RESUMEN
The problem of comparing two proportions in a 2 x 2 matched-pairs design with binary responses is considered. We consider one-sided null and alternative hypotheses. The problem has two nuisance parameters. Using the monotonicity of the multinomial distribution, four exact unconditional tests based on p-values are proposed by reducing the dimension of the nuisance parameter space from two to one in computation. The size and power of the four exact tests and two other tests, the exact conditional binomial test and the asymptotic McNemar's test, are considered. It is shown that the tests based on the confidence interval p-value are more powerful than the tests based on the standard p-value. In addition, it is found that the exact conditional binomial test is conservative and not powerful for testing the hypothesis. Moreover, the asymptotic McNemar's test is shown to have incorrect size; that is, its size is larger than the nominal level of the test. Overall, the test based on McNemar's statistic and the confidence interval p-value is found to be the most powerful test with the correct size among the tests in this comparison.
Asunto(s)
Interpretación Estadística de Datos , Análisis por Apareamiento , Distribución Binomial , Investigación Biomédica , Biometría , Estudios de Casos y Controles , Intervalos de Confianza , Proyectos de Investigación , Estados UnidosRESUMEN
Enhanced DNA repair is an important factor in drug resistance in cancer. Using cell-free extracts derived from the fission yeast, Schizosaccharomyces pombe, we demonstrate in an in vitro system DNA repair system that increased cAMP levels, which activates cAMP-dependent protein kinase (PKA), inhibits repair of ultraviolet (UV)-damaged DNA. Supplementing the cell-free system with the catalytic kinase subunit of PKA also inhibits DNA repair. In contrast, addition of the PKA inhibitor H-89 enhances repair activity. These results show that PKA regulates DNA repair synthesis, thus implicating the cAMP signaling pathway in DNA damage response and repair of UV-damaged DNA lesions.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Reparación del ADN/fisiología , Sulfonamidas , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Sistema Libre de Células , AMP Cíclico/metabolismo , Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/inmunología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , ADN/efectos de la radiación , Daño del ADN , Reparación del ADN/efectos de los fármacos , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Sueros Inmunes , Isoquinolinas/farmacología , Schizosaccharomyces/química , Transducción de Señal/fisiologíaRESUMEN
DNA repair is essential for the surveillance and maintenance of the integrity of the genome in response to various insults that damage DNA. The development of cell-free repair systems using radiolabeled nucleotide to monitor repair synthesis of exogenously introduced damaged-plasmid DNA has enabled the analysis of specific proteins required for repair synthesis. However, the hazards and the burgeoning cost of using radioisotopes have become significant factors in the laboratory. We describe here the use of digoxigenin-dUTP in place of radioactivity in a nonradioactive cell-free repair assay to detect DNA repair.
Asunto(s)
Reparación del ADN , Animales , Células CHO , Cricetinae , Daño del ADN , Plásmidos , Schizosaccharomyces/genética , Sensibilidad y Especificidad , Rayos UltravioletaRESUMEN
Nonparametric versions of normal theory step-down multiple-test procedures for inferring minimum effective dose (see Tamhane et al. (1)) were developed and studied by Monte Carlo simulation. Two types of step-down testing procedures were examined. For both procedures, pairwise, linear, or Helmert contrasts of mean ranks were studied. All nonparametric step-down procedures controlled familywise error rate under normal, double exponential, and exponential distributions. Specific recommendations based on power and bias comparisons of nonparametric methods among themselves, with Shirley's (2) test, and with parametric step-down multiple-test procedures are given.
Asunto(s)
Preparaciones Farmacéuticas/administración & dosificación , Estadísticas no Paramétricas , Simulación por Computador , Relación Dosis-Respuesta a Droga , Cómputos Matemáticos , Método de Montecarlo , Farmacología/métodos , Toxicología/métodosRESUMEN
In addition to nucleotide excision repair (NER), the fission yeast Schizosaccharomyces pombe possesses a UV damage endonuclease (UVDE) for the excision of cyclobutane pyrimidine dimers and 6-4 pyrimidine pyrimidones. We have previously described UVDE as part of an alternative excision repair pathway, UVDR, for UV damage repair. The existence of two excision repair processes has long been postulated to exist in S.pombe, as NER-deficient mutants are still proficient in the excision of UV photoproducts. UVDE recognizes the phosphodiester bond immediately 5'of the UV photoproducts as the initiating event in this process. We show here that UVDE activity is inducible at both the level of uve1+ mRNA and UVDE enzyme activity. Further, we show that UVDE activity is regulated by the product of the rad12 gene.
Asunto(s)
Reparación del ADN , ADN de Hongos/genética , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/genética , Rayos Ultravioleta , Secuencia de Bases , Ciclo Celular , Daño del ADN , ADN de Hongos/efectos de la radiación , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Inducción Enzimática , Genes Fúngicos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Schizosaccharomyces/enzimologíaRESUMEN
We have discovered a new DNA endonuclease in the fission yeast Schizosaccharomyces pombe which recognizes cyclobutane pyrimidine dimers and (6-4) pyrimidine-pyrimidone photoproducts. S. pombe DNA endonuclease (SPDE) catalyzes a single ATP-independent incision immediately 5' to the UV photoproduct and generates termini containing 3' hydroxyl and 5' phosphoryl groups. Based on these properties, we propose that SPDE may function in a DNA repair capacity, representing the initial recognition/cleavage step of a DNA excision repair pathway.
Asunto(s)
Adenosina Trifosfato/farmacología , Reparación del ADN , Endodesoxirribonucleasas/metabolismo , Dímeros de Pirimidina/metabolismo , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/enzimología , Secuencia de Bases , ADN/química , ADN/metabolismo , Daño del ADN , Datos de Secuencia Molecular , Fotoquímica , Especificidad por Sustrato , Rayos UltravioletaRESUMEN
A whole cell extract prepared from Schizosaccharomyces pombe was shown to be active in an assay for repair of plasmid DNA damaged by either ultraviolet (UV) light or gamma-radiation. The assay allows for analysis of repair synthesis at single-strand nicks generated by gamma-rays and analysis of the incision step and repair synthesis in UV-light-damaged DNA. Repair synthesis of DNA damaged by either UV light or gamma-rays was shown to depend on the presence of ATP in the reaction mixture. However, incision at pyrimidine dimers did not require the addition of exogenous ATP. These studies showed that plasmid DNA containing a single pyrimidine dimer or one single-strand nick is a suitable substrate in this assay system. S. pombe is a genetically well-defined eukaryotic organism and many radiation-sensitive mutant derivatives have already been described, making this a powerful system in which to study DNA excision repair.
Asunto(s)
Daño del ADN , Reparación del ADN , Schizosaccharomyces/genética , Adenosina Trifosfato/metabolismo , Sistema Libre de Células , Daño del ADN/efectos de la radiación , ADN Glicosilasas , Relación Dosis-Respuesta en la Radiación , Rayos gamma , Magnesio/metabolismo , N-Glicosil Hidrolasas/metabolismo , Plásmidos , Rayos UltravioletaRESUMEN
We have examined ligation and nucleosome rearrangement of repair patches in chromatin of human fibroblasts damaged with bleomycin (BLM), UV radiation and methyl methanesulfonate (MMS) to follow completion of excision repair involving different combinations of repair enzymes. Conditions were used that allowed analysis of the correlation between these two events over a large range (i.e. from 5% to greater than 99% ligated). Cells exposed to BLM were reversibly permeabilized with L-alpha-lysophosphatidylcholine and pulse-labeled with either [3H]dTTP or [3H]dThd to label selectively cells that 'reseal their membranes' at different rates. A striking difference is observed in the rates of ligation of these nascent repair patches, in that those labeled with [3H]dTTP are ligated much slower (25-50% unligated after 24 h) than those labeled with [3H]dThd (less than 5% unligated after 6 h). The nuclease sensitivity of [3H]dTTP-labeled patches also decreases more slowly, indicating that the rate of nucleosome rearrangement decreases compared to that of repair patches labeled with [3H]dThd. The rates of repair patch ligation and loss of nuclease sensitivity were also modulated in intact cells exposed to UV radiation or MMS by treatment with aphidicolin and/or hydroxyurea. A plot of relative nuclease sensitivity versus fraction of ligated repair patches yields an overall linear correlation of greater than 0.8 in each case, indicating that these two features of nascent repair patches are 'moderately coupled' events. These results support the idea that ligation of repair patches is a prerequisite for nucleosome rearrangement following three different modes of excision repair.
Asunto(s)
Bleomicina/farmacología , Reparación del ADN , Metilmetanosulfonato/farmacología , Nucleosomas/efectos de los fármacos , Afidicolina/farmacología , Células Cultivadas , Humanos , Nucleosomas/efectos de la radiación , Nucleótidos de Timina/metabolismo , Rayos UltravioletaRESUMEN
We have examined the structure of newly repaired regions of chromatin in intact and permeabilized human cells following exposure to bleomycin (BLM). The average repair patch size (in permeabilized cells) was six to nine bases, following doses of 1-25 micrograms/mL BLM, and greater than 80% of the total repair synthesis was resistant to aphidicolin. In both intact and permeabilized cells, nascent repair patches were initially very sensitive to staphylococcal nuclease, analogous to repair induced by "long patch" agents, and are nearly absent from isolated nucleosome core DNA. Unlike long patch repair, however, the loss of nuclease sensitivity during subsequent chase periods was very slow in intact cells, or in permeabilized cells treated with a low dose of BLM (1 microgram/mL), and was abolished by treatment with hydroxyurea (HU) or aphidicolin (APC). The rate of repair patch ligation did not correlate with this slow rate of chromatin rearrangement since greater than 95% of the patches were ligated within 6 h after incorporation (even in the presence of HU or APC). In permeabilized cells, repair patches induced by either 5 or 25 micrograms/mL BLM, where significant levels of strand breaks occur in compact regions of chromatin, lost the enhanced nuclease sensitivity at a rate similar to that observed following long patch repair. This rapid rate of rearrangement was not affected by APC. These results indicate that short patch repair in linker regions of nucleosomes, and/or "open" regions of chromatin, involves much less nucleosome rearrangement than long patch repair or short patch repair in condensed chromatin domains.
Asunto(s)
Bleomicina/farmacología , Daño del ADN , Reparación del ADN , ADN/efectos de los fármacos , Nucleosomas/efectos de los fármacos , Alquilantes/farmacología , Permeabilidad de la Membrana Celular , Células Cultivadas , Cromatina/fisiología , ADN/efectos de la radiación , ADN Polimerasa I/metabolismo , Replicación del ADN/efectos de los fármacos , Humanos , Mutación , Nucleosomas/efectos de la radiación , Sensibilidad y Especificidad , Rayos UltravioletaRESUMEN
We have examined bleomycin-induced DNA damage and repair in confluent human fibroblasts that were reversibly permeabilized to small molecules (e.g., deoxynucleotide triphosphates and trypan blue) by a short exposure to 80 micrograms/ml lysophosphatidylcholine. We found that this treatment dramatically increases the dose effectiveness of bleomycin in inducing DNA strand breaks and DNA repair synthesis in these cells. For example, when intact cells (not treated with lysophosphatidylcholine) were incubated with 100 micrograms/ml bleomycin, only about 5% of the cell population was observed to have undergone measurable DNA repair synthesis (by autoradiography). On the other hand, when these cells were reversibly permeabilized with lysophosphatidylcholine before treatment, we observed significant repair synthesis in greater than 80% of the cells using a bleomycin dose of only 5 micrograms/ml. Furthermore, sufficient levels of single- and double-strand breaks were introduced into nucleosome linker DNA of permeabilized cells to yield a nucleosomal repeat pattern in alkaline and neutral agarose gels. However, no change in the amount of DNA less than 23 kilobases was observed on these gels when intact cells were incubated with bleomycin.
Asunto(s)
Bleomicina/farmacología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Daño del ADN , Reparación del ADN/efectos de los fármacos , Lisofosfatidilcolinas/farmacología , Autorradiografía , Bleomicina/metabolismo , ADN/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Factores de TiempoRESUMEN
Rearrangements of chromatin structure during excision repair were examined in xeroderma pigmentosum (XP; complementation group A) human fibroblasts treated with the small-molecule alkylating agent methyl methanesulfonate (MMS). In agreement with past reports, we observed normal levels of repair synthesis in these cells during the first 12 h after exposure to 1.5 mM MMS, in contrast to the near zero incorporation of repair patches following exposure to 12 J/m2 u.v. light. Our results indicate that the relative nuclease sensitivity of newly repaired regions in MMS-treated nuclease sensitivity of newly repaired regions in MMS-treated XP (group A) cells is quantitatively similar to that of newly repaired regions in MMS-treated normal human fibroblasts. This enhanced sensitivity is accompanied by a marked under-representation of repair-incorporated nucleotides in isolated nucleosome core DNA. Pulse-chase experiments demonstrated that these regions rapidly undergo rearrangements in chromatin structure, and both the rate and extent of these rearrangements are similar (but not identical) to those observed in normal cells. This was also the case for the rate and extent of ligation of repair patches, as measured by the sensitivity of these regions to exonuclease III digestion. If the changes in nuclease sensitivity of newly repaired regions in DNA reflect an unfolding of nucleosome structure during excision repair, then these results indicate that the activity associated with this unfolding is present in XP (group A) cells.
Asunto(s)
Reparación del ADN , Nucleosomas/metabolismo , Xerodermia Pigmentosa/genética , ADN/biosíntesis , Daño del ADN , Fibroblastos/metabolismo , Humanos , Metilmetanosulfonato/farmacología , Xerodermia Pigmentosa/metabolismoRESUMEN
We have examined both the initial nuclease sensitivity and subsequent nucleosome rearrangement of newly repaired regions of chromatin in human diploid fibroblasts treated with methyl methanesulfonate (MMS) and methylnitrosourea (MNU). We initially examined the effect of these two alkylating agents on DNA replicative synthesis. The results indicate that immediately following damage by MMS or MNU, at a concentration of 2 mM, the level of replicative synthesis is 20-25% of the level in untreated cells. In the MMS-treated cells, this suppression of replicative synthesis is short lived and by 15 h after damage the level of replicative synthesis is approximately 3-fold greater than that in untreated cells. This 'latent stimulation' of replicative synthesis was not observed in the cells treated with 2 mM MNU, although the level of replicative synthesis in these cells did approach the level of untreated cells at later times. When these contributions were corrected for, it was found that the nucleotides incorporated by repair synthesis are initially (i.e., immediately following repair synthesis) both staphylococcal nuclease and DNase I sensitive, and are underrepresented in isolated nucleosome core DNA. Using methods previously described by us, we show that the relative nuclease sensitivity of these regions is quantitatively similar to that of newly repaired DNA following damage by u.v. radiation. Furthermore, the relative nuclease sensitivity of newly repaired DNA is initially high regardless of the time after damage that repair occurs (at least for 13 h after damage). This feature is also similar to u.v. induced repair synthesis. Finally, pulse-chase experiments demonstrated that following repair synthesis induced by MMS or MNU rearrangements of chromatin structure take place, and both the rate and extent of these rearrangements are similar to that observed for cells treated with u.v. radiation or bulky chemical carcinogens. Thus, our results indicate that the excision repair induced by these two small alkylating agents is associated with the same overall chromatin structural features as the excision repair of DNA damage induced by u.v. radiation and 'u.v.-mimetic' chemicals.