel-Lindau syndrome (VHL), Li-Fraumeni syndrome (LFS) and a family with high incidence of breast and ovarian cancer. Cells from AN, DNS or VHL patients were found to exhibit heterogeneity in HCR. Cells from individuals belonging to an LFS family show reduced HCR in all cases where the cells were derived from persons carrying one mutated p53 allele, whereas cells derived from members with two wild-type alleles show normal HCR. LFS cells with reduced HCR also reveal reduced genome overall repair, and a slower gene-specific repair of the active adenosine deaminase (ADA) gene, but little if any repair of the inactive 754 gene. In the breast/ovarian cancer family, reduced HCR is observed in skin fibroblasts derived from both afflicted and unaffected individuals. In addition, these cells display lower survival after exposure to UV-C and exhibit higher levels of SCEs than those in normal cells. These observations indicate that various hereditary cancer-prone syndromes, carrying mutations in different tumor-suppressor genes, exhibit an unexplained impairment of the capacity to repair UV-damaged DNA.
Asunto(s)
Reparación del ADN/genética , Enfermedades de la Piel/metabolismo , Neoplasias Cutáneas/metabolismo , Piel/citología , Aniridia/genética , Aniridia/metabolismo , Aniridia/virología , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/virología , Supervivencia Celular/efectos de la radiación , Células Cultivadas , Reparación del ADN/efectos de la radiación , Síndrome del Nevo Displásico/genética , Síndrome del Nevo Displásico/metabolismo , Síndrome del Nevo Displásico/virología , Células Eucariotas/metabolismo , Células Eucariotas/efectos de la radiación , Células Eucariotas/virología , Salud de la Familia , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Genes p53/genética , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/efectos de la radiación , Humanos , Síndrome de Li-Fraumeni/genética , Síndrome de Li-Fraumeni/metabolismo , Mutación/genética , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/virología , Linaje , Piel/metabolismo , Piel/patología , Enfermedades de la Piel/genética , Neoplasias Cutáneas/genética , Replicación Viral/genética , Replicación Viral/efectos de la radiación , Enfermedad de von Hippel-Lindau/genética , Enfermedad de von Hippel-Lindau/metabolismo , Enfermedad de von Hippel-Lindau/virologíaRESUMEN
A patient with xeroderma pigmentosum group C was extensively examined for mutations in the p53 gene in normal skin exposed to varying degrees of sunlight and in excisional biopsies of basal cell cancer, squamous cell cancer, and squamous cell dysplasia. Seventy-three samples were analyzed by microdissection of small cell clusters, followed by PCR and direct DNA sequencing. In skin taken from areas that most likely had never been exposed to the sun, no mutations were found. However, in skin exposed to the sun, we observed a multitude of mutations in the p53 gene. UV light-induced mutations were found in all types of lesions, as well as in clusters of morphologically normal epidermal cells. Twenty-nine distinct mutations were found in exons 5-8, all missense or nonsense, of which 27 (93%) were UV-specific C --> T or CC --> TT transitions at dipyrimidine sites of the nontranscribed strand. Two types of normal skin areas containing p53 mutations were observed: areas that stain strongly with p53 antibody (p53 patches) and those that do not stain. Because no silent or intron mutations were found in these cell clusters, the alterations in the p53 gene of morphologically normal cells are likely to have resulted in a selective growth advantage. The poor correlation between mutations and morphological phenotypes demonstrates that p53 mutations alone do not determine the phenotypes observed.