Asunto(s)
Apoptosis/efectos de la radiación , Daño del ADN/genética , Reparación del ADN/genética , Queratinocitos/efectos de la radiación , Rayos Ultravioleta/efectos adversos , Apoptosis/genética , Ciclo Celular/genética , Ciclo Celular/efectos de la radiación , Células Cultivadas , ADN/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Fibroblastos/metabolismo , Fibroblastos/efectos de la radiación , Humanos , Queratinocitos/metabolismo , Dímeros de Pirimidina/metabolismo , Dímeros de Pirimidina/efectos de la radiación , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/efectos de la radiaciónRESUMEN
When a recA strain of Escherichia coli was transformed with the multicopy plasmid pSF11 carrying the uvrA gene of E. coli, its extreme ultraviolet (UV) sensitivity was decreased. The sensitivity of the lexA1 (Ind(-)) strain to UV was also decreased by pSF11. The recA cells expressing Neurospora crassa UV damage endonuclease (UVDE), encoding UV-endonuclease, show UV resistance. On the other hand, only partial amelioration of UV sensitivity of the recA strain was observed in the presence of the plasmid pNP10 carrying the uvrB gene. Host cell reactivation of UV-irradiated lambda phage in recA cells with pSF11 was as efficient as that in wild-type cells. Using an antibody to detect cyclobutane pyrimidine dimers, we found that UV-irradiated recA cells removed dimers from their DNA more rapidly if they carried pSF11 than if they carried a vacant control plasmid. Using anti-UvrA antibody, we observed that the expression level of UvrA protein was about 20-fold higher in the recA strain with pSF11 than in the recA strain without pSF11. Our results were consistent with the idea that constitutive level of UvrA protein in the recA cells results in constitutive levels of active UvrABC nuclease which is not enough to operate full nucleotide excision repair (NER), thus leading to extreme UV sensitivity.
Asunto(s)
Adenosina Trifosfatasas/fisiología , Proteínas Bacterianas/fisiología , Daño del ADN , Reparación del ADN , Proteínas de Unión al ADN/fisiología , Proteínas de Escherichia coli/fisiología , Escherichia coli/efectos de la radiación , Rayos Ultravioleta , Adenosina Trifosfatasas/deficiencia , Adenosina Trifosfatasas/genética , Proteínas Bacterianas/genética , Bacteriófago lambda/crecimiento & desarrollo , Bacteriófago lambda/efectos de la radiación , ADN Helicasas/deficiencia , ADN Helicasas/genética , ADN Helicasas/fisiología , ADN Viral/genética , ADN Viral/fisiología , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Endonucleasas/fisiología , Escherichia coli/genética , Escherichia coli/virología , Proteínas de Escherichia coli/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiología , Prueba de Complementación Genética , Vectores Genéticos/genética , Neurospora crassa/enzimología , Neurospora crassa/genética , Dímeros de Pirimidina/metabolismo , Rec A Recombinasas/genética , Proteínas Recombinantes de Fusión/fisiología , Respuesta SOS en Genética/genética , Serina Endopeptidasas/genética , Serina Endopeptidasas/fisiología , Activación ViralRESUMEN
UV-induced DNA damage causes cells to repress RNA synthesis and to initiate nucleotide excision repair (NER). NER and transcription are intimately linked processes. Evidence has been presented that, in addition to damaged genes, undamaged loci are transcriptionally inhibited. We investigated whether RNA synthesis from undamaged genes is affected by the presence of UV damage elsewhere in the same nucleus, using a novel technique to UV irradiate only part of a nucleus. We show that the basal transcription/repair factor TFIIH is recruited to the damaged nuclear area, partially depleting the undamaged nuclear area. Remarkably, this sequestration has no effect on RNA synthesis. This result was obtained for cells that are able to carry out NER and for cells deficient in NER. We conclude that cross talk between NER and transcription occurs only over short distances in nuclei of living cells.
Asunto(s)
Núcleo Celular/efectos de la radiación , Daño del ADN , Factores de Transcripción TFII , Transcripción Genética/efectos de la radiación , Rayos Ultravioleta , Células Cultivadas , Reparación del ADN , Fibroblastos/efectos de la radiación , Humanos , Microscopía Fluorescente , Plásmidos/metabolismo , Factores de Tiempo , Factor de Transcripción TFIIH , Factores de Transcripción/metabolismoRESUMEN
Decreased cell viability and increased formation of cyclobutane-type pyrimidine dimers (CPDs) in DNA of UVB-irradiated keratinocytes were shown to be appreciably restored by the addition of w/o emulsion of microcorpuscular zinc oxide (mcZnO) with a corpuscle diameter of 0.15 microm. The cytoprotection was exerted only by 20 wt/wt% mcZnO at levels equivalent to 40- to 100-microm-thick emulsion layers, which screened 90-92% of the incident UVB. However, protection was not seen by mcZnO below 20-microm thickness, which, unexpectedly, screened 79% of the incident radiation. This suggests that thorough UVB screening is necessary for cytoprotection. This may be attributable to involvement of intracellular reactive oxygen species (ROS) secondarily generated from UVB-irradiated mcZnO. Intracellular ROS was increased in mcZnO-added cells in a time-dependent manner even after UVB irradiation, contrasting with reduction of intracellular ROS in ascorbic acid-added cells. UVB-induced disruption of cell membrane integrity was reduced by mcZnO at 100-microm thickness, equivalent to the addition of ascorbic acid of 50 microM. Thus, mcZnO was thought to be cytoprotective through reductions of intracellular ROS generation, CPD formation and cell membrane disintegration when added so abundantly so as to achieve UVB-screening more than 90%.
Asunto(s)
Ácido Ascórbico/farmacología , Daño del ADN/efectos de la radiación , Queratinocitos/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Fármacos Sensibilizantes a Radiaciones/farmacología , Protectores Solares/farmacología , Rayos Ultravioleta , Óxido de Zinc/farmacología , Animales , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Células Cultivadas , Daño del ADN/efectos de los fármacos , Relación Dosis-Respuesta en la Radiación , Queratinocitos/citología , Queratinocitos/efectos de la radiación , Ratones , Estrés Oxidativo/efectos de la radiación , Dímeros de Pirimidina/efectos de la radiación , Piel/citología , Piel/efectos de los fármacos , Piel/efectos de la radiaciónRESUMEN
We have developed a novel method that uses a microfilter mask to produce ultraviolet-induced DNA lesions in localized areas of the cell nucleus. This technique allows us to visualize localized DNA repair in situ using immunologic probes. Two major types of DNA photoproducts [cyclobutane pyrimidine dimers and (6-4) photoproducts] were indeed detected in several foci per nucleus in normal human fibroblasts. They were repaired at those localized sites at different speeds, indicating that DNA photoproducts remain in relatively fixed nuclear positions during repair. A nucleotide excision repair protein, proliferating cell nuclear antigen, was recruited to the sites of DNA damage within 30 min after ultraviolet exposure. The level of proliferating cell nuclear antigen varied with DNA repair activity and diminished within 24 h. In contrast, almost no proliferating cell nuclear antigen fluorescence was observed within 3 h in xeroderma pigmentosum fibroblasts, which could not repair either type of photolesion. These results demonstrate that this technique is useful for visualizing the normal nucleotide excision repair process in vivo. Interestingly, however, in xeroderma pigmentosum cells, proliferating cell nuclear antigen appeared at ultraviolet damage sites after a delay and persisted as late as 72 h after ultraviolet exposure. This result suggests that this technique is also valuable for examining an incomplete or stalled nucleotide excision repair process caused by the lack of a single functional nucleotide excision repair protein. Thus, the technique provides a powerful approach to understanding the temporal and spatial interactions between DNA damage and damage-binding proteins in vivo.
Asunto(s)
Daño del ADN/efectos de la radiación , Reparación del ADN , Fibroblastos/fisiología , Fibroblastos/efectos de la radiación , Rayos Ultravioleta , Línea Celular , Núcleo Celular/fisiología , Núcleo Celular/efectos de la radiación , Proteínas de Unión al ADN/deficiencia , Detergentes , Humanos , Antígeno Nuclear de Célula en Proliferación/metabolismo , Solubilidad , Factores de Tiempo , Proteína de la Xerodermia Pigmentosa del Grupo ARESUMEN
p53 mutation is one of the major results of ultraviolet (UV) radiation. UV photoproducts of cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts (64PPs) also play an important role in skin cancer development. Atypical fibroxanthoma (AFX), which mimics malignant fibrous histiocytoma (MFH) histologically, occurs in the sun-exposed skin of the elderly, and therefore, an association with UV has long been suspected. Eighteen fibrohistiocytic skin lesions comprising AFX (n = 7), storiform-pleomorphic type MFH centered in the subcutis (superficial MFH; S-MFH; n = 4) and benign fibrous histiocytoma (BFH; n = 7) were used for immunohistochemical and molecular analysis. Eight cases of deep MFH (D-MFH) were also analyzed for UV photoproduct expression for the purposes of comparison. Immunohistochemically, the CPD scores of AFX (3.6 +/- 0.4) were significantly higher than those of S-MFH (1.3 +/- 0.8), D-MFH (0.8 +/- 0.5), or BHF (1.4 +/- 0.7); however, the 64PP scores were extremely low in all these tumors (AFX, 0.1 +/- 0.1; S-MFH, 0.0 +/- 0.0; D-MFH, 0.0 +/- 0.0; and BHF, 0.0 +/- 0.0). AFX, S-MFH, and BFH showed immunoexpression for p53 (2/7, 2/4, and 0/7), respectively. p53 mutations were detected in AFX (4/6; 67%) and S-MFH (1/4; 25%), but not in BFH (0/5; 0%) using polymerase chain reaction-single-strand conformation polymorphism, and all of the mutations in AFX were either C-T transitions or at dipyrimidine sites. In conclusion, AFX and S-MFH are both similar fibrohistocytic lesions; however, AFX has high immunoreactivity for CPDs compared with S-MFH, D-MFH, or BFH. These data suggest that CPDs may play an important role in the pathogenesis of AFX.
Asunto(s)
Histiocitoma Fibroso Benigno/patología , Dímeros de Pirimidina/análisis , Neoplasias Cutáneas/patología , Proteína p53 Supresora de Tumor/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Bases , ADN/química , ADN/genética , Análisis Mutacional de ADN , Femenino , Histiocitoma Fibroso Benigno/genética , Histiocitoma Fibroso Benigno/metabolismo , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Mutación , Polimorfismo Conformacional Retorcido-Simple , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismoRESUMEN
Human cells contain a protein that binds to UV-irradiated DNA with high affinity. This protein, damaged DNA-binding protein (DDB), is a heterodimer of two polypeptides, p127 and p48. Recent in vivo studies suggested that DDB is involved in global genome repair of cyclobutane pyrimidine dimers (CPDs), but the mechanism remains unclear. Here, we show that in vitro DDB directly stimulates the excision of CPDs but not (6-4)photoproducts. The excision activity of cell-free extracts from Chinese hamster AA8 cell line that lacks DDB activity was increased 3-4-fold by recombinant DDB heterodimer but not p127 subunit alone. Moreover, the addition of XPA or XPA + replication protein A (RPA), which themselves enhanced excision, also enhanced the excision in the presence of DDB. DDB was found to elevate the binding of XPA to damaged DNA and to make a complex with damaged DNA and XPA or XPA + RPA as judged by both electrophoretic mobility shift assays and DNase I protection assays. These results suggest that DDB assists in the recognition of CPDs by core NER factors, possibly through the efficient recruitment of XPA or XPA.RPA, and thus stimulates the excision reaction of CPDs.
Asunto(s)
Daño del ADN , Proteínas de Unión al ADN/fisiología , Dímeros de Pirimidina , Proteínas de Unión al ARN/fisiología , Secuencia de Bases , Cartilla de ADN , Dimerización , Células HeLa , Humanos , Proteínas Recombinantes/metabolismo , Proteína de Replicación A , Proteína de la Xerodermia Pigmentosa del Grupo ARESUMEN
Using purified photolyases for pyrimidine (6-4) pyrimidone photoproducts [(6-4)PP] and cyclobutane pyrimidine dimers (CPD), the effects of photoreactivation on mutagenesis were examined in the supF gene on a plasmid transfected into repair-deficient SOS-induced Escherichia coli host cells. More than 95% of CPD and (6-4)PP were removed from plasmid DNA by treatment with CPD photolyase and (6-4)photolyase, respectively. In each photolyase treatment, base substitutions at dipyrimidine sequences were predominantly observed. Of the single base substitutions observed after CPD photoreactivation, 83% were A:T-->G:C transitions at 5'-TT-3' sites. After (6-4)photolyase treatment, 81% were G:C-->A:T transitions at 5'-CC-3' and 5'-TC-3' sequences. Thus, the major mutagenic photoproducts of single-base substitutions were CPD at 5'-CC-3' or 5'-TC-3' sites and (6-4)PP at 5'-TT-3' sites. Tandem double mutations occurred mainly at 5'-CC-3' sites and were CPD-photoreactivated, suggesting that CPD at 5'-CC-3' was responsible for tandem double mutations. After photoreactivation of both CPD and (6-4)PP, single-base substitutions were primarily G:C-->A:T transitions at 5'-CC-3' or 5'-TC-3' sites and A:T-->G:C transitions at 5'-TT-3' sites, and secondarily G:C-->T:A transversions at 5'-CC-3' sites, G:C-->C:G transversions at 5'-CC-3' sites and A:T-->T:A transversions at 5'-TT-3' sites, which were essentially the same as those observed after photoreactivation of CPD alone, (6-4)PP alone and without photoreactivation. Thus, these transversions were not derived from unknown UV adducts but from incompletely repaired CPD and (6-4)PP.
Asunto(s)
Reparación del ADN/genética , ADN Bacteriano/química , Escherichia coli/genética , Escherichia coli/efectos de la radiación , Mutagénesis , Dímeros de Pirimidina/química , Dímeros de Pirimidina/efectos de la radiación , Respuesta SOS en Genética/genética , Respuesta SOS en Genética/efectos de la radiación , Rayos Ultravioleta , Secuencia de Bases , Reparación del ADN/efectos de la radiación , ADN Bacteriano/genética , ADN Bacteriano/efectos de la radiación , Desoxirribodipirimidina Fotoliasa/metabolismo , Escherichia coli/enzimología , Genes Supresores , Datos de Secuencia Molecular , Fotoquímica , Plásmidos/genética , Plásmidos/metabolismo , Plásmidos/efectos de la radiación , Dímeros de Pirimidina/genética , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , ARN de Transferencia/efectos de la radiaciónRESUMEN
Nucleotide excision repair is a major mechanism of defense against the carcinogenic effects of ultraviolet light. Ultraviolet B causes sunburn and DNA damage in human skin. Nucleotide excision repair has been studied extensively and described in detail at the molecular level, including identification of many nucleotide excision repair-specific proteins and the genes encoding nucleotide excision repair proteins. In this study, normal human keratinocytes were exposed to increasing doses of ultraviolet B from fluorescent sunlamps, and the effect of this exposure on expression of nucleotide excision repair genes was examined. An RNase protection assay was performed to quantify transcripts from nucleotide excision repair genes, and a slot blot DNA repair activity assay was used to assess induction of the nucleotide excision repair pathway. The activity assay demonstrated that cyclobutane pyrimidine dimers were removed efficiently after exposure to low doses of ultraviolet B, but this activity was delayed significantly at higher doses. All nucleotide excision repair genes examined demonstrated a similar trend: ultraviolet B induces expression of nucleotide excision repair genes at low doses, but downregulates expression at higher doses. In addition, we show that pre-exposure of cells to low-dose ultraviolet protected keratinocytes from apoptosis following high-dose exposure. These data support the notion that nucleotide excision repair is induced in cells exposed to low doses of ultraviolet B, which may protect damaged keratinocytes from cell death; however, exposure to high doses of ultraviolet B downregulates nucleotide excision repair genes and is associated with cell death.
Asunto(s)
Reparación del ADN/genética , Reparación del ADN/efectos de la radiación , Queratinocitos/fisiología , Apoptosis/efectos de la radiación , Células Cultivadas , Dimerización , Relación Dosis-Respuesta en la Radiación , Citometría de Flujo , Regulación de la Expresión Génica/efectos de la radiación , Humanos , Queratinocitos/citología , Antígeno Nuclear de Célula en Proliferación/genética , ARN Mensajero/análisis , Piel/efectos de la radiación , Timina/química , Transcripción Genética/efectos de la radiación , Rayos UltravioletaRESUMEN
Reactive oxygen species have been shown to play a role in ultraviolet light (UV)-induced skin carcinogenesis. Vitamin E and green tea polyphenols reduce experimental skin cancers in mice mainly because of their antioxidant properties. Since olive oil has also been reported to be a potent antioxidant, we examined its effect on UVB-induced skin carcinogenesis in hairless mice. Extra-virgin olive oil was applied topically before or after repeated exposure of mice to UVB. The onset of UVB-induced skin tumors was delayed in mice painted with olive oil compared with UVB control mice. However, with increasing numbers of UVB exposures, differences in the mean number of tumors between UVB control mice and mice pretreated with olive oil before UVB exposure (pre-UVB group) were lost. In contrast, mice that received olive oil after UVB exposure (post-UVB group) showed significantly lower numbers of tumors per mouse than those in the UVB control group throughout the experimental period. The mean number of tumors per mouse in the UVB control, pre-UVB and post-UVB groups was 7.33, 6.69 and 2.64, respectively, in the first experiment, and 8.53, 9.53 and 3.36 in the second experiment. Camellia oil was also applied, using the same experimental protocol, but did not have a suppressive effect. Immunohistochemical analysis of DNA damage in the form of cyclobutane pyrimidine dimers (CPD), (6-4) photoproducts and 8-hydroxy-2'-deoxyguanosine (8-OHdG) in samples taken 30 min after a single exposure of UVB showed no significant difference between UVB-irradiated control mice and the pre-UVB group. In the post-UVB group, there were lower levels of 8-OHdG in epidermal nuclei, but the formation of CPD and (6-4) photoproducts did not differ. Exposure of olive oil to UVB before application abrogated the protective effect on 8-OHdG formation. These results indicate that olive oil topically applied after UVB exposure can effectively reduce UVB-induced murine skin tumors, possibly via its antioxidant effects in reducing DNA damage by reactive oxygen species, and that the effective component may be labile to UVB.
Asunto(s)
Anticarcinógenos/farmacología , Desoxiguanosina/análogos & derivados , Aceites de Plantas/farmacología , Neoplasias Cutáneas/prevención & control , Rayos Ultravioleta/efectos adversos , 8-Hidroxi-2'-Desoxicoguanosina , Administración Tópica , Animales , Desoxiguanosina/biosíntesis , Femenino , Ratones , Ratones Pelados , Ratones Endogámicos BALB C , Aceite de Oliva , Dímeros de Pirimidina/biosíntesis , Piel/efectos de los fármacos , Piel/metabolismo , Piel/efectos de la radiación , Neoplasias Cutáneas/etiología , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Proteína p53 Supresora de Tumor/biosíntesisRESUMEN
We and colleagues have suggested that deletions of mitochondrial DNA may be useful as a biomarker of ultraviolet radiation exposure in skin. In this study using a southwestern approach involving monoclonal antibodies against thymine dimers we provide direct evidence for the presence of ultraviolet-induced damage in mitochondrial DNA purified from any nuclear DNA contamination. Previous studies have been limited, as they have focused on the frequency of a single mitochondrial DNA deletion. Therefore we have addressed the question of the spectrum of mitochondrial DNA deletions in skin and whether this can be used as an index of overall DNA damage. We have used a long polymerase chain reaction technique to determine the mitochondrial DNA deletion spectrum of almost the entire mitochondrial genome in 71 split skin samples in relation to sun exposure. There was a significant increase in the number of deletions with increasing ultraviolet exposure in the epidermis (Kruskal-Wallis test, p = 0.0015) but not the dermis (p = 0.6376). The findings in the epidermis are not confounded by any age-dependent increases in mitochondrial DNA deletions also detected by the long polymerase chain reaction technique. The large spectrum of deletions identified in our study highlights the ubiquitous nature and the high mutational load of mitochondrial DNA associated with ultraviolet exposure and chronologic aging. Compared with the detection of single deletions using competitive polymerase chain reaction, we show that long polymerase chain reaction is a sensitive technique and may therefore provide a more comprehensive, although not quantitative, index of overall mitochondrial DNA damage in skin.
Asunto(s)
ADN Mitocondrial/genética , Piel/efectos de la radiación , Rayos Ultravioleta , Biomarcadores/análisis , Eliminación de Gen , Prueba de Complementación Genética , Humanos , Mutación/fisiología , Dímeros de Pirimidina/efectos de la radiación , Neoplasias Cutáneas/química , Neoplasias Cutáneas/genética , Xerodermia Pigmentosa/genéticaRESUMEN
To assess the photoprotective role of melanocytes in the epidermis, we studied the effects of ultraviolet B on an epidermis reconstructed with and without melanocytes. To address more specifically the role of melanin in fair-skinned individuals, experiments were done with cells obtained from human skin of low phototypes (II-III). To study the effect of constitutive melanin and possibly that of newly synthesized melanin precursors, a single dose of ultraviolet B (0.10 or 0.15 J per cm2, corresponding to a 4-5 minimal erythema dose in vivo) was administered to reconstructs and the effects were monitored over the first 24 h. When reconstructs with and without melanocytes were compared, no difference was found for DNA damage/repair assessed with antibodies to cyclobutane pyrimidine dimers and 6-4 photoproducts. More necrotic/apoptotic cells, however, were noted 24 h following ultraviolet B irradiation in reconstructs lacking melanocytes. Twenty-four hours following ultraviolet B irradiation the number of necrotic/apoptotic cells and the number of cyclobutane pyrimidine dimer positive cells was coarsely concentration-dependent. The number of cyclobutane pyrimidine dimer positive cells, however, was independent of the type of reconstruct used (with/without melanocytes). In conclusion, low phototype melanocytes seem to protect epidermal basal cells against ultraviolet B-induced apoptosis/necrosis and may preserve the overall integrity of the epidermis after ultraviolet B irradiation. On the contrary, such melanocytes do not seem to have a protective role against DNA damage and may not prevent cancer.
Asunto(s)
Daño del ADN/efectos de los fármacos , Melanocitos/fisiología , Pigmentación de la Piel , Quemadura Solar/prevención & control , Población Blanca , Adulto , Técnicas de Cocultivo , Células Epidérmicas , Epidermis/fisiología , Humanos , Recién Nacido , Melanocitos/clasificación , Melanocitos/citología , Quemadura Solar/patologíaRESUMEN
DNA photoproducts with (6-4) pyrimidine-pyrimidone adducts formed by ultraviolet radiation are implicated in mutagenesis and cancer, particularly skin cancer. The crystal structure of the Fab fragment of the murine 64M-2 antibody specific to DNA T(6-4)T photoproducts is determined as a complex with dT(6-4)T, a (6-4) pyrimidine-pyrimidone photodimer of dTpT, at 2.4 A resolution to a crystallographic R-factor of 0.199 and an R(free) value of 0.279. The 64M-2 Fab molecule is in an extended arrangement with an elbow angle of 174 degrees, and its five complementarity-determining regions, except L2, are involved in the ligand binding. The bound dT(6-4)T ligand adopting a ring structure with (6-4) linked 5' thymine-3' pyrimidone bases is fully accommodated in an antigen-binding pocket of about 15 Ax10 A. The 5'-thymine and 3'-pyrimidone bases are in half-chair and planar conformations, respectively, and are nearly perpendicular to each other. The 5'-thymine base is hydrogen-bonded to Arg95H and Ser96H, and is in van der Waals contact with Tyr100iH. The 3'-pyrimidone base is hydrogen-bonded to His35H, and is in contact with Trp33H. Three water molecules are located at the interface between the bases and the Fab residues. Hydrogen bonds involving these water molecules also contribute to Fab recognition of the dT(6-4)T bases. The sugar-phosphate backbone connecting the bases is surrounded by residues His27dL, Tyr32L, Ser92L, Trp33H, and Ser58H, but is not hydrogen-bonded to these residues.
Asunto(s)
Anticuerpos Antinucleares/química , ADN/química , ADN/inmunología , Fragmentos Fab de Inmunoglobulinas/química , Conformación de Ácido Nucleico/efectos de la radiación , Rayos Ultravioleta , Animales , Anticuerpos Antinucleares/inmunología , Especificidad de Anticuerpos , Sitios de Unión de Anticuerpos , Bovinos , Cristalografía por Rayos X , ADN/genética , ADN/efectos de la radiación , Daño del ADN/genética , Daño del ADN/inmunología , Daño del ADN/efectos de la radiación , ADN de Cadena Simple/química , ADN de Cadena Simple/genética , ADN de Cadena Simple/inmunología , ADN de Cadena Simple/efectos de la radiación , Epítopos/química , Epítopos/genética , Epítopos/inmunología , Epítopos/efectos de la radiación , Enlace de Hidrógeno , Fragmentos Fab de Inmunoglobulinas/inmunología , Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/inmunología , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Datos de Secuencia Molecular , Nucleótidos/química , Nucleótidos/genética , Nucleótidos/inmunología , Conformación Proteica , Dímeros de Pirimidina/química , Dímeros de Pirimidina/genética , Dímeros de Pirimidina/inmunología , Dímeros de Pirimidina/efectos de la radiación , Electricidad Estática , Agua/metabolismoRESUMEN
In order to better understand the relative contribution of the different UV components of sunlight to solar mutagenesis, the distribution of the bipyrimidine photolesions, cyclobutane pyrimidine dimers (CPD), (6-4) photoproducts ((6-4)PP), and their Dewar valence photoisomers (DewarPP) was examined in Chinese hamster ovary cells irradiated with UVC, UVB, or UVA radiation or simulated sunlight. The absolute amount of each type of photoproduct was measured by using a calibrated and sensitive immuno-dot-blot assay. As already established for UVC and UVB, we report the production of CPD by UVA radiation, at a yield in accordance with the DNA absorption spectrum. At biologically relevant doses, DewarPP were more efficiently produced by simulated solar light than by UVB (ratios of DewarPP to (6-4)PP of 1:3 and 1:8, respectively), but were detected neither after UVA nor after UVC radiation. The comparative rates of formation for CPD, (6-4)PP and DewarPP are 1:0.25 for UVC, 1:0. 12:0.014 for UVB, and 1:0.18:0.06 for simulated sunlight. The repair rates of these photoproducts were also studied in nucleotide excision repair-proficient cells irradiated with UVB, UVA radiation, or simulated sunlight. Interestingly, DewarPP were eliminated slowly, inefficiently, and at the same rate as CPD. In contrast, removal of (6-4)PP photoproducts was rapid and completed 24 h after exposure. Altogether, our results indicate that, in addition to CPD and (6-4)PP, DewarPP may play a role in solar cytotoxicity and mutagenesis.
Asunto(s)
Daño del ADN , Reparación del ADN , ADN/efectos de la radiación , Dímeros de Pirimidina/efectos de la radiación , Luz Solar , Animales , Células CHO , Cricetinae , MutagénesisRESUMEN
We previously developed a highly sensitive method to assess in situ repair kinetics of ultraviolet (UV)-induced DNA photoproducts in epidermal cells using monoclonal antibodies specific for cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts (64PPs) by immunohistochemistry. In order to determine whether nucleotide excision repair capacity is operative in postmitotic mature neurons, brain surfaces of adult mice were exposed to UVB, and induction and removal of CPDs and 64PPs in Purkinje cell DNA were assessed immunohistochemically. UVB penetrated brain tissue to a depth sufficient to allow quantitative study. CPDs but not 64PPs were clearly detectable in the nuclei of Purkinje cells at doses >500 J/m2, in a dose-dependent manner. A time course experiment showed a statistically significant decrease of CPDs with time after irradiation. Although there was no apparent removal on Day 1, about half of CPDs were removed within 5 days, and the repair was essentially completed by Day 10. We conclude that non-dividing cerebellar neuronal cells can indeed repair UV-induced DNA damage, but with relatively low efficiency as compared with dividing epidermal cells.
Asunto(s)
Daño del ADN/efectos de la radiación , Reparación del ADN , Células de Purkinje/fisiología , Células de Purkinje/efectos de la radiación , Animales , Ratones , Rayos UltravioletaRESUMEN
The role of UV light-induced photoproducts in initiating base substitution mutation in human cells was examined by determining the frequency and spectrum of mutation in a supF tRNA gene in a shuttle vector plasmid transfected into DNA repair deficient cells (xeroderma pigmentosum complementation group A). To compare the role of two major UV-induced photoproducts, cis-syn cyclobutane-type pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs), each photoproduct was removed from UV-irradiated plasmid by photoreactivation before transfection. Removal of either CPDs or 6-4PPs by in vitro photoreactivation reduced the mutation frequency while keeping the mutation distribution and the predominance of G:C-A:T transitions as UV-irradiated plasmid without photoreactivation, indicating that both cytosine-containing CPDs and 6-4PPs were premutagenic lesions for G:C-A:T transitions. On the other hand, A:T-G:C transitions were not recovered from plasmids after the removal of 6-4PPs, whereas this type of mutation occurred at a significant level (11%) after the removal of CPDs. Thus, the premutagenic lesions for the A:T-G:C transition are 6-4PPs. Removal of both CPDs and 6-4PPs resulted in the disappearance of mutational hot spots and random distribution of mutation as observed in unirradiated control plasmids. However, the mutational spectrum of photoreactivated plasmids differed significantly from that of unirradiated plasmids. A characteristic feature is a high portion of A:T-T:A transversions (11%) in the photoreactivated plasmid. This mutation is due to nondipyrimidinic "minor" photoproducts, and the mutation spectrum suggests that TA*, the major photoproduct of thymidylyl-(3'-5')-deoxyadenosine, is the premutagenic lesion for this mutation. This is the first report revealing the distinct mutagenic roles of the major UV photoproducts and "minor" photoproducts by the use of (6-4)photolyase.
Asunto(s)
Reparación del ADN , ADN/efectos de la radiación , Dímeros de Pirimidina/metabolismo , Rayos Ultravioleta , Secuencia de Bases , Línea Celular Transformada , ADN/química , ADN/genética , Análisis Mutacional de ADN , Ensayo de Inmunoadsorción Enzimática , Genes Supresores/genética , Humanos , Datos de Secuencia Molecular , Mutagénesis , Mutación , Plásmidos/química , Plásmidos/genética , Plásmidos/efectos de la radiación , Mutación Puntual , Dímeros de Pirimidina/fisiología , ARN de Transferencia/genética , Homología de Secuencia de Ácido Nucleico , Xerodermia Pigmentosa/genética , Xerodermia Pigmentosa/patologíaRESUMEN
Monoclonal antibodies specific for the cyclobutane pyrimidine dimer (CPD) are widely used for detection and quantification of DNA photolesions. However, the mechanisms of antigen binding by anti-CPD antibodies are little understood. Here we report NMR analyses of antigen recognition by TDM-2, which is a mouse monoclonal antibody specific for the cis - syn -cyclobutane thymine dimer (T[ c, s ]T). (31)P NMR and surface plasmon resonance data indicated that the epitope recognized by TDM-2 comprises hexadeoxynucleotides centered on the CPD. Chemical shift perturbations observed for TDM-2 Fab upon binding to d(T[ c, s ]T) and d(TAT[ c, s ]TAT) were examined in order to identify the binding sites for these antigen analogs. It was revealed that d(T[ c, s ]T) binds to the central part of the antibody-combining site, while the CPD-flanking nucleotides bind to the positively charged area of the V(H)domain via electrostatic interactions. By applying a novel NMR method utilizing a pair of spin-labeled DNA analogs, the orientation of DNA with respect to the antigen-binding site was determined: CPD-containing oligonucleotides bind to TDM-2 in a crooked form, draping the 3'-side of the nucleotides onto the H1 and H3 segments, with the 5'-side on the H2 and L3 segments. These data provide valuable information for antibody engineering of TDM-2.
Asunto(s)
Anticuerpos Monoclonales/inmunología , ADN/metabolismo , Fragmentos Fab de Inmunoglobulinas/metabolismo , Dímeros de Pirimidina/inmunología , Animales , Simulación por Computador , Espectroscopía de Resonancia Magnética , Ratones , Modelos Moleculares , Isótopos de Fósforo , Unión Proteica , Marcadores de SpinRESUMEN
We investigated the protective effects of commercial sunscreening agents against UVB-induced photoresponses in group A xeroderma pigmentosum (XPA) model mice. XPA gene-deficient mice are defective in nucleotide excision repair and show a high incidence of skin tumors and severe acute inflammation in response to UVB irradiation, in a similar manner to XP patients. SPF 10 and SPF 60 sunscreens protected partially and almost completely, respectively, ear swelling responses produced by UVB up to 200 mJ/cm2 in (-/-) mice. XPA (-/-) mice were irradiated three times a week to a cumulative dose of 2.6 J/cm2 UVB for a period of 24 weeks with or without SPF 10 or SPF 60 sunscreen. UV-induced skin tumors had developed in all unprotected (-/-) mice (13.3 tumors per mouse) at the completion of UVB irradiation. The SPF 60 sunscreen afforded stronger protection against photocarcinogenesis (1.0 tumors per mouse) than the SPF 10 sunscreen (4.4 tumors per mouse). Regarding photoaging, SPF 60 sunscreen also protected against mast cell infiltration (79% inhibition), elastic fiber accumulation, and dermal cyst proliferation in XPA (-/-) mice compared with unprotected (-/-) mice. In (-/-) mice, the SPF 60 sunscreen provided stronger protection against cyclobutane pyrimidine dimer formation shown immunohistologically following irradiation with 200 mJ/cm2 UVB than the SPF 10 sunscreen. The XPA model mouse is a useful animal for the evaluation of the photoprotective ability of sunscreens because photoresponses, even chronic changes, can be easily and quickly induced experimentally.
Asunto(s)
Proteínas de Unión al ADN/genética , Protectores Solares/uso terapéutico , Xerodermia Pigmentosa/tratamiento farmacológico , Envejecimiento/efectos de los fármacos , Envejecimiento/efectos de la radiación , Animales , Proteínas de Unión al ADN/deficiencia , Modelos Animales de Enfermedad , Oído/efectos de la radiación , Técnica del Anticuerpo Fluorescente , Mastocitos/efectos de los fármacos , Mastocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Noqueados , Neoplasias Inducidas por Radiación/prevención & control , Dímeros de Pirimidina/análisis , Dímeros de Pirimidina/uso terapéutico , Piel/efectos de los fármacos , Piel/patología , Neoplasias Cutáneas/etiología , Neoplasias Cutáneas/prevención & control , Rayos Ultravioleta , Xerodermia Pigmentosa/inmunología , Xerodermia Pigmentosa/patología , Proteína de la Xerodermia Pigmentosa del Grupo ARESUMEN
In this paper, we report the development and characterization of a solar ultraviolet (UV) dosimetry system that can be used as a film badge for radiation monitoring. DNA molecules are coated on a thin nylon membrane as a UV dosimeter. The membrane is sealed in a polyethylene filter envelope with silica gel to keep the humidity low. After exposure to UV or solar light, induced DNA damage is measured by an immunochemical reaction. The intensity of color developed during the immunological reaction can be correlated linearly with the irradiated UV dose delivered by an Oriel solar simulator within a limited dose range. We observe no effects of temperature on the level of damage induction. The membrane is proficient for measuring DNA damage for more than 21 days when stored at either 37 or 4 degrees C. The induced damage remains stable on the membrane for at least 22 days at both 37 and 4 degrees C. In addition to these indoor experiments, we report measurements of solar UV dose in outdoor experiments.
Asunto(s)
ADN/efectos de la radiación , Dosimetría por Película/métodos , Luz Solar , Rayos Ultravioleta , Relación Dosis-Respuesta en la Radiación , Estabilidad de Medicamentos , Dosimetría por Película/instrumentaciónRESUMEN
The antigen binding site of monoclonal antibody 64M5, which possesses a high degree of affinity for DNA containing pyrimidine (6-4) pyrimidone photoproducts, were investigated by use of stable-isotope-assisted NMR spectroscopy. A variety of 64M5 Fab fragments specifically labeled with 13C and 15N at backbone amide groups were prepared. Extensive assignments of amide resonances originating from the variable region of 64M5 were made by using 2D-HN(CO) measurements along with recombination of the heavy and light chains of 64M5. On the basis of chemical shift changes of the amide resonances caused upon addition of d(T[6-4]T) and d(GTAT[6-4]TATG), the binding sites of 64M5 Fab for the (6-4) photodimer and for the oligodeoxynucleotides flanking it were identified. It was revealed that the L1 and L3 segments, which are responsible for the binding to (6-4) photodimer, exhibit conformational multiplicities in the absence of antigens, and take different conformations between the d(T[6-4]T) and d(GTAT[6-4]TATG)-bound forms. On the basis of spectral comparison with another Fab fragment with a similarity in the amino acid sequence of the VL domain of 64M5, we suggest that the conformational multiplicities observed in the present study is caused by a substitution of an amino acid residue at the position of a key residue in L3 canonical structure, which leads to a preferable effect on the antigen binding, and by a specific combination of L1 and L3 canonical structures.