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

The radA gene is an archaeal homolog of bacterial recA and eukaryotic RAD51 genes, which are critical components in homologous recombination and recom binational DNA repair. We cloned the radA gene from a hyperthermophilic arc haeon, Pyrobaculum islandicum, overproduced the radA gene product in Escher ichia 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 kca l.mol(-1) (76.5 kJ.mol(-1)) at 25 degrees C and neutral pH. P. islandicum R adA 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 an d to catalyze DNA strand exchange. At 75 degrees C, all DNAs tested stimula ted ATPase activity of the RadA. The protein exhibited a break in the Arrhe nius plot of ATP hydrolysis at 75 degrees C. The cooperativity of ATP hydro lysis and ssDNA-binding ability of the protein above 75 degrees C were high er than at lower temperatures, and the activation energy of ATP hydrolysis was lower above this break point temperature. These results suggest that th e ssDNA-dependent ATPase activity of P. islandicum RadA displays a temperat ure-dependent capacity to exist in two different catalytic modes, with 75 d egrees C being the critical threshold temperature.