RESUMEN
Mutations in the DYNAMIN2 (DNM2) gene are frequently detected in human acute T-cell lymphoblastic leukemia (T-ALL), although the mechanisms linking these mutations to disease pathogenesis remain unknown. Using an ENU-based forward genetic screen for mice with erythroid phenotypes, we identified a heterozygous mouse line carrying a mutation in the GTPase domain of Dnm2 (Dnm2V265G) that induced a microcytic anemia. In vitro assays using the V265G mutant demonstrated loss of GTPase activity and impaired endocytosis that was comparable to other DNM2 mutants identified in human T-ALL. To determine the effects of DNM2 mutations in T-ALL, we bred the Dnm2V265G mice with the Lmo2 transgenic mouse model of T-ALL. Heterozygous Dnm2 mutants lacking the Lmo2 transgene displayed normal T-cell development, and did not develop T-ALL. In contrast, compound heterozygotes displayed an accelerated onset of T-ALL compared with mice carrying the Lmo2 oncogene alone. The leukemias from these mice exhibited a more immature immunophenotype and an expansion in leukemic stem cell numbers. Mechanistically, the Dnm2 mutation impaired clathrin-mediated endocytosis of the interleukin (IL)-7 receptor resulting in increased receptor density on the surface of leukemic stem cells. These findings suggest that DNM2 mutations cooperate with T-cell oncogenes by enhancing IL-7 signalling.
Asunto(s)
Dinamina II/genética , Interleucina-7/metabolismo , Leucemia de Células T/etiología , Mutación , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Endocitosis/genética , GTP Fosfohidrolasas/metabolismo , Humanos , Proteínas con Dominio LIM/genética , Leucemia de Células T/genética , Leucemia de Células T/metabolismo , Ratones , Oncogenes , Transducción de SeñalRESUMEN
This was a two-phase prospective intervention study in the cardiology intensive care unit (CICU) and medical intensive care unit (MICU) and of a public 1800-bed medical centre in Taiwan. In phase I, cleaning efficacy was monitored by ATP bioluminescence after daily morning cleaning, and only 43.9% of 221 tested surfaces passed. The baseline data were used to define an intervention consisting of a new cleaning protocol as well as a new education/training programme. In phase II, following the intervention, 88.1% of 270 surfaces were found to be clean. The combined infection rate in the CICU and MICU showed a statistically significant decrease of 49.7%.
Asunto(s)
Infección Hospitalaria/epidemiología , Infección Hospitalaria/prevención & control , Desinfección/métodos , Contaminación de Equipos/prevención & control , Servicio de Limpieza en Hospital/métodos , Centros Médicos Académicos , Servicio de Cardiología en Hospital , Equipos Desechables , Equipos y Suministros de Hospitales/microbiología , Personal de Salud/educación , Humanos , Unidades de Cuidados Intensivos , Mediciones Luminiscentes , Estudios Prospectivos , Taiwán/epidemiologíaRESUMEN
Studies by different groups on the rescue effect, where unirradiated bystander cells mitigated the damages in the irradiated cells, since its discovery by the authors' group in 2011 were first reviewed. The properties of the rescue effect were then examined using a novel experimental set-up to physically separate the rescue signals from the bystander signals. The authors' results showed that the rescue effect was mediated through activation of the nuclear factor-κB (NF-κB) response pathway in the irradiated cells, and that the NF-κB activation inhibitor BAY-11-7082 did not affect the activation of this response pathway in the irradiated cells induced by direct irradiation.
Asunto(s)
Adaptación Fisiológica/fisiología , Efecto Espectador/fisiología , Supervivencia Celular/fisiología , Regulación de la Expresión Génica/fisiología , FN-kappa B/metabolismo , Transducción de Señal/fisiología , Adaptación Fisiológica/efectos de la radiación , Partículas alfa , Efecto Espectador/efectos de la radiación , Supervivencia Celular/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Regulación de la Expresión Génica/efectos de la radiación , Células HeLa , Humanos , Dosis de Radiación , Transducción de Señal/efectos de la radiaciónRESUMEN
The common infectious agents in the chest wall include Mycobacterium tuberculosis, Actinomyces, fungi, Nocardia, Entamoeba histolytica, and other aerobes and anaerobes. Klebsiella pneumoniae is an uncommon etiological agent. We describe a case of ankylosing spondylitis in a 45-year-old man, who had exhibited a painless lump in the left posterior chest wall for 3 months and who presented with acute-onset pain, erythematous change, and fever in the 2 weeks before admission. Cultures of the blood and chest wall abscess both showed Gram-negative bacilli, which were classified as K. pneumoniae. A contrast-enhanced computed tomography scan of the abdomen revealed a nonenhancing cystic abscess measuring 4.9 × 6.5 × 6.4 cm in segment 6 of the liver and communicating with the chest wall. Drainage of the liver abscess under ultrasound guidance and open surgical drainage of the chest wall abscess combined with adequate antibiotic treatment resolved the abscess.
Asunto(s)
Absceso/microbiología , Infecciones por Klebsiella/microbiología , Klebsiella pneumoniae/aislamiento & purificación , Absceso Hepático/microbiología , Neoplasias Torácicas/microbiología , Absceso/patología , Humanos , Infecciones por Klebsiella/patología , Absceso Hepático/patología , Masculino , Persona de Mediana Edad , Neoplasias Torácicas/patología , Pared Torácica/microbiología , Pared Torácica/patología , Pared Torácica/cirugíaAsunto(s)
Antineoplásicos Fitogénicos/farmacología , Carcinoma Hepatocelular/tratamiento farmacológico , Puntos de Control del Ciclo Celular/efectos de los fármacos , Diterpenos/farmacología , Neoplasias Hepáticas/tratamiento farmacológico , Andrographis/química , Carcinoma Hepatocelular/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Células HeLa , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Mitosis/efectos de los fármacos , FN-kappa B/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Proteoma , Proteómica , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Mammalian cells respond to ionization radiation by sending out extracellular signals to affect non-irradiated neighboring cells, which is referred to as radiation induced bystander effect. In the present paper, we described a phenomenon entitled the "rescue effects", where the bystander cells rescued the irradiated cells through intercellular signal feedback. The effect was observed in both human primary fibroblast (NHLF) and cancer cells (HeLa) using two-cell co-culture systems. After co-culturing irradiated cells with unirradiated bystander cells for 24h, the numbers of 53BP1 foci, corresponding to the number of DNA double-strand breaks in the irradiated cells were less than those in the irradiated cells that were not co-cultured with the bystander cells (0.78±0.04foci/cell vs. 0.90±0.04foci/cell) at a statistically significant level. Similarly, both micronucleus formation and extent of apoptosis in the irradiated cells were different at statistically significant levels if they were co-cultured with the bystander cells. Furthermore, it was found that unirradiated normal cells would also reduce the micronucleus formation in irradiated cancer cells. These results suggested that the rescue effects could participate in repairing the radiation-induced DNA damages through a media-mediated signaling feedback, thereby mitigating the cytotoxicity and genotoxicity of ionizing radiation.
Asunto(s)
Efecto Espectador/efectos de la radiación , Comunicación Celular/efectos de la radiación , Retroalimentación Fisiológica/efectos de la radiación , Transducción de Señal/efectos de la radiación , Partículas alfa , Apoptosis/efectos de la radiación , Células Cultivadas , Técnicas de Cocultivo , Roturas del ADN de Doble Cadena/efectos de la radiación , Fibroblastos/metabolismo , Fibroblastos/efectos de la radiación , Células HeLa , Humanos , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Micronúcleos con Defecto Cromosómico/efectos de la radiación , Pruebas de Micronúcleos , Microscopía Confocal , Radiobiología/instrumentación , Radiobiología/métodos , Proteína 1 de Unión al Supresor Tumoral P53RESUMEN
AIMS: To assess the dosimetric effect of using a split-organ delineation approach during intensity-modulated radiotherapy (IMRT) treatment planning for advanced T-stage nasopharyngeal carcinoma (NPC). MATERIALS AND METHODS: Twenty NPC patients with T3-4 tumours were studied. A reference (REF) IMRT plan was generated based on a standard treatment planning protocol, with a set of user-defined dose constraints for optimisation. An investigative (INV) IMRT plan was then generated based on the same protocol, but treating several organs at risk (OARs; parotid glands, temporal lobes, cochlea, auditory nerves and planning organ at risk volume [PRV] of the brainstem) as split organs consisting of target-overlapping and non-target-overlapping sub-segments. These sub-segments were assigned independent dose constraints. The REF and INV plans were compared with respect to target coverage and OAR sparing. Target coverage was evaluated by the Dmin (minimum dose), V66/V60 (percentage volume of gross target volume [GTV]/planning target volume [PTV] receiving 66 Gy/60 Gy), target conformity index (CI), and tumour control probability (TCP). The sparing of OARs was evaluated by the commonly used dose end points for the respective OAR, and normal tissue complication probability (NTCP). RESULTS: For PTV coverage, the INV plan was superior to the REF plan in terms of Dmin (P=0.000), CI (P=0.005) and TCP (P=0.002). This is attributed to an increase in dose to the PTV-OAR overlapping sub-segments. Regarding the sparing of OARs, there was a significant reduction in the mean dose of the parotid glands (P=0.002), and a slight, but non-significant, increase in NTCP of the temporal lobes, cochlea and brainstem. CONCLUSIONS: Using a split-organ delineation approach in IMRT treatment planning for advanced T-stage NPC, a significant improvement in the target coverage and TCP could be achieved, whereas the mean dose of the parotid was reduced significantly. There was insignificant change in the NTCP of the temporal lobe, parotid gland, cochlea and brainstem, but a significant change in the NTCP of the auditory nerve. The approach provides the planner extra room to manipulate the dose constraints during optimisation, and to obtain the desired result in less attempts. This approach also has the potential to be used in a broader context for IMRT planning for other tumour sites.
Asunto(s)
Tronco Encefálico/efectos de la radiación , Neoplasias Nasofaríngeas/radioterapia , Radioterapia de Intensidad Modulada , Cóclea/efectos de la radiación , Nervio Coclear/efectos de la radiación , Humanos , Glándula Parótida/efectos de la radiación , Radiometría , Dosificación Radioterapéutica , Radioterapia de Intensidad Modulada/métodos , Lóbulo Temporal/efectos de la radiaciónRESUMEN
AIMS: The role of adjuvant chemoradiation for gastric cancer after curative R0 gastrectomy was first established by the US Intergroup 0116 study. Although confirmatory studies are in progress, few data are available regarding its application to the Chinese population. We describe our radiotherapy technique and report the treatment results in Hong Kong. MATERIALS AND METHODS: This was a single centre retrospective study on 63 Chinese patients who underwent adjuvant chemoradiation for gastric adenocarcinoma between June 2000 and December 2004. The treatment protocol was based on that of the Intergroup study. Computed tomography planned anteroposterior opposing field arrangement and treatment under breath hold at deep inspiration position were adopted. RESULTS: In total, 63 patients, mean age 50 years, with gastric cancer stage IB to limited metastatic IV disease were analysed. The median follow-up time was 27.2 months. The relapse-free survival and overall survival at 3 years were 50 and 54%, respectively. The recurrence pattern was dominated by distant failure and only one patient developed isolated locoregional recurrence. Of the 10 patients who had positive microscopic surgical margins after surgery, seven had recurred and died. On multivariate analysis, margin status was the only significant prognosticator for survival. Thirty per cent of patients experienced grade 3 or above acute toxicity (24% haematological, 14% gastrointestinal) and one patient died of neutropenic sepsis. There was one case of grade 3 late toxicity. CONCLUSIONS: The outcome after adjuvant chemoradiation for gastric cancer seemed to be favourable, with manageable toxicities, in the Chinese population. Locoregional failure was uncommon. Patients with microscopic surgical margin involvement had a very high failure rate despite adjuvant chemoradiation.
Asunto(s)
Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/radioterapia , Adulto , Anciano , Pueblo Asiatico , Quimioterapia Adyuvante , Terapia Combinada , Supervivencia sin Enfermedad , Femenino , Hong Kong/etnología , Humanos , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia , Radioterapia Adyuvante , Neoplasias Gástricas/cirugía , Tasa de Supervivencia , Resultado del TratamientoRESUMEN
Tien-Hua-Hu Reservoir is currently under planning by the Water Resources Agency, Taiwan to meet the increasing water demands of central Taiwan arising from rapid growth of domestic water supply, and high-tech industrial parks. This study develops a simulation model for the ten-day period reservoir operation to calculate the ten-day water shortage index under varying rule curves. A genetic algorithm is coupled to the simulation model to find the optimal rule curves using the minimum ten-day water shortage index as an objective function. This study generates many sets of synthetic streamflows for risk, reliability, resiliency, and vulnerability analyses of reservoir operation. ARMA and disaggregation models are developed and applied to the synthetic streamflow generation. The optimal rule curves obtained from this study perform better in the ten-day shortage index when compared to the originally designed rule curves from a previous study. The optimal rule curves are also superior to the originally designed rule curves in terms of vulnerability. However, in terms of reliability and resiliency, the optimal rule curves are inferior to the those originally designed. Results from this study have provided in general a set of improved rule curves for operation of the Tien-Hua-Hu Reservoir. Furthermore, results from reliability, resiliency and vulnerability analyses offer much useful information for decision making in reservoir operation.
Asunto(s)
Conservación de los Recursos Naturales , Modelos Teóricos , Abastecimiento de Agua , Algoritmos , Simulación por Computador , Riesgo , TaiwánRESUMEN
AIMS: To define epidemiology, clinical disease, and outcome of gemella bacteraemia by 16S rRNA gene sequencing. To examine the usefulness of the Vitek, API, and ATB systems in identifying two gemella species. METHODS: All alpha haemolytic streptococci other than Streptococcus pneumoniae isolated from blood cultures during a six year period were identified by conventional biochemical methods, the Vitek system, and the API system. 16S rRNA gene sequencing was performed on all isolates identified by both kits as gemella with >or= 95% confidence or by either kit as any bacterial species with < 95% confidence. The ATB expression system was used to identify the two isolates that were defined as gemella species by 16S rRNA gene sequencing. RESULTS: Of the 302 alpha haemolytic streptococci other than S pneumoniae isolated, one was identified as Gemella morbillorum, and another as Gemella haemolysans by 16S rRNA gene sequencing. The patient with monomicrobial G morbillorum bacteraemia was a 66 year old man with community acquired infective endocarditis with septic thromboemboli. The patient with G haemolysans bacteraemia was a 41 year old woman with hospital acquired polymicrobial bacteraemia during the neutropenic period of an autologous bone marrow transplant for non-Hodgkin's lymphoma, the first case of its kind in the English literature. The API and ATB expression systems only identified the second strain as G haemolysans at 94% and 99% confidence, respectively, whereas the Vitek system identified none of the two strains correctly at > 70% confidence. CONCLUSIONS: Gemella bacteraemia is uncommon. 16S rRNA gene sequencing is the method of choice for identification of gemella and gemella-like isolates.
Asunto(s)
Bacteriemia/diagnóstico , Infección Hospitalaria/diagnóstico , ARN Ribosómico 16S/análisis , Staphylococcaceae/genética , Infecciones Estafilocócicas/diagnóstico , Adulto , Anciano , Trasplante de Médula Ósea , Endocarditis Bacteriana/microbiología , Femenino , Humanos , Huésped Inmunocomprometido , Masculino , Reacción en Cadena de la Polimerasa/métodos , Sensibilidad y Especificidad , Alineación de SecuenciaRESUMEN
Escherichia coli RecA protein pairs homologous DNA molecules to form paranemic joints when there is an absence of a free end in the region of homologous contact. Paranemic joints are a key intermediate in homologous recombination and are important in understanding the mechanism for a search of homology. The efficiency of paranemic joint formation depended on the length of homology and the topological forms of the duplex DNA. The presence of negative superhelicity increased the pairing efficiency and reduced the minimal length of homology required for paranemic joint formation. Negative superhelicity stimulated joint formation by favoring the initial unwinding of duplex DNA that occurred during the homology search and was not essential in the maintenance of the paired structure. Regardless of length of homology, formation of paranemic joints using circular duplex DNA required the presence of more than six negative supercoils. Above six negative turns, an increasing degree of negative superhelicity resulted in a linear increase in the pairing efficiency. These results support a model of two distinct kinds of DNA unwinding occurring in paranemic joint formation: an initial unwinding caused by heterologous contacts during synapsis and a later one during pairing of the homologous molecules.
Asunto(s)
ADN Bacteriano , Rec A Recombinasas/metabolismo , Recombinación Genética , Bacteriófago phi X 174/genética , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , ADN Circular/genética , ADN Circular/metabolismo , Proteínas de Unión al ADN/metabolismo , Escherichia coli/genética , Microvirus/genética , Homología de Secuencia de Ácido NucleicoRESUMEN
Bacteriophage lambda encodes a 28 kDa protein called beta that binds to single-stranded DNA and promotes the renaturation of complementary single strands. beta Protein fails to bind directly to duplex DNA but remains bound to the DNA product of renaturation that beta itself catalyzes. These observations led to an examination of the ability of beta protein to promote strand exchange. beta Protein caused the replacement of a 43-mer oligonucleotide annealed to M13 circular single-stranded DNA by a homologous 63-mer whose 20 extra nucleotide residues were complementary to the adjacent 3' region of M13 DNA. The role of beta protein in this reaction was manifested in several ways: beta protein pushed the exchange through four to eight mismatches, which blocked exchange mediated by spontaneous renaturation and branch migration; beta imposed a polarity on the strand exchange that was lacking in the spontaneous reaction; and beta remained bound to the heteroduplex product of strand exchange. These observations reveal a mechanism by which a protein can drive strand exchange in one direction without using ATP or any other exogenous source of energy.
Asunto(s)
Bacteriófago lambda/metabolismo , ADN Viral/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Virales/metabolismo , Bacteriófago M13/genética , Bacteriófago lambda/genética , Secuencia de Bases , ADN de Cadena Simple/química , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , ADN Viral/química , ADN Viral/genética , Sustancias Macromoleculares , Modelos Biológicos , Datos de Secuencia Molecular , Ácidos Nucleicos Heterodúplex/química , Ácidos Nucleicos Heterodúplex/genética , Ácidos Nucleicos Heterodúplex/metabolismo , Renaturación de Ácido Nucleico , Unión Proteica , Recombinación GenéticaRESUMEN
Phage lambda encodes two recombination proteins that are required for homologous recombination in a recA- host strain. Of these two recombination proteins, one is an exonuclease whose action on double-stranded DNA produces 3' single-stranded ends; the other, called beta protein, is a DNA binding protein that promotes the renaturation of complementary single strands. The enzymes of phage lambda provide a model for understanding a recombination pathway called "single-strand annealing". Further investigation of the binding of beta protein to DNA has revealed a new mechanism of renaturation. As reported before, beta protein binds directly to single-stranded DNA, but not to double-stranded DNA. However, in the experiments reported here, we observed that beta protein bound more strongly to a presumed intermediate in the renaturation reaction that beta itself catalyzed, and beta thereby protected all of a renatured duplex 83-mer oligonucleotide from nuclease digestion.
Asunto(s)
Bacteriófago lambda/metabolismo , ADN Viral/química , ADN Viral/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Virales/metabolismo , Bacteriófago lambda/genética , Sitios de Unión , ADN de Cadena Simple/química , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , ADN Viral/genética , Proteínas de Unión al ADN/química , Concentración de Iones de Hidrógeno , Sustancias Macromoleculares , Datos de Secuencia Molecular , Renaturación de Ácido Nucleico , Oligonucleótidos/química , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Unión Proteica , Recombinación Genética , Proteínas Virales/químicaRESUMEN
The aim of this study was to determine the efficacy and toxicity of combination paclitaxel and carboplatin chemotherapy in patients with metastatic and/or locoregionally advanced nasopharyngeal carcinoma (NPC). Patients with metastatic and/or locoregionally advanced NPC were treated with carboplatin calculated according to an AUC of 6 mg ml/min (based on Calvert formula) given as an intravenous (i.v.) bolus, followed by paclitaxel 135 mg/ml2 given as an i.v. infusion over 3 h with standard premedication. Cycles were given 3 weekly to a maximum of six. From January 1996 to November 1997, 27 patients were entered and assessable for response and toxicity. A total of 122 cycles were given and the median number of cycles given was five. The overall response rate was 59% (16/27). There were 3 (11%) complete responses, 13 (48%) partial responses, 5 (19%) static disease and 6 (22%) progressive disease. Toxicity was mainly haematological including: grade 3/4 neutropenia (39 cycles, 32%), grade 3/4 anaemia (nine cycles, 7%), grade 3/4 thrombocytopenia (eight cycles, 7%). There were three episodes of neutropenic fever (3%). Non-haematological toxicities were mild and infrequent. Paclitaxel and carboplatin combination chemotherapy is active in NPC and has tolerable toxicity. Further study with dose escalation is required to assess its optimal efficacy.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Nasofaríngeas/tratamiento farmacológico , Adulto , Anciano , Carboplatino/administración & dosificación , Carboplatino/efectos adversos , Femenino , Enfermedades Hematológicas/inducido químicamente , Humanos , Masculino , Persona de Mediana Edad , Neoplasias Nasofaríngeas/secundario , Paclitaxel/administración & dosificación , Paclitaxel/efectos adversos , Estudios Prospectivos , Análisis de Supervivencia , Resultado del TratamientoRESUMEN
The nucleoprotein filament formed on a circular single strand by Escherichia coli RecA protein in vitro can pair with homologous duplex DNA even when the latter lacks a free homologous end, but subsequent progression of the reaction through strand exchange requires an end in at least one strand of the duplex DNA. We purified from E. coli an endonuclease activity that cleaves the outgoing strand of duplex DNA at the junction of homologous and heterologous sequences in three-stranded RecA-recombination intermediates. This endonuclease activity also cleaves specifically at the junctions of duplex and single-stranded regions in synthetic double-stranded oligonucleotides whose central portion consists of unpaired heterologous sequences. These activities are consistent with a role in recombination and repair of DNA.
Asunto(s)
Endonucleasas/metabolismo , Escherichia coli/metabolismo , Rec A Recombinasas/metabolismo , Recombinación Genética , Secuencia de Bases , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Reparación del ADN , Electroforesis en Gel de Poliacrilamida , Endonucleasas/aislamiento & purificación , Escherichia coli/genética , Genotipo , Modelos Estructurales , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/metabolismo , Especificidad por SustratoRESUMEN
The Escherichia coli RecA protein promotes homologous recognition in base triplets via non-Watson-Crick bonds that differ from those formed nonenzymically from DNA consisting of runs of purines or pyrimidines. Base substitutions reveal recognition to be permissive, consistent with a search for homology that achieves speed at the cost of precision.
Asunto(s)
Recombinación Genética , Sitios de Unión , ADN/química , ADN/metabolismo , Rec A Recombinasas/metabolismo , Homología de Secuencia de Ácido NucleicoRESUMEN
RecA protein from Escherichia coli has been used to form a triple-stranded DNA structure from either single-stranded M13 DNA or a single-stranded oligonucleotide plus a duplex oligonucleotide with a hairpin loop. The secondary structure of purified deproteinized triplex was examined by probing with DNase I, P1 nuclease, potassium permanganate, and diethyl pyrocarbonate. The two strands destined to form heteroduplex DNA showed the same patterns of chemical modification and enzymatic digestion as control duplex DNA, indicating that they formed a normal duplex substructure. However, the nascent outgoing strand showed properties consistent with a novel triplex structure: most of its purine residues, especially adenines, were hyperreactive to all probes. The patterns of digestion by DNase I and P1 nuclease indicated that the nascent outgoing strand was not a freely mobile or single-stranded branch but rather was still interacting with the newly formed heteroduplex DNA. On the basis of the planar base triads proposed previously (Rao et al., 1993) and energy minimization of a third strand in the major groove of B-form DNA, we derived a model that helps to rationalize the properties revealed by chemical and enzymatic probing.
Asunto(s)
Oligodesoxirribonucleótidos/química , Rec A Recombinasas/química , Bacteriófago M13/química , Secuencia de Bases , ADN/química , ADN Viral/química , Dietil Pirocarbonato/química , Enlace de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Permanganato de Potasio/química , Ésteres del Ácido Sulfúrico/químicaRESUMEN
RecA protein formed a stable triplex from a 33 bp duplex oligonucleotide and a circular plus strand of M13 DNA when a hairpin connection at the proximal end of the homologous duplex oligonucleotide blocked displacement of the 5' end of its own plus strand. An oligonucleotide with a hairpin connection at the other end yielded five times fewer joints that survived deproteinization, and an ordinary duplex oligonucleotide yielded none. The stability of the three-stranded structure was not attributable to exonucleolytic nibbling of the 3' end of the hairpin oligonucleotide, which could generate a region of stable duplex DNA. In the triplexes, the hairpin duplex became more accessible to copper phenanthroline, exhibited novel sites of cleavage by DNase I, and resisted digestion by Escherichia coli exonuclease I. The enzymatic methylation of only two residues at N-6 adenine and two at N-4 cytosine in the hairpin duplex prior to the pairing reaction lowered the tm of triplexes by 8 deg.C, whereas extensive methylation at N-7 guanine by dimethyl sulfate had no effect. These results are discussed in relation to possible models of triplex DNA.
Asunto(s)
ADN de Cadena Simple/metabolismo , Oligodesoxirribonucleótidos/metabolismo , Rec A Recombinasas/metabolismo , Adenina/metabolismo , Secuencia de Bases , Citosina/metabolismo , ADN Bacteriano/química , ADN Bacteriano/metabolismo , ADN de Cadena Simple/química , Escherichia coli/genética , Exonucleasas/metabolismo , Guanina/metabolismo , Enlace de Hidrógeno , Cinética , Metilación , Datos de Secuencia Molecular , Estructura Molecular , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/química , Fenantrolinas , Rec A Recombinasas/química , Especificidad por Sustrato , TemperaturaRESUMEN
In the pairing reaction between circular gapped and fully duplex DNA, RecA protein first polymerizes on the gapped DNA to form a nucleoprotein filament. Conditions that removed the formation of secondary structure in the gapped DNA, such as addition of Escherichia coli single-stranded DNA binding protein or preincubation in 1 mM-MgCl2, optimized the binding of RecA protein and increased the formation of joint molecules. The gapped duplex formed stable joints with fully duplex DNA that had a 5' or 3' terminus complementary to the single-stranded region of the gapped molecule. However, the joints formed had distinct properties and structures depending on whether the complementary terminus was at the 5' or 3' end. Pairing between gapped DNA and fully duplex linear DNA with a 3' complementary terminus resulted in strand displacement, symmetric strand exchange and formation of complete strand exchange products. By contrast, pairing between gapped and fully duplex DNA with a 5' complementary terminus produced a joint that was restricted to the gapped region; there was no strand displacement or symmetric strand exchange. The joint formed in the latter reaction was likely a three-stranded intermediate rather than a heteroduplex with the classical Watson-Crick structure. We conclude that, as in the three-strand reaction, the process of strand exchange in the four-strand reaction is polar and progresses in a 5' to 3' direction with respect to the initiating strand. The present study provides further evidence that in both three-strand and four-strand systems the pairing and strand exchange reactions share a common mechanism.
Asunto(s)
Rec A Recombinasas/metabolismo , Recombinación Genética , Adenosina Trifosfatasas/metabolismo , Proteínas Bacterianas/metabolismo , ADN de Cadena Simple/química , Proteínas de Unión al ADN/metabolismo , Escherichia coli , Técnicas In Vitro , Magnesio/metabolismo , Conformación de Ácido Nucleico , Relación Estructura-ActividadRESUMEN
RecA protein promotes homologous pairing and symmetrical strand exchange between partially single-stranded duplex DNA and fully duplex molecules. We constructed circular gapped DNA with a defined gap length and studied the pairing reaction between the gapped substrate and fully duplex DNA. RecA protein polymerizes onto the single-stranded and duplex regions of the gapped DNA to form a nucleoprotein filament. The formation of such filaments requires a stoichiometric amount of RecA protein. Both the rate and yield of joint molecule formation were reduced when the pairing reaction was carried out in the presence of a sub-saturating amount of RecA protein. The amount of RecA protein required for optimal pairing corresponds to the binding site size of RecA protein at saturation on duplex DNA. The result suggests that in the 4-stranded system the single-stranded as well as the duplex regions are involved in pairing. By using fully duplex DNA that shares different lengths and regions of homology with the gapped molecule, we directly showed that the duplex region of the gapped DNA increased both the rate and yield of joint molecule formation. The present study indicates that even though strand exchange in the 4-stranded system must require the presence of a single-stranded region, the pairing that occurs in duplex regions between DNA molecules is functionally significant and contributes to the overall activity of the gapped DNA.