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 °C

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 8C. It also showed high stability of 18.3 kcal´mol 21(76.5 kJ´mol 21) at 25 8C and neutral pH. P. islandicum RadA exhibited activities typical of the family of RecAlike proteins, such as the ability to bind ssDNA, to hydrolyze ATP in a DNA-dependent manner and to catalyze DNA strand exchange. At 75 8C, all DNAs tested stimulated ATPase activity of the RadA. The protein exhibited a break in the Arrhenius plot of ATP hydrolysis at 75 8C. The cooperativity of ATP hydrolysis and ssDNA-binding ability of the protein above 75 8C 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 8C being the critical threshold temperature.Keywords: Arrhenius plot; DNA-dependent ATPase; homologous genetic recombination; Pyrobaculum islandicum; RadA.Homologous or general recombination is a fundamental mechanism and a vital process in most living organisms. The RecA-like recombinational proteins play a central role in the process of genetic recombination by bringing two interacting DNA molecules together, searching for homology and exchanging the DNA strands [1,2]. Upon binding ATP, RecA protein in bacteria and Rad51 protein in eukaryotes organize a highly structured nucleoprotein filament which makes the DNA available for homologous pairing [1,3±7]. Recently, the RecA paradigm of homologous recombination was extended to all three domains of life, Bacteria, Eukarya and Archaea [8±11]. The mechanisms of homologous recombination and DNA repair have been extensively described for prokaryotes [1±5] and eukaryotes [7,12,13], but little is known about these mechanisms in archaea. Many RecA homologs have been isolated from prokaryotes and eukaryotes, but only recently have sequences encoding proteins similar to RecA and Rad51 been found in the third phylogenetically divergent group, Archaea [8,11,14±17]. Two types of sequence encoding putative RecA-like proteins have been discovered in different archaeal species: polypeptides of 320±350 amino acids (RadA proteins) [8,11], and those of 210±230 amino acids (RadB proteins) [14]. In addition, both types of sequence have been found in archaeal strains, complete genomes of which are now available [15±17]. The reported putative proteins encoded by radA genes are more similar in sequence to Rad51 and Dmc1 proteins than to RecA proteins [8,9,11]. Within the minimal core region, in whic...

Characteristic Thermodependence of the RadA Recombinase from the Hyperthermophilic Archaeon Desulfurococcus amylolyticus

“…1). This concentration is fivefold lower than that used for RadA Pi (17). Moreover, the addition of 100 mM NaCl resulted in a 3.2-fold decrease of the monomer k cat value.…”

“…Like other members of this family, RadA polymerizes on single-stranded DNA (ssDNA) in the presence of ATP, forming a presynaptic complex (PC) which possesses the ATPase activity. PC interacts with double-stranded DNA (dsDNA) to promote homologous pairing and strand exchange, the two critical steps of recombination (4, 15).The biochemical and recombination activities of five RadA proteins from different hyperthermophilic crenarchaeons and euryarchaeons have been described (6,7,10,14,17). One of them, RadA from Pyrobaculum islandicum (RadA Pi ), has been studied in more detail than the others.…”

“…The biochemical and recombination activities of five RadA proteins from different hyperthermophilic crenarchaeons and euryarchaeons have been described (6,7,10,14,17). One of them, RadA from Pyrobaculum islandicum (RadA Pi ), has been studied in more detail than the others.…”

“…One of them, RadA from Pyrobaculum islandicum (RadA Pi ), has been studied in more detail than the others. The ATPase of its PC exhibits two disparate catalytic modes, and its PC is active within a wide temperature range (from 37 to 90°C) (17).…”

Rad51 Protein from the Thermotolerant Yeast Pichia angusta as a Typical but Thermodependent Member of the Rad51 Family

“…This recombinase is a RecARadA-like protein, found in all three domains of life (4), which forms filaments on single-stranded DNA (ssDNA) in the presence of ATP (and thus possesses ATPase activity) and promotes homologous pairing and strand exchange, which are the two main steps in the initiation of homologous recombination and recombination repair, as well (6,16,27). All members of the RecA-RadA-Rad51 recombinase superfamily form nucleoprotein filaments of similar structures and stoichiometries, and they display similar preferences in DNA substrates for DNAdependent ATP hydrolysis (26,30,32). Although similar in ATP-dependent recombination functions, these members are divided into two subfamilies: RecA-like and RadA-Rad51-like proteins.…”