RESUMEN
To investigate the repairability of X-ray induced DNA damage, particularly non-double-strand breaks in living cells, enhanced green fluorescent protein (EGFP)-expressing plasmids X-ray irradiated and then transfected into nonirradiated human cells, MCF7 and MCF10A. Live-cell imaging of EGFP fluorescence was performed to measure the efficiency of plasmid repair in cells. The number of EGFP-expressing cells significantly decreased with increasing X-ray dose for both cell lines. The obtained kinetic curves of EGFP expression indicating plasmid repair were quantitatively compared against algebraically calculated ones based on the values of the transfected plasmids that had been treated with nicking or restriction enzymes. Then, assuming a Poisson distribution of single-strand breaks (SSBs), the number of cells carrying these nicked plasmids that could express EGFP were estimated. Our experimental results revealed considerably fewer cells expressing EGFP compared to the expected values we had calculated. These results suggest that the lower proportion of cells expressing EGFP as a measure of plasmid repair was due not only to the complex chemical structures of termini created by SSBs compared to those created by enzyme treatments, but also that base lesions or AP sites proximately arising at the strand-break termini might compromise EGFP expression. These results emphasize that radiation-induced DNA breaks are less repairable than enzymatically induced DNA breaks, which is not apparent when using conventional gel electrophoresis assays of plasmid DNA.
Asunto(s)
Genes Reporteros/efectos de la radiación , Proteínas Fluorescentes Verdes/genética , Plásmidos/efectos de la radiación , Línea Celular , Daño del ADN , Reparación del ADN , ADN Recombinante/efectos de la radiación , Células Epiteliales/efectos de la radiación , Genes BRCA1 , Proteínas Fluorescentes Verdes/biosíntesis , Humanos , Microscopía Intravital , Células MCF-7 , Microscopía Fluorescente , Conformación de Ácido Nucleico/efectos de la radiación , Plásmidos/genética , Imagen de Lapso de Tiempo , TransfecciónRESUMEN
PURPOSE: In an effort to assess the characteristics of mutation induced by different linear energy transfer (LET) radiation in higher plants, the mutational effects of carbon-ion beams and gamma-rays were investigated in Arabidopsis. MATERIALS AND METHODS: The rpsL (Escherichia coli ribosomal protein small subunit S12) transgenic Arabidopsis (Arabidopsis/rpsL) mutation detection system was adopted. Dry seeds of Arabidopsis/rpsL were irradiated with gamma-rays and 208-MeV carbon ions (208-MeV (12)C(5+)), and the mutation frequency and mutation spectrum were examined. RESULTS: The frequency of mutant clones increased following irradiation with 208-MeV (12)C(5+) and gamma-rays. Mutation spectrum analysis showed that G:C to A:T transitions and >2 bp deletions/insertions were significantly induced by both 208-MeV (12)C(5+) and gamma-rays. -1 and -2 frameshift mutations were characteristic in the gamma-ray irradiated group. CONCLUSIONS: 208-MeV (12)C(5+) and gamma-rays induced different intragenic mutations in respect to the size of deletions, reflecting differences in the nature of the DNA damage induced. Our results also suggested that base substitutions derived from the generation of 8-oxoguanine were low in dry seeds. The mutation spectrum obtained in this study might have reflected the characteristic conditions of plant dry seeds such as low water content and cell proliferation activity.
Asunto(s)
Arabidopsis/genética , Arabidopsis/efectos de la radiación , Transferencia Lineal de Energía , Mutación , Secuencia de Bases , Carbono , Reparación del ADN/genética , Reparación del ADN/efectos de la radiación , ADN Recombinante/genética , ADN Recombinante/efectos de la radiación , Proteínas de Escherichia coli , Rayos gamma , Genes Bacterianos/efectos de la radiación , Plantas Modificadas Genéticamente , Tolerancia a Radiación , Proteína Ribosómica S9 , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/efectos de la radiaciónRESUMEN
In biomaterials applications there exists a need to protect against the environmental release of recombinant microorganisms and transmissible genetic material and to prevent the recovery of proprietary genetic information. Irradiation technologies have long been used to eliminate microorganisms associated with spoilage and contamination and recent studies have demonstrated that moderate doses of irradiation may be used to sterilize medically important proteins without causing adverse effects in their desirable biological properties. Recombinant Escherichia coli cells expressing organophosphate hydrolase (OPH, E.C. 3.1.8.1), an important enzyme for the detection and decontamination of neurotoxic pesticides and chemical warfare agents, were subjected to electron beam irradiation to gauge its effect on enzymatic activity, cell viability and DNA recoverability. Bacterial samples were irradiated at 2, 20 and 200 kGy using a 10 MeV electron source. Irradiation levels of 2 to 20 kGy were sufficient to eliminate viable cells without affecting OPH enzymatic activity. Biologically active DNA was recovered via PCR from all samples through the 20 kGy irradiation level. While DNA was not recovered from samples at the 200 kGy exposure level, protein activity was reduced by 19 to 78%, depending on the method of cell preparation. These results demonstrate that irradiation can be effective in preventing the release of recombinant organisms intended for use in biomaterials applications without eliminating enzymatic activity and suggests that further research may indicate specific conditions whereby DNA recovery can be eliminated while retaining sufficient enzymatic activity for targeted biomaterials applications.
Asunto(s)
Biotecnología/métodos , ADN Recombinante/efectos de la radiación , Electrones , Ambiente , Ingeniería Genética/métodos , Proteínas Recombinantes/efectos de la radiación , Arildialquilfosfatasa/metabolismo , Supervivencia Celular/efectos de la radiación , ADN Recombinante/genética , ADN Recombinante/metabolismo , Escherichia coli/citología , Escherichia coli/enzimología , Escherichia coli/genética , Escherichia coli/efectos de la radiación , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Monoéster Fosfórico Hidrolasas/efectos de la radiación , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
The wide range of sensitivities of stimulated T-cells from different individuals to radiation-induced chromatid breakage indicates the involvement of several low penetrance genes that appear to link elevated chromatid breakage to cancer susceptibility. The mechanisms of chromatid breakage are not yet fully understood. However, evidence is accumulating that suggests chromatid breaks are not simply expanded DNA double-strand breaks (DSB). Three models of chromatid breakage are considered. The classical breakage-first and the Revell "exchange" models do not accord with current evidence. Therefore a derivative of Revell's model has been proposed whereby both spontaneous and radiation-induced chromatid breaks result from DSB signaling and rearrangement processes from within large looped chromatin domains. Examples of such rearrangements can be observed by harlequin staining whereby an exchange of strands occurs immediately adjacent to the break site. However, these interchromatid rearrangements comprise less than 20% of the total breaks. The rest are thought to result from intrachromatid rearrangements, including a very small proportion involving complete excision of a looped domain. Work is in progress with the aim of revealing these rearrangements, which may involve the formation of inversions adjacent to the break sites. It is postulated that the disappearance of chromatid breaks with time results from the completion of such rearrangements, rather than from the rejoining of DSB. Elevated frequencies of chromatid breaks occur in irradiated cells with defects in both nonhomologous end-joining (NHEJ) and homologous recombination (HR) pathways, however there is little evidence of a correlation between reduced DSB rejoining and disappearance of chromatid breaks. Moreover, at least one treatment which abrogates the disappearance of chromatid breaks with time leaves DSB rejoining unaffected. The I-SceI DSB system holds considerable promise for the elucidation of these mechanisms, although the break frequency is relatively low in the cell lines so far derived. Techniques to study and improve such systems are under way in different cell lines. Clearly, much remains to be done to clarify the mechanisms involved in chromatid breakage, but the experimental models are becoming available with which we can begin to answer some of the key questions.
Asunto(s)
Cromátides/ultraestructura , Rotura Cromosómica , Animales , Ciclo Celular , Cromátides/efectos de la radiación , Cromosomas/efectos de los fármacos , Cromosomas/efectos de la radiación , Cromosomas/ultraestructura , Cricetinae , Cricetulus , ADN/efectos de los fármacos , ADN/genética , ADN/ultraestructura , ADN de Hongos/genética , ADN Recombinante/efectos de la radiación , ADN Recombinante/ultraestructura , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo II/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/efectos de la radiación , Fibroblastos/ultraestructura , Humanos , Intrones/genética , Modelos Genéticos , Ciervo Muntjac , Neoplasias/genética , Neoplasias/ultraestructura , Tolerancia a Radiación , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/farmacología , Intercambio de Cromátides Hermanas , Coloración y Etiquetado , TransfecciónRESUMEN
Many commonly used drugs, such as psoralen and cisplatin, can generate a very unique type of DNA damage, namely ICL (interstrand cross-link). An ICL can severely block DNA replication and transcription and cause programmed cell death. The molecular mechanism of repairing the ICL damage has not been well established. We have studied the role of XPF (xeroderma pigmentosum group F) protein in psoralen-induced ICL-mediated DNA repair and mutagenesis. The results obtained from our mutagenesis studies revealed a very similar mutation frequency in both human normal fibroblast cells and XPF cells. The mutation spectra generated in both cells, however, were very different: most of the mutations generated in the normal fibroblast cells were T167-->A transversions, whereas most of the mutations generated in the XPF cells were T167-->G transversions. When a wild-type XPF gene cDNA was stably transfected into the XPF cells, the T167-->A mutations were increased and the T167-->G mutations were decreased. We also determined the DNA repair capability of the XPF cells using both the host-cell reactivation and the in vitro DNA repair assays. The results obtained from the host-cell reactivation experiments revealed an effective reactivation of a luciferase reporter gene from the psoralen-damaged plasmid in the XPF cells. The results obtained from the in vitro DNA repair experiments demonstrated that the XPF nuclear extract is normal in introducing dual incisions during the nucleotide excision repair process. These results suggest that the XPF protein has important roles in the psoralen ICL-mediated DNA repair and mutagenesis.
Asunto(s)
Reactivos de Enlaces Cruzados/farmacología , Daño del ADN , Reparación del ADN/fisiología , ADN Recombinante/efectos de los fármacos , Proteínas de Unión al ADN/fisiología , Mutagénesis , Oligonucleótidos/farmacología , Trioxsaleno/farmacología , Xerodermia Pigmentosa/genética , Secuencia de Bases , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Células Cultivadas/efectos de la radiación , Aductos de ADN/metabolismo , Reparación del ADN/genética , ADN Complementario/genética , ADN Recombinante/genética , ADN Recombinante/efectos de la radiación , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/efectos de la radiación , Genes Reporteros , Genes Supresores , Genes Sintéticos/efectos de los fármacos , Genes Sintéticos/efectos de la radiación , Humanos , Luciferasas/biosíntesis , Luciferasas/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fotoquímica , Plásmidos/efectos de los fármacos , Plásmidos/efectos de la radiación , ARN de Transferencia/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Transfección , Trioxsaleno/análogos & derivados , Rayos Ultravioleta , Xerodermia Pigmentosa/metabolismo , Xerodermia Pigmentosa/patologíaRESUMEN
The large RBE (approximately 7) measured for the killing of Chinese hamster V79 cells by 340 eV ultrasoft X rays, which preferentially ionize the K shell of carbon atoms (Hervé du Penhoat et al., Radiat. Res. 151, 649-658, 1999), was used to investigate the location of sensitive sites for cell inactivation and the physical modes of action of radiation. The enhancement of the RBE above the carbon K-shell edge either may indicate a high intrinsic efficiency of carbon K-shell ionizations (due, for example, to a specific physical or chemical effect) or may be related to the preferential localization of these ionizations on the DNA. The second interpretation would indicate a strong local (within 3 nm) action of K-shell ionizations and consequently the importance of a direct mechanism for radiation lethality (without excluding an action in conjunction with an indirect component). To distinguish between these two hypotheses, the efficiencies of core ionizations in DNA atoms (phosphorus L-shell, carbon K-shell, and oxygen K-shell ionizations) to induce damages were investigated by measuring their capacities to produce DNA double-strand breaks (DSBs). The effect of photoionizations in isolated DNA was studied using pBS plasmids in a partially hydrated state. No enhancement of the efficiency of DSB induction by carbon K-shell ionizations compared to oxygen K-shell ionizations was found, supporting the hypothesis that it is the localization of these carbon K-shell events on DNA which gives to the 340 eV photons their high killing efficiency. In agreement with this interpretation, cell inactivation and DSB induction, which do not appear to be correlated when expressed in terms of yields per unit dose in the sample, exhibit a rather good correlation when expressed in terms of efficiencies per core event in the DNA. These results suggest that core ionizations in DNA, through core-hole relaxation in conjunction with localized effects of spatially correlated secondary and Auger electrons, may be the major critical events for cell inactivation, and that the resulting DSBs (or a constant fraction of these DSBs) may be a major class of unrepairable lesions.
Asunto(s)
Daño del ADN/efectos de la radiación , ADN/efectos de la radiación , Fibroblastos/efectos de la radiación , Rayos X/efectos adversos , Animales , Carbono/efectos de la radiación , Línea Celular/efectos de la radiación , Núcleo Celular/efectos de la radiación , Supervivencia Celular/efectos de la radiación , Cricetinae , Cricetulus , ADN Bacteriano/efectos de la radiación , ADN Recombinante/efectos de la radiación , ADN de Cadena Simple/efectos de la radiación , ADN Superhelicoidal/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Electrones , Rayos gamma , Iones , Pulmón/citología , Modelos Biológicos , Oxígeno/efectos de la radiación , Fósforo/efectos de la radiación , Fotones , Plásmidos/efectos de la radiación , Efectividad Biológica RelativaRESUMEN
The toxic and mutagenic effects of ionizing radiation are believed to be caused by damage to cellular DNA. We have made use of a novel immunoassay for thymine glycol to examine the removal of this lesion from the DNA of irradiated human cells. Because of the sensitivity of the assay, we have been able to keep the radiation doses at or below the standard clinical dose of 2 Gy. Our initial observations indicated that although removal of thymine glycol is > 80% complete by 4 h post-irradiation with 2 Gy, there is a lag of 30-60 min before repair commences. However, if cells are irradiated with 0.25 Gy 4 h prior to the 2-Gy dose, removal of the thymine glycols commences immediately after the second irradiation, suggesting that repair of thymine glycol is inducible. Our current studies are directed at two aspects of the repair process, (1) factors involved in the repair process leading up to and including glycosylase-mediated removal of thymine glycol and (2) the control of the inducible response. We have observed that mutation of the XPG gene drastically reduced the level and rate of global removal of thymine glycol (induced by 2-Gy irradiation), and there was no evidence for an inducible response. Similar results were seen with a Cockayne syndrome B (CSB) cell line. We have also examined repair in quiescent and phytohemagglutinin-stimulated human lymphocytes. Both show similar kinetics for the rate of removal of thymine glycol under induced and noninduced conditions.
Asunto(s)
Reparación del ADN , ADN/metabolismo , Timina/análogos & derivados , Timina/metabolismo , Animales , Células Cultivadas/efectos de la radiación , Síndrome de Cockayne/genética , Síndrome de Cockayne/patología , ADN/efectos de la radiación , Daño del ADN , ADN Circular/efectos de la radiación , ADN Recombinante/efectos de la radiación , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Relación Dosis-Respuesta en la Radiación , Electroforesis Capilar , Endonucleasas , Técnica del Anticuerpo Fluorescente Indirecta , Rayos gamma , Humanos , Activación de Linfocitos/genética , Linfocitos/metabolismo , Linfocitos/efectos de la radiación , Ratones , Proteínas Nucleares , Plásmidos/efectos de la radiación , Ratas , Fase de Descanso del Ciclo Celular , Sensibilidad y Especificidad , Timina/análisis , Factores de Transcripción , Xerodermia Pigmentosa/genética , Xerodermia Pigmentosa/patologíaRESUMEN
The chromatin remodeling process that takes place during spermiogenesis in mammals is characterized by a transient increase in DNA single-strand breaks (SSB). The mammalian transition proteins (TPs) are expressed at a high level at mid-spermiogenesis steps coincident with chromatin remodeling and could be involved in the repair of these lesions since SSB are no longer detected in terminally differentiated spermatids. We report that TP1 can stimulate the repair of SSB in vitro and demonstrate that in vivo repair of UV-induced DNA lesions is enhanced in mammalian cells stably expressing TP1. These results suggest that, aside from its role in DNA compaction, this major transition protein may contribute to the yet unidentified enzymatic activity responsible for the repair of SSB at mid-spermiogenesis steps. These results also suggest that the TP1 proteins have the potential to participate in the repair process following genotoxic insults and therefore may play an active role in the maintenance of the integrity of the male haploid genome during spermiogenesis.
Asunto(s)
Proteínas Cromosómicas no Histona/fisiología , Daño del ADN , Reparación del ADN , ADN de Cadena Simple/genética , Espermatogénesis/fisiología , Animales , Células COS , Chlorocebus aethiops , ADN/efectos de la radiación , ADN Ligasas/fisiología , ADN Circular/genética , ADN Circular/metabolismo , ADN Circular/efectos de la radiación , ADN Recombinante/genética , ADN Recombinante/metabolismo , ADN Recombinante/efectos de la radiación , ADN de Cadena Simple/metabolismo , Genes Reporteros , Infertilidad Masculina/genética , Luciferasas/genética , Masculino , Nucleosomas/efectos de los fármacos , Nucleosomas/efectos de la radiación , Proteínas Recombinantes de Fusión/fisiología , Espermatogénesis/genética , Transfección , Rayos UltravioletaRESUMEN
BACKGROUND: Most non-viral gene therapy vectors deliver transgenes into cells through the endocytic pathway. Lack of escape from endocytic vesicles in many cases constitutes a major barrier for delivery of the functional gene. We have developed a new technology named photochemical internalisation (PCI) to achieve light-inducible cytosolic delivery of the transgene. The technology is based on a photochemical treatment employing photosensitisers localised in endocytic vesicles. In this work mechanisms involved in PCI-mediated transfection (photochemical transfection) were studied. METHODS: Human melanoma or colon carcinoma cells were pre-incubated with the photosensitiser aluminium phthalocyanine disulfonate (AlPcS2a) followed by treatment with plasmid encoding enhanced green fluorescent protein (EGFP) complexed with poly-L-lysine, N-(1-(2,3-dioleoxyloxy)propyl)-N,N,N,-trimethylammonium-methyl-sulfate (DOTAP) or polyethylenimine (PEI) and light exposure. The expression of the EGFP-gene was scored by fluorescence microscopy and flow cytometry. RESULTS: The photochemical treatment using light doses corresponding to D50 substantially improves the efficiency of transfection mediated by poly-L-lysine and PEI, but not by DOTAP. The treatment does not enhance the delivery of the plasmid complex across the plasma membrane, since the amount of internalised plasmid is similar for irradiated and non-irradiated cells. Light-inducible transfection occurs only under temperature conditions allowing endocytic uptake and is not improved by chloroquine or ammonium chloride, but is inhibited by bafilomycin A1 (agents that increase vesicular pH and interfere with the endocytic transport). CONCLUSIONS: Photochemical transfection occurs through endocytosis, followed by cytosolic release of the transfecting DNA from photochemically permeabilised endocytic vesicles. Release of plasmid from early endosomes seems to be of importance in photochemical transfection, although a role of later endocytic vesicles can, however, not be ruled out.
Asunto(s)
Endosomas/fisiología , Luz , Macrólidos , Transfección/métodos , Cloruro de Amonio/farmacología , Antibacterianos/farmacología , Cloroquina/farmacología , ADN/genética , ADN Recombinante/genética , ADN Recombinante/metabolismo , ADN Recombinante/efectos de la radiación , Endocitosis/efectos de los fármacos , Endocitosis/efectos de la radiación , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/efectos de la radiación , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Proteínas Luminiscentes/efectos de la radiación , Plásmidos/genética , Temperatura , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
Ionizing radiation (IR) triggers apoptosis, cell-cycle arrest, and DNA-repair induction in mammalian cells. These responses are mediated by proteins, including p53, which are activated or induced by IR. To determine the role of p53 in double-strand break (DSB) repair following irradiation of mammalian cells, we compared the abilities of unirradiated and irradiated TK6 human lymphoblast line and its derivatives TK6-E6-20C and TK6-E6-5E to repair restriction-enzyme-linearized shuttle pZ189 and the luciferase-reporter plasmid pGL3-control. TK6-E6-20C expresses wild-type p53 like the parental TK6 line, while TK6-E6-5E is p53 null. DSB-rejoining capacity was determined from the ratio of viable progenies arising from DSB-containing plasmids (linDNA) to the number of viable progenies from undamaged, supercoiled plasmids (scDNA). The ratio from the p53wt hosts was two- to three-fold higher than that from the p53null host, using either pZ189 or pGL3-control plasmid. After exposure of both hosts to 0.5 Gy gamma-radiation, DSB-rejoining capacity of p53null increased two-fold compared to unirradiated null controls, if transfection occurred immediately after irradiation. In contrast, the DSB-rejoining capacity of p53wt was unaffected by irradiation. If transfection was delayed for 2 h following irradiation, however, DSB-rejoining declined in both p53wt and p53null hosts. Irradiation also altered DSB-rejoining fidelity, measured from the mutation frequencies, among progenies of pZ189 linDNA. But, unlike rejoining capacity, changes in DSB-rejoining fidelity were similar in p53wt and p53null hosts. Changes in cell-cycle distribution in p53wt and p53null hosts were also similar following irradiation. These findings show that IR increases DSB-rejoining capacity in mammalian cells without functional p53, suggesting that p53 participates in suppressing DSB-rejoining following exposure of mammalian cells to IR.
Asunto(s)
Daño del ADN , Reparación del ADN/efectos de la radiación , ADN/efectos de la radiación , Rayos gamma/efectos adversos , Genes p53 , Linfocitos/efectos de la radiación , Proteína p53 Supresora de Tumor/fisiología , Ciclo Celular/efectos de la radiación , Línea Celular , ADN/metabolismo , Reparación del ADN/genética , ADN Recombinante/metabolismo , ADN Recombinante/efectos de la radiación , Humanos , Linfocitos/ultraestructura , Mutagénesis , Plásmidos/efectos de la radiación , ARN de Transferencia/genética , TransfecciónRESUMEN
The genetic disorders xeroderma pigmentosum (XP) and Cockayne syndrome (CS) exhibit deficiencies in the repair of UV-induced DNA damage. CS fibroblasts retain proficient nucleotide excision repair (NER) of inactive (or bulk) DNA, but are deficient in the transcription-coupled repair (TCR) of active genes. In contrast, XP complementation group C (XP-C) fibroblasts retain proficient TCR, but are deficient in bulk DNA repair. The remaining NER-deficient XP groups exhibit deficiencies in both repair pathways. Ad5HCMVsp1lacZ is a recombinant adenovirus vector that is unable to replicate in human fibroblasts, but can efficiently infect and express the beta-galactosidase reporter gene in these cells. We have examined the host cell reactivation (HCR) of beta-galactosidase activity for UV-irradiated Ad5HCMVsp1lacZ in non-irradiated and UV-irradiated normal, XP-B, XP-C, XP-D, XP-F, XP-G, CS-A and CS-B fibroblasts. HCR of beta-galactosidase activity for UV-irradiated Ad5HCMVsp1lacZ was reduced in non-irradiated cells from each of the repair-deficient groups examined (including XP-C) relative to that in non-irradiated normal cells. Prior irradiation of cells with low UV fluences resulted in an enhancement of HCR for normal and XP-C strains, but not for the remaining XP and CS strains. HCR of the UV-damaged reporter gene in UV-irradiated XP and CS strains was similar to measurements of TCR reported previously for these cells. These results suggest that UV treatment results in an induced repair of UV-damaged DNA in the transcribed strand of an active gene in XP-C and normal cells through an enhancement of TCR or a mechanism which involves the TCR pathway.
Asunto(s)
Síndrome de Cockayne/patología , Reparación del ADN/efectos de la radiación , Genes Reporteros/efectos de la radiación , Operón Lac/efectos de la radiación , Transcripción Genética/efectos de la radiación , Rayos Ultravioleta , Xerodermia Pigmentosa/patología , Adenovirus Humanos/genética , Adenovirus Humanos/efectos de la radiación , Línea Celular , Síndrome de Cockayne/genética , ADN Recombinante/genética , ADN Recombinante/efectos de la radiación , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Vectores Genéticos/genética , Vectores Genéticos/efectos de la radiación , Humanos , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Transfección , Rayos Ultravioleta/efectos adversos , Xerodermia Pigmentosa/genética , beta-Galactosidasa/biosíntesisRESUMEN
Restriction fragments of pUC19 DNA were irradiated by various doses of UV light and analyzed by denaturing (alkaline) agarose gel electrophoresis. The irradiation generated retarded species whose mobility indicated two crosslinked DNA strands. Quantitative analysis of the experimental data provided an empirical equation relating the fraction of crosslinked DNA molecules to their length and to the dose of their irradiation by UV light. This equation can be used to predict the crosslinking behavior of pUC19-like DNA molecules whose primary structures do not much differ from a random nucleotide sequence. The amount of interstrand crosslinks increased with the (A+T) content of the pUC19 DNA fragments but the dependence was not clear-cut to indicate that oligonucleotide composition of DNA played a significant role as well.
Asunto(s)
ADN Recombinante/efectos de la radiación , Plásmidos/efectos de la radiación , Rayos Ultravioleta , Enzimas de Restricción del ADN , FotoquímicaRESUMEN
Irradiation of aqueous solutions of plasmid DNA (pUC18) at pH 7.6 with 193 nm laser light results in low yields of prompt single strand breakage (air-saturated sample phi ssh = [1.5 +/- 0.1] x 10(4), argon-saturated sample phi ssh = [0.9 +/- 0.1] x 10(4). Treatment of the irradiated DNA samples with Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg) protein results in an approximate 20-fold increase in the yield of single strand break-age (air-saturated sample phi fpg = [33.1 +/- 3.1] x 10(-4), argon-saturated sample phi fpg = [23.8 +/- 2.6] x 10(-4). This result indicates that 193 nm light induces other modification(s) (most likely of the purine moieties) that are 20 times more abundant than prompt strand breakage within the DNA matrix.
Asunto(s)
ADN Recombinante/efectos de la radiación , Proteínas de Escherichia coli , N-Glicosil Hidrolasas/biosíntesis , Rayos Ultravioleta , Aductos de ADN , ADN-Formamidopirimidina Glicosilasa , Escherichia coli/enzimologíaRESUMEN
Transcription when coupled to nucleotide excision repair specifies the location in active genes where preferential DNA repair is to take place. During DNA damage-induced recruitment of RNA polymerase (RNAP), there is a physical association of the beta subunit of Escherichia coli RNAP and the UvrA component of the repair apparatus (G. C. Lin and L. Grossman, submitted for publication). This molecular affinity is reflected in the ability of the RNAP to increase, in a promoter-dependent manner, DNA supercoiling by the UvrAB complex. In the presence of the RNAP, the UvrAB complex is able to bind to promoter regions and to translocate in a 5' to 3' direction along the non-transcribed strand. As a consequence of this helicase-catalyzed translocation, preferential incision of DNA damaged sites occurs downstream on the transcribed strand. Because of the helicase directionality, the initial binding of the UvrAB complex to the transcribed strand would inevitably lead to its collision with the RNAP. These results imply that the RNAP-induced DNA structure in the vicinity of the transcription start site signals a landing or entry site for the UvrAB complex on DNA.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas Bacterianas/metabolismo , ADN Helicasas , Proteínas de Unión al ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Proteínas de Escherichia coli , Transducción de Señal , Adenosina Trifosfatasas/química , Proteínas Bacterianas/química , Secuencia de Bases , Sitios de Unión , Reparación del ADN , ADN-Topoisomerasas de Tipo I/metabolismo , ADN Recombinante/metabolismo , ADN Recombinante/efectos de la radiación , Proteínas de Unión al ADN/química , Escherichia coli/enzimología , Datos de Secuencia Molecular , Permanganato de Potasio , Regiones Promotoras Genéticas , Conformación Proteica , Rayos UltravioletaRESUMEN
Based on the binding of the UvrAB complex to a promoter region in transcription open complexes (Ahn, B., and Grossman, L. (1996) J. Biol. Chem. 271, 21453-21461) and the requirement of a single-stranded region for UvrAB helicase activity, we examined the binding of UvrAB proteins to synthetic bubble or loop regions in duplex DNA and the role of these regions in translocation of the UvrAB complex as well as incision of DNA damage. We found that the UvrAB complex was able to bind to bubble and loop regions with an affinity similar to that for damaged DNA in the absence of RNAP. The preferential recognition and incision of damaged sites by the UvrAB complex was observed downstream of the bubble or loop region in the strand complementary to the strand along which the UvrAB complex translocates. These results imply that the bubble region generated in duplex DNA by RNAP serves as a preferred entry site for the translocation of the UvrAB complex, and that preferential binding and unidirectional translocation of the UvrAB complex predetermine where incision is to occur.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas Bacterianas/metabolismo , ADN Helicasas , ADN Recombinante/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Escherichia coli , Secuencia de Bases , Transporte Biológico , Western Blotting , Daño del ADN , ADN Recombinante/efectos de la radiación , ARN Polimerasas Dirigidas por ADN/metabolismo , Cinética , Datos de Secuencia Molecular , Permanganato de Potasio/química , Unión Proteica , Rayos UltravioletaRESUMEN
Capillary electrophoresis (CE) was used to monitor the laser-induced conversion of supercoiled pKOL8UV5 plasmid DNA into nicked conformers. The plasmid samples (0.1 mg/ml) were incubated in the absence or presence of 110 mumol/l ethidium bromide (EB) and then exposed to 100 J of argon laser radiation (488 nm). The nicked, open circular conformers were separated from the supercoiled DNA by a 15% increase in retention time. Approximately 90% of the control DNA was in the supercoiled form. Laser radiation in the presence of EB caused complete conversion of the supercoiled plasmid DNA into nicked conformers. Laser-induced fluorescence CE (LIF-CE) was about 100-fold more sensitive than UV-CE in the detection of these conformers. Agarose gel electrophoresis confirmed these findings and showed the presence of the nicked plasmid conformers. Based on these comparisons, CE is an efficient analytical tool for the identification of laser-induced conformational changes in plasmid DNA.
Asunto(s)
ADN Recombinante/efectos de la radiación , Electroforesis Capilar/métodos , Daño del ADN , ADN Recombinante/metabolismo , Rayos Láser , Fotólisis , Espectrofotometría UltravioletaRESUMEN
A newly-developed polyomavirus-based shuttle vector, pPySLPT-2, was used to analyze mutations induced by UV radiation in the two related mouse lymphoma cell lines, L5178Y-R (LY-R) and L5178Y-S (LY-S). These well-studied cell lines differ in sensitivity to UV radiation, apparently due to differences in DNA repair capacity. Consistent with these differences, we found that replication of UV-irradiated vector is inhibited to a greater extent in the UV sensitive LY-R cells than in the LY-S cells. Mutations were induced in the vector's supF target gene at a higher frequency in the LY-R cells than in the LY-S cells, but the sequence characteristics of the base substitution mutations were similar in the two cell lines. The pattern of UV-induced mutations in the supF target gene of this polyomavirus-based vector, replicated in the mouse cells, was very similar to the pattern observed in the same target gene in the analogous simian virus 40-based vector, pZ189, replicated in monkey and human cells.
Asunto(s)
Vectores Genéticos , Mutagénesis , Poliomavirus/genética , Rayos Ultravioleta , Animales , Secuencia de Bases , Daño del ADN , Replicación del ADN , ADN Recombinante/efectos de la radiación , Linfoma , Ratones , Datos de Secuencia Molecular , Células Tumorales CultivadasRESUMEN
The Kluyveromyces linear plasmids, pGKL1 and pGKL2, carrying terminal protein (TP), are located in the cytoplasm and have a unique gene expression system with the plasmid-specific promoter element termed UCS, which functions only in the cytoplasm. In this study we have developed an in vivo assay system in Saccharomyces cerevisiae which enables the detection of a rare migration of the yeast cytoplasmic plasmid to the nucleus, using a pGKL1-derived cytoplasmic linear plasmid pCLU1. pCLU1 had both the UCS-fused LEU2 gene (a cytoplasmic marker) and the native URA3 gene (a nuclear marker) and therefore its cytoplasmic-nucleo localized could be determined by the phenotypic analysis of the marker. The nuclearly migrated plasmids were often detected as linear plasmids having the telomere sequence of the host yeast at both ends, although circular plasmids were also found. The circular form was produced by the the terminal fusion of pCLU1. Insertion of a Ty element into a nuclearly migrated plasmid was observed, allowing the ROAM-regulated expression of the adjacent nuclearly silent UCS-fused LEU2 gene. The nuclearly located plasmids, whether linear or circular, were less sensitive to UV-mediated curing than pGKL and pCLU1.
Asunto(s)
Núcleo Celular/metabolismo , Citoplasma/metabolismo , ADN de Hongos/metabolismo , Plásmidos/genética , Saccharomyces cerevisiae/metabolismo , Secuencia de Bases , Transporte Biológico , Núcleo Celular/efectos de la radiación , ADN Circular/genética , ADN Circular/metabolismo , ADN Circular/efectos de la radiación , ADN de Hongos/efectos de la radiación , ADN Recombinante/genética , ADN Recombinante/metabolismo , ADN Recombinante/efectos de la radiación , Kluyveromyces/genética , Datos de Secuencia Molecular , Fenotipo , Plásmidos/metabolismo , Plásmidos/efectos de la radiación , Regiones Promotoras Genéticas , Saccharomyces cerevisiae/efectos de la radiación , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , Telómero , Rayos UltravioletaRESUMEN
Nucleotide excision repair (NER) functions to remove DNA damage caused by ultraviolet light and by other agents that distort the DNA helix. The NER machinery has been conserved in structure and function from yeast to humans, and in humans, defective NER is the underlying cause of the cancer-prone disease xeroderma pigmentosum. Here, we reconstitute the incision reaction of NER in Saccharomyces cerevisiae using purified protein factors. The Rad14 protein, the Rad4-Rad23 complex, the Rad2 nuclease, the Rad1-Rad10 nuclease, replication protein A, and the RNA polymerase II transcription factor TFIIH were purified to near homogeneity from yeast. We show that these protein factors are both necessary and sufficient for dual incision of DNA damaged by either ultraviolet light or N-acetoxy-2-aminoacetylfluorene. Incision in the reconstituted system requires ATP, which cannot be substituted by adenosine 5'-O-(3-thiotriphosphate), suggesting that the hydrolysis of ATP is indispensable for the incision reaction. The excision DNA fragments formed as a result of dual incision are in the 24-27-nucleotide range.
Asunto(s)
Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Factores Asociados con la Proteína de Unión a TATA , Factor de Transcripción TFIID , Factores de Transcripción TFII , Factores de Transcripción/metabolismo , 2-Acetilaminofluoreno/toxicidad , Daño del ADN , Enzimas Reparadoras del ADN , Replicación del ADN , ADN Recombinante/efectos de los fármacos , ADN Recombinante/aislamiento & purificación , ADN Recombinante/efectos de la radiación , Proteína de Replicación A , Factor de Transcripción TFIIH , Rayos UltravioletaRESUMEN
We have compared the effectiveness in causing DNA strand breaks of 111In bound to DNA or free in aqueous solution with that of gamma rays. Supercoiled DNA from pBR322 plasmid labeled with [3H]thymidine was purified and mixed with 111InCl3 in the absence or presence of diethylenetriaminepentaacetic dianhydride (DTPA), a metal chelator which prevents the binding of indium to DNA. The reaction mixtures were stored at 4 degrees C to accumulate radiation dose from the decay of 111In. The DNA was then resolved by gel electrophoresis into supercoiled, nicked circular and linear forms, representing undamaged DNA, single-strand breaks (SSBs) and double-strand breaks (DSBs), respectively. The D0 values of pBR322 DNA exposed to gamma radiation from an external 137Cs source and the decay of 111In dispersed in solution (+DTPA) are 3.1 +/- 0.1 and 2.8 +/- 0.1 Gy, respectively. In terms of accumulated 111In disintegrations cm-3 of plasmid DNA solution, the D0 value is 15.3 (+/- 0.7) x 10(10) disintegrations in the absence of DTPA and 38.2 (+/- 1.1) x 10(10) disintegrations in its presence. Since only 14.6 +/- 5% of the 111In was bound to DNA in the absence of DTPA, an effective D0 for bound 111In of 3.4 (+/- 1.1) x 10(10) disintegrations is obtained. The 11-fold (range 9- to 17-fold) increased effectiveness of this Auger electron emitter when in proximity to DNA appears to be due mainly to the higher yield of SSBs.