Efficient Strand Transfer by the RadA Recombinase from the Hyperthermophilic Archaeon Desulfurococcus amylolyticus

Abstract

The radA gene predicted to be responsible for homologous recombination in a hyperthermophilic archaeon, Desulfurococcus amylolyticus, was cloned, sequenced, and overexpressed in Escherichia coli cells. The deduced amino acid sequence of the gene product, RadA, was more similar to the human Rad51 protein (65% homology) than to the E. coli RecA protein (35%). A highly purified RadA protein was shown to exclusively catalyze single-stranded DNA-dependent ATP hydrolysis, which monitored presynaptic recombinational complex formation, at temperatures above 65°C (catalytic rate constant of 1.2 to 2.5 min ؊1 at 80 to 95°C). The RadA protein alone efficiently promoted the strand exchange reaction at the range of temperatures from 80 to 90°C, i.e., at temperatures approaching the melting point of DNA. It is noteworthy that both ATP hydrolysis and strand exchange are very efficient at temperatures optimal for host cell growth (90 to 92°C).RecA protein is a key enzyme of homologous recombination in eubacteria and is also essential to many other aspects of DNA metabolism (for review, see references 3, 11, and 18). To make recombination possible, the RecA protein is polymerized on single-stranded DNA (ssDNA) in the presence of ATP and Mg 2ϩ , forming a helical nucleoprotein filament. This presynaptic recombination complex interacts with double-stranded DNA (dsDNA), aligns homologous sequences between the ssand dsDNA, and promotes the DNA strand exchange by switching DNA pairing within the triple-stranded DNA complex.Although ATP hydrolysis has been shown to not be required for the presynaptic complex formation (10), the latter possesses Mg 2ϩ -and ssDNA-dependent ATPase activity. This activity can easily be used to monitor the RecA protein nucleation as well as saturation of the RecA binding to ssDNA that results in formation of the complex activated for recombination. The in vivo strand exchange process can be modelled in vitro by the transfer of one strand of linear dsDNA to the complementary circular ssDNA (for example, M13 phage); this transfer results in branched DNA recombination intermediates (joint molecules) that are gradually converted into nicked circular heteroduplex dsDNA molecules (final products) and linear ssDNA. As a rule, the circular ssDNA is introduced into the strand exchange reaction in the form of the presynaptic complex. In principle, the strand exchange can proceed in the presence of nonhydrolyzable analogues of ATP, ATP␥S, or ADP-A1F 4 Ϫ (12, 14), but the hydrolysis is required to exchange molecules longer than 1,500 bp as well as to dissociate RecA from the heteroduplex products, to facilitate the bypass of heterologous sequences, and to maintain the reaction polarity (for references, see reference 7).SSB protein is another important component of the strand exchange reaction proceeding under physiological temperature (37°C). This protein affects both the binding of RecA to ssDNA by removing secondary structures from the latter and the completion of strand exchange (2) by covering the linear ssDNA rep...

Replication Protein A in Pyrococcus furiosus Is Involved in Homologous DNA Recombination

“…In the eukaryotic recombination system, RPA strikingly stimulates the Rad51-mediated strand-exchange reaction in vitro (26 -28). Furthermore, various proteins, including Rad52, Rad55, Rad57, and Rad54, stimulate the RPA-associated Rad51 reaction (29 -34).Recently, several laboratories have reported a RecA/Rad51 family protein, called RadA, in Archaea, the third domain of life (35)(36)(37)(38). The archaeal RadA protein sequence is more similar to the eukaryotic Rad51 than to the bacterial RecA and can mediate the strand-exchange in vitro.…”

“…Recently, several laboratories have reported a RecA/Rad51 family protein, called RadA, in Archaea, the third domain of life (35)(36)(37)(38). The archaeal RadA protein sequence is more similar to the eukaryotic Rad51 than to the bacterial RecA and can mediate the strand-exchange in vitro.…”

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

“…Earlier, we described a distinguishing property of RadA protein from crenarchaeon Desulfurococcus amylolyticus (RadA Da ), namely, its ability to promote efficient strand exchange even at 95°C (6). Here, we continued the analysis of biochemical activities of RadA Da and presented its additional characteristics, which include both expected and distinguishing properties of the protein with regard to known activities of the RecA and RadA recombinases.…”

“…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.…”