RESUMO
This paper describes for the first time the isolation and characterization of a high-molecular-weight predominant DNA polymerase from the genus Leishmania, which are parasitic flagellated protozoa. Like mammalian DNA polymerase alpha, the leishmanial DNA polymerase, designated DNA polymerase A, is of high-molecular-weight, is sensitive to N-ethylmaleimide and is inhibited by high ionic strength. Unlike mammalian DNA polymerase alpha, but similar to the predominant DNA polymerase isolated from the related lower eukaryotic organisms, Trypanosoma cruzi and Crithidia fasciculata, the leishmanial DNA polymerase A is resistant to inhibition by aphidicolin, a potent inhibitor of DNA replication in mammalian cells and of DNA polymerase alpha. The DNA polymerase A was purified 28,000-fold and properties such as pH optimum, salt sensitivity, template requirements and response to DNA polymerase inhibitors were determined. A low-molecular-weight DNA polymerase was detected during the isolation procedures, but was separated from the polymerase A activity. Differences in responses to specific antisera and specific mammalian DNA polymerase alpha inhibitors suggest that the leishmanial high-molecular-weight A enzyme is sufficiently different to suggest this enzyme as a chemotherapeutic target.
Assuntos
DNA Polimerase Dirigida por DNA/isolamento & purificação , Leishmania mexicana/enzimologia , Proteínas de Protozoários/isolamento & purificação , Animais , Afidicolina/farmacologia , DNA Polimerase Dirigida por DNA/química , Estabilidade Enzimática , Temperatura Alta , Humanos , Focalização Isoelétrica , Leishmania mexicana/genética , Peso Molecular , Inibidores da Síntese de Ácido Nucleico , Concentração Osmolar , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/química , Moldes GenéticosRESUMO
1. The replicase gene of foot-and-mouth disease virus (FMDV) was expressed in Escherichia coli under the control of a tac promoter. The recombinant enzyme was purified by inclusion body precipitation, elution, and poly(U) Sepharose chromatography. 2. The enzyme exhibits poly(A)-dependent oligo(U)-primed poly(U) polymerase activity. The specific activity of the purified replicase is 1.3 x 10(5). The recombinant replicase synthesizes RNA using FMDV RNA as template, as well as heterologous RNAs, such as globin RNA and synthetic RNAs, polyadenylated or not. In all polymerization reactions, RNA products twice the size of the template are formed, both in the presence and absence of an oligo(U) primer. The enzyme is also capable of incorporating [alpha 32P]UTP in all RNAs tested except the viral template. This activity does not seem to be related to the primer independent polymerization activity. 3. The products from polymerization reactions were characterized by hybridization. In the absence of primer they consist of the template and a complementary strand covalently attached, while in the presence of primer they consist of two complementary strands synthesized de novo. 4. We propose mechanisms of RNA synthesis by the recombinant FMDV replicase in the absence and presence of primer. These mechanisms are discussed in terms of models for in vitro RNA synthesis of other picornaviruses.
Assuntos
Aphthovirus/genética , Replicação do DNA , DNA Viral/genética , DNA Polimerase Dirigida por DNA/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , Regulação Enzimológica da Expressão Gênica/genética , Regulação Viral da Expressão Gênica/genética , Replicação Viral , Aphthovirus/enzimologia , Aphthovirus/fisiologia , DNA Viral/biossíntese , DNA Polimerase Dirigida por DNA/análise , DNA Polimerase Dirigida por DNA/isolamento & purificação , DNA Polimerase Dirigida por DNA/metabolismo , Escherichia coli/enzimologia , Regulação Bacteriana da Expressão Gênica/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Regulação Viral da Expressão Gênica/fisiologia , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , RNA Viral/genética , Proteínas Recombinantes/análise , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Moldes GenéticosRESUMO
A thermostable DNA polymerase, isolated from the thermophilic strain Thermus thermophilus HB 8 was purified by a five-step procedure which provides a high yield and a homogeneous preparation. The molecular weight was estimated to be 67,000 daltons and the extension rate was determined to be 1500 nucleotides per minute. The enzyme works in polymerase chain reaction conditions similar to those used for Taq polymerase from Thermus aquaticus.