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The RadA protein from a hyperthermophilic archaeon Pyrobaculum islandicum is a DNA-dependent ATPase that exhibits two disparate catalytic modes, with a transition temperature at 75 degrees C
Journal article   Open access

The RadA protein from a hyperthermophilic archaeon Pyrobaculum islandicum is a DNA-dependent ATPase that exhibits two disparate catalytic modes, with a transition temperature at 75 degrees C

M Spies, Y Kil, R Masui, R Kato, C Kujo, T Ohshima, S Kuramitsu and V Lanzov
European journal of biochemistry, Vol.267(4), pp.1125-1137
02/2000
DOI: 10.1046/j.1432-1327.2000.01108.x
PMID: 10672022
url
https://doi.org/10.1046/j.1432-1327.2000.01108.xView
Published (Version of record) Open Access

Abstract

The radA gene is an archaeal homolog of bacterial recA and eukaryotic RAD51 genes, which are critical components in homologous recombination and recombinational DNA repair. We cloned the radA gene from a hyperthermophilic archaeon, Pyrobaculum islandicum, overproduced the radA gene product in Escherichia coli and purified it to homogeneity. The purified P. islandicum RadA protein maintained its secondary structure and activities in vitro at high temperatures, up to 87 degrees C. It also showed high stability of 18.3 kcal.mol-1 (76.5 kJ.mol-1) at 25 degrees C and neutral pH. P. islandicum RadA exhibited activities typical of the family of RecA-like proteins, such as the ability to bind ssDNA, to hydrolyze ATP in a DNA-dependent manner and to catalyze DNA strand exchange. At 75 degrees C, all DNAs tested stimulated ATPase activity of the RadA. The protein exhibited a break in the Arrhenius plot of ATP hydrolysis at 75 degrees C. The cooperativity of ATP hydrolysis and ssDNA-binding ability of the protein above 75 degrees C were higher than at lower temperatures, and the activation energy of ATP hydrolysis was lower above this break point temperature. These results suggest that the ssDNA-dependent ATPase activity of P. islandicum RadA displays a temperature-dependent capacity to exist in two different catalytic modes, with 75 degrees C being the critical threshold temperature.
 Adenosine Triphosphatases - isolation & purification Archaeal Proteins - isolation & purification Temperature Archaeal Proteins - chemistry Molecular Sequence Data Thermoproteaceae - genetics Cations, Divalent - pharmacology DNA, Single-Stranded - genetics DNA-Binding Proteins - metabolism Recombinant Proteins - isolation & purification Thermodynamics Adenosine Triphosphate - metabolism Cloning, Molecular Archaeal Proteins - genetics Rec A Recombinases - genetics Archaeal Proteins - metabolism Recombinant Proteins - metabolism Amino Acid Sequence Rec A Recombinases - metabolism Protein Structure, Secondary DNA, Single-Stranded - metabolism Enzyme Stability Adenosine Triphosphatases - metabolism Recombinant Proteins - chemistry Recombinant Proteins - genetics DNA - metabolism Thermoproteaceae - enzymology DNA-Binding Proteins - genetics DNA-Binding Proteins - isolation & purification DNA-Binding Proteins - chemistry DNA, Single-Stranded - chemistry DNA - genetics Catalysis - drug effects DNA - chemistry Sequence Alignment Escherichia coli - genetics Adenosine Triphosphatases - chemistry Rec A Recombinases - chemistry Adenosine Triphosphatases - genetics Enzyme Activation Kinetics

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