RESUMEN
A DNA polymerase was purified to near homogeneity from Trypanosoma cruzi epimastigotes. This preparation had a major polypeptide of 50 kDa and a minor band of 45 kDa. SDS-PAGE studies and a novel colorimetric activity gel technique demonstrated that the 50-kDa polypeptide chain is the catalytic subunit of this T. cruzi DNA polymerase. Western blot analysis of different purification stage fractions strongly suggests that this 50-kDa protein is the intact catalytic subunit and does not correspond to a degradation product from a larger one. This T. cruzi DNA polymerase is insensitive to aphidicolin, butylphenyldeoxyguanosine triphosphate, berenil, ethidium bromide and N-ethylmaleimide, but is markedly inhibited by the dideoxythymidine triphosphate analogue. Studies with different DNA templates showed that the DNA polymerase prefers activated DNA as substrate and that it cannot elongate oligoriboadenylate primers. The data presented in this paper are consistent with the hypothesis that this enzyme corresponds to a beta-like DNA polymerase present in the parasitic protozoon T. cruzi.
Asunto(s)
ADN Polimerasa I/aislamiento & purificación , Trypanosoma cruzi/enzimología , Animales , Western Blotting , ADN Polimerasa I/antagonistas & inhibidores , ADN Polimerasa I/metabolismo , Cartilla de ADN , Didesoxinucleótidos , Peso Molecular , Polidesoxirribonucleótidos , Conformación Proteica , Especificidad por Sustrato , Nucleótidos de Timina/farmacologíaRESUMEN
Two previously isolated DNA polymerases from the parasitic protozoan Leishmania mexicana were further characterized by exposure to inhibitors of mammalian DNA polymerases. DNA polymerase A, a high molecular mass enzyme, and DNA polymerase B, a beta-like DNA polymerase were compared to each other and to their mammalian counterparts regarding pH optimum, utilization of templates, and response to various inhibitors and ionic strengths. The results suggest that the DNA polymerases from L. mexicana differ from the host enzymes and may offer a target for chemotherapeutic intervention.
Asunto(s)
ADN Polimerasa III/metabolismo , ADN Polimerasa I/metabolismo , Leishmania mexicana/enzimología , Animales , ADN Polimerasa I/antagonistas & inhibidores , ADN Polimerasa I/aislamiento & purificación , ADN Polimerasa III/antagonistas & inhibidores , ADN Polimerasa III/aislamiento & purificación , Concentración de Iones de Hidrógeno , Peso Molecular , Cloruro de Potasio/farmacología , Cloruro de Sodio/farmacología , Moldes GenéticosRESUMEN
Deoxyribonucleic acid polymerase beta (EC 2.7.7.7) from the lower eukaryotic parasitic protozoan Leishmania mexicana has been partially purified over 9,000 fold and characterized for the very first time. Like mammalian DNA polymerase beta the protozoan enzyme is of low molecular weight (40,000), has a broad pH range, and is resistant to inhibition by N-ethylmaleimide and aphidicolin. It is unlike mammalian DNA polymerase beta in utilization of various templates and response to various inhibitors and sensitivity to high ionic strength, but similar to a beta-like enzyme from a related organism Crithidia fasciculata. It is estimated that this enzyme constitutes 20% of the polymerase activity of the crude cell extract.
Asunto(s)
ADN Polimerasa I/aislamiento & purificación , Leishmania mexicana/enzimología , Animales , Centrifugación por Gradiente de Densidad , Cromatografía en Gel , Reacciones Cruzadas/inmunología , ADN Polimerasa I/efectos de los fármacos , Mamíferos , Peso MolecularRESUMEN
El fago Lambda polA (Qam 73, Sam 7) fue aislado y purificado de un lisógeno de la E. coli 594, y posteriormente se multiplicó en una cepa SupE, SupF. Se obtuvo una cantidad considerable de DNA polimerasa I multiplicándolo por vía ciclo lítico, tras la infección a alto MOI (Multiplicity of Infection) de una cepa libre de supresores amber (E. coli WK6). En este trabajo se muestran los resultados de la estandarización de las condiciones para obtener un nivel adecuado de expresión del gen polA, el esquema de purificación utilizado y se discuten las modificaciones realizadas para la obtención de la DNA polimerasa I a gran escala. La DNA pol I purificada incorporó P32-dATP por Nick translation en fragmentos de DNA entre 800-50 000 bp con un alto marcaje específico