RESUMO
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.
Assuntos
Genes Reporter/efeitos da radiação , Proteínas de Fluorescência Verde/genética , Plasmídeos/efeitos da radiação , Linhagem Celular , Dano ao DNA , Reparo do DNA , DNA Recombinante/efeitos da radiação , Células Epiteliais/efeitos da radiação , Genes BRCA1 , Proteínas de Fluorescência Verde/biossíntese , Humanos , Microscopia Intravital , Células MCF-7 , Microscopia de Fluorescência , Conformação de Ácido Nucleico/efeitos da radiação , Plasmídeos/genética , Imagem com Lapso de Tempo , TransfecçãoRESUMO
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.
Assuntos
Arabidopsis/genética , Arabidopsis/efeitos da radiação , Transferência Linear de Energia , Mutação , Sequência de Bases , Carbono , Reparo do DNA/genética , Reparo do DNA/efeitos da radiação , DNA Recombinante/genética , DNA Recombinante/efeitos da radiação , Proteínas de Escherichia coli , Raios gama , Genes Bacterianos/efeitos da radiação , Plantas Geneticamente Modificadas , Tolerância a Radiação , Proteína S9 Ribossômica , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/efeitos da radiaçãoRESUMO
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.
Assuntos
Biotecnologia/métodos , DNA Recombinante/efeitos da radiação , Elétrons , Meio Ambiente , Engenharia Genética/métodos , Proteínas Recombinantes/efeitos da radiação , Arildialquilfosfatase/metabolismo , Sobrevivência Celular/efeitos da radiação , DNA Recombinante/genética , DNA Recombinante/metabolismo , Escherichia coli/citologia , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/efeitos da radiação , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Monoéster Fosfórico Hidrolases/efeitos da radiação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
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.
Assuntos
Cromátides/ultraestrutura , Quebra Cromossômica , Animais , Ciclo Celular , Cromátides/efeitos da radiação , Cromossomos/efeitos dos fármacos , Cromossomos/efeitos da radiação , Cromossomos/ultraestrutura , Cricetinae , Cricetulus , DNA/efeitos dos fármacos , DNA/genética , DNA/ultraestrutura , DNA Fúngico/genética , DNA Recombinante/efeitos da radiação , DNA Recombinante/ultraestrutura , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/efeitos da radiação , Fibroblastos/ultraestrutura , Humanos , Íntrons/genética , Modelos Genéticos , Cervo Muntjac , Neoplasias/genética , Neoplasias/ultraestrutura , Tolerância a Radiação , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/farmacologia , Troca de Cromátide Irmã , Coloração e Rotulagem , TransfecçãoRESUMO
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.
Assuntos
Reagentes de Ligações Cruzadas/farmacologia , Dano ao DNA , Reparo do DNA/fisiologia , DNA Recombinante/efeitos dos fármacos , Proteínas de Ligação a DNA/fisiologia , Mutagênese , Oligonucleotídeos/farmacologia , Trioxsaleno/farmacologia , Xeroderma Pigmentoso/genética , Sequência de Bases , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Células Cultivadas/efeitos da radiação , Adutos de DNA/metabolismo , Reparo do DNA/genética , DNA Complementar/genética , DNA Recombinante/genética , DNA Recombinante/efeitos da radiação , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Genes Reporter , Genes Supressores , Genes Sintéticos/efeitos dos fármacos , Genes Sintéticos/efeitos da radiação , Humanos , Luciferases/biossíntese , Luciferases/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fotoquímica , Plasmídeos/efeitos dos fármacos , Plasmídeos/efeitos da radiação , RNA de Transferência/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Transfecção , Trioxsaleno/análogos & derivados , Raios Ultravioleta , Xeroderma Pigmentoso/metabolismo , Xeroderma Pigmentoso/patologiaRESUMO
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.
Assuntos
Dano ao DNA/efeitos da radiação , DNA/efeitos da radiação , Fibroblastos/efeitos da radiação , Raios X/efeitos adversos , Animais , Carbono/efeitos da radiação , Linhagem Celular/efeitos da radiação , Núcleo Celular/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Cricetinae , Cricetulus , DNA Bacteriano/efeitos da radiação , DNA Recombinante/efeitos da radiação , DNA de Cadeia Simples/efeitos da radiação , DNA Super-Helicoidal/efeitos da radiação , Relação Dose-Resposta à Radiação , Elétrons , Raios gama , Íons , Pulmão/citologia , Modelos Biológicos , Oxigênio/efeitos da radiação , Fósforo/efeitos da radiação , Fótons , Plasmídeos/efeitos da radiação , Eficiência Biológica RelativaRESUMO
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.
Assuntos
Reparo do DNA , DNA/metabolismo , Timina/análogos & derivados , Timina/metabolismo , Animais , Células Cultivadas/efeitos da radiação , Síndrome de Cockayne/genética , Síndrome de Cockayne/patologia , DNA/efeitos da radiação , Dano ao DNA , DNA Circular/efeitos da radiação , DNA Recombinante/efeitos da radiação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Relação Dose-Resposta à Radiação , Eletroforese Capilar , Endonucleases , Técnica Indireta de Fluorescência para Anticorpo , Raios gama , Humanos , Ativação Linfocitária/genética , Linfócitos/metabolismo , Linfócitos/efeitos da radiação , Camundongos , Proteínas Nucleares , Plasmídeos/efeitos da radiação , Ratos , Fase de Repouso do Ciclo Celular , Sensibilidade e Especificidade , Timina/análise , Fatores de Transcrição , Xeroderma Pigmentoso/genética , Xeroderma Pigmentoso/patologiaRESUMO
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.
Assuntos
Proteínas Cromossômicas não Histona/fisiologia , Dano ao DNA , Reparo do DNA , DNA de Cadeia Simples/genética , Espermatogênese/fisiologia , Animais , Células COS , Chlorocebus aethiops , DNA/efeitos da radiação , DNA Ligases/fisiologia , DNA Circular/genética , DNA Circular/metabolismo , DNA Circular/efeitos da radiação , DNA Recombinante/genética , DNA Recombinante/metabolismo , DNA Recombinante/efeitos da radiação , DNA de Cadeia Simples/metabolismo , Genes Reporter , Infertilidade Masculina/genética , Luciferases/genética , Masculino , Nucleossomos/efeitos dos fármacos , Nucleossomos/efeitos da radiação , Proteínas Recombinantes de Fusão/fisiologia , Espermatogênese/genética , Transfecção , Raios UltravioletaRESUMO
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.
Assuntos
Endossomos/fisiologia , Luz , Macrolídeos , Transfecção/métodos , Cloreto de Amônio/farmacologia , Antibacterianos/farmacologia , Cloroquina/farmacologia , DNA/genética , DNA Recombinante/genética , DNA Recombinante/metabolismo , DNA Recombinante/efeitos da radiação , Endocitose/efeitos dos fármacos , Endocitose/efeitos da radiação , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos da radiação , Proteínas de Fluorescência Verde , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas Luminescentes/efeitos da radiação , Plasmídeos/genética , Temperatura , Fatores de Tempo , Células Tumorais CultivadasRESUMO
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.
Assuntos
Dano ao DNA , Reparo do DNA/efeitos da radiação , DNA/efeitos da radiação , Raios gama/efeitos adversos , Genes p53 , Linfócitos/efeitos da radiação , Proteína Supressora de Tumor p53/fisiologia , Ciclo Celular/efeitos da radiação , Linhagem Celular , DNA/metabolismo , Reparo do DNA/genética , DNA Recombinante/metabolismo , DNA Recombinante/efeitos da radiação , Humanos , Linfócitos/ultraestrutura , Mutagênese , Plasmídeos/efeitos da radiação , RNA de Transferência/genética , TransfecçãoRESUMO
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.
Assuntos
Síndrome de Cockayne/patologia , Reparo do DNA/efeitos da radiação , Genes Reporter/efeitos da radiação , Óperon Lac/efeitos da radiação , Transcrição Gênica/efeitos da radiação , Raios Ultravioleta , Xeroderma Pigmentoso/patologia , Adenovírus Humanos/genética , Adenovírus Humanos/efeitos da radiação , Linhagem Celular , Síndrome de Cockayne/genética , DNA Recombinante/genética , DNA Recombinante/efeitos da radiação , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Vetores Genéticos/genética , Vetores Genéticos/efeitos da radiação , Humanos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Transfecção , Raios Ultravioleta/efeitos adversos , Xeroderma Pigmentoso/genética , beta-Galactosidase/biossínteseRESUMO
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.
Assuntos
DNA Recombinante/efeitos da radiação , Plasmídeos/efeitos da radiação , Raios Ultravioleta , Enzimas de Restrição do DNA , FotoquímicaRESUMO
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.
Assuntos
DNA Recombinante/efeitos da radiação , Proteínas de Escherichia coli , N-Glicosil Hidrolases/biossíntese , Raios Ultravioleta , Adutos de DNA , DNA-Formamidopirimidina Glicosilase , Escherichia coli/enzimologiaRESUMO
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.
Assuntos
DNA Recombinante/efeitos da radiação , Eletroforese Capilar/métodos , Dano ao DNA , DNA Recombinante/metabolismo , Lasers , Fotólise , Espectrofotometria UltravioletaRESUMO
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.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , DNA Helicases , Proteínas de Ligação a DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli , Transdução de Sinais , Adenosina Trifosfatases/química , Proteínas de Bactérias/química , Sequência de Bases , Sítios de Ligação , Reparo do DNA , DNA Topoisomerases Tipo I/metabolismo , DNA Recombinante/metabolismo , DNA Recombinante/efeitos da radiação , Proteínas de Ligação a DNA/química , Escherichia coli/enzimologia , Dados de Sequência Molecular , Permanganato de Potássio , Regiões Promotoras Genéticas , Conformação Proteica , Raios UltravioletaRESUMO
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.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , DNA Helicases , DNA Recombinante/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli , Sequência de Bases , Transporte Biológico , Western Blotting , Dano ao DNA , DNA Recombinante/efeitos da radiação , RNA Polimerases Dirigidas por DNA/metabolismo , Cinética , Dados de Sequência Molecular , Permanganato de Potássio/química , Ligação Proteica , Raios UltravioletaRESUMO
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.
Assuntos
Vetores Genéticos , Mutagênese , Polyomavirus/genética , Raios Ultravioleta , Animais , Sequência de Bases , Dano ao DNA , Replicação do DNA , DNA Recombinante/efeitos da radiação , Linfoma , Camundongos , Dados de Sequência Molecular , Células Tumorais CultivadasRESUMO
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.
Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , DNA Fúngico/metabolismo , Plasmídeos/genética , Saccharomyces cerevisiae/metabolismo , Sequência de Bases , Transporte Biológico , Núcleo Celular/efeitos da radiação , DNA Circular/genética , DNA Circular/metabolismo , DNA Circular/efeitos da radiação , DNA Fúngico/efeitos da radiação , DNA Recombinante/genética , DNA Recombinante/metabolismo , DNA Recombinante/efeitos da radiação , Kluyveromyces/genética , Dados de Sequência Molecular , Fenótipo , Plasmídeos/metabolismo , Plasmídeos/efeitos da radiação , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/efeitos da radiação , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Telômero , Raios UltravioletaRESUMO
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.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Fatores Associados à Proteína de Ligação a TATA , Fator de Transcrição TFIID , Fatores de Transcrição TFII , Fatores de Transcrição/metabolismo , 2-Acetilaminofluoreno/toxicidade , Dano ao DNA , Enzimas Reparadoras do DNA , Replicação do DNA , DNA Recombinante/efeitos dos fármacos , DNA Recombinante/isolamento & purificação , DNA Recombinante/efeitos da radiação , Proteína de Replicação A , Fator de Transcrição TFIIH , Raios UltravioletaRESUMO
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.