RESUMO
A gene (No. AF0497 GenBank, USA) was cloned from the archaeon Archaeoglobus fulgidus strain found in the water of hot springs. This gene contains an open reading frame of 2346 base pairs which encodes a thermostable DNA-polymerase (762 amino acid residues). A recombinant protein Afu-pol with molecular weight of 94 kD was isolated in an Escherichia coli strain used as a producer and characterized. By site-directed mutagenesis in the afu-pol gene the amino acid residue Glu170 was replaced with Ala; this resulted in a complete loss of the 3;-5;-exonuclease activity of the enzyme. Thus, the Glu170 residue was suggested to be directly involved in formation of the 3;-5;-exonuclease site. Physicochemical features of the exodeficient enzyme form were studied, and the possible use of Afu(exo(-))-pol in the polymerase chain reaction is shown.
Assuntos
Archaeoglobus fulgidus/enzimologia , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Substituição de Aminoácidos , Archaeoglobus fulgidus/genética , Autorradiografia , Clonagem Molecular , Primers do DNA/genética , DNA Polimerase Dirigida por DNA/química , Estabilidade Enzimática , Escherichia coli/metabolismo , Exodesoxirribonucleases/metabolismo , Genes Arqueais , Concentração de Íons de Hidrogênio , Magnésio/química , Magnésio/farmacologia , Mutagênese Sítio-Dirigida , Reação em Cadeia da Polimerase , Cloreto de Potássio/farmacologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TemperaturaAssuntos
Archaeoglobus fulgidus/enzimologia , DNA Polimerase Dirigida por DNA/genética , Clonagem Molecular , DNA Polimerase Dirigida por DNA/metabolismo , Estabilidade Enzimática/fisiologia , Escherichia coli/genética , Escherichia coli/metabolismo , Temperatura Alta , Filogenia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Site-directed mutagenesis of the ecoRII gene has been used to search for the active site of the EcoRII restriction endonuclease. Plasmids with point mutations in ecoRII gene resulting in substitutions of amino acid residues in the Asp110-Glu112 region of the EcoRII endonuclease (Asp110 --> Lys, Asn, Thr, Val, or Ile; Pro111 --> Arg, His, Ala, or Leu; Glu112 --> Lys, Gln, or Asp) have been constructed. When expressed in E. coli, all these plasmids displayed EcoRII endonuclease activity. We also constructed a plasmid containing a mutant ecoRII gene with deletion of the sequence coding the Gln109-Pro111 region of the protein. This mutant protein had no EcoRII endonuclease activity. The data suggest that Asp110, Pro111, and Glu112 residues do not participate in the formation of the EcoRII active site. However, this region seems to be relevant for the formation of the tertiary structure of the EcoRII endonuclease.
