INTRODUCTION

Top Abstract Introduction Materials and Methods Results and Discussion References

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 Mg²⁺, forming a helical nucleoprotein filament. This presynaptic recombination complex interacts with double-stranded DNA (dsDNA), aligns homologous sequences between the ss- and 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 Mg²⁺- 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, ATPS, or ADP-A1F₄ (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 replaced from the reaction (26).

The recA gene of Escherichia coli is a prototypic member of a large family of genes found ubiquitously (18). These recA-like genes have been described in two domains of life other than Bacteria: Eucarya (RAD51 [16]) and Archaea (radA genes [20, 21]). Evolutionary comparisons of the deduced sequences of known RecA-like proteins show that the archaeal proteins are structurally more closely related to their eukaryal analogues (5).

Recently, the archaeal RadA protein from Sulfolobus solfataricus (RadA_Ss) has been characterized (22). Its ssDNA-dependent ATPase has been found to be weak (catalytic rate constant [k_cat] of 0.1 to 0.2 min¹ at a temperature range from 65 to 85°C) and more comparable to that observed for the Saccharomyces cerevisiae Rad51 (Rad51_Sc) protein (k_cat of 0.7 min¹ [25]) or human Rad51 (Rad51_Hs) protein (k_cat of 0.16 min¹ [1]) than for the E. coli RecA (RecA_Ec) protein (k_cat of 30 min¹ [7]). The strand exchange reaction measured at 65°C for 90 min has demonstrated the ability of the RadA protein alone to convert 13% X174 dsDNA into final products.

Desulfurococcus amylolyticus (strain Z533) is a hyperthermophilic and obligatory anaerobic archaeon that has been isolated from hot volcanic vents of Kamchatka and Kunashir (4). Like most members of the Crenarchaeota group (9), D. amylolyticus shares an extraordinary resistance to high temperatures. The strain grows at a pH range from 5.7 to 7.5, with an optimum pH of 6.4, at temperatures between 68 and 97°C, with an optimum temperature of 90 to 92°C.

Here, we describe the cloning and sequencing of the recA/RAD51-like gene (named here radA_Da) from the archaeon D. amylolyticus, the purification of RadA (named here RadA_Da) protein, and the characterization of its recombination activities. As is shown, the temperature optimum for the two activities analyzed is similar to that described for the host cells.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results and Discussion References

Materials. Restriction endonucleases were obtained from MBI-Fermentas, ATP and ATPS were obtained from Sigma, [-³²P]ATP was obtained from ICN, isopropyl--D-()-thiogalactopyranoside (IPTG) was obtained from Wako Pure Chemical, DEAE-cellulose (DE-52) and phosphocellulose (P11) were obtained from Whatman Biochemicals, and proteinase K was obtained from Fisher Biotech. Both supercoiled circular dsDNA (replication form [RFI]) and the circular ssDNA of bacteriophage M13mp18 were obtained from Sigma. Bacteriophage X174 DNA (RFI and circular ssDNA) was obtained from New England Biolabs. Linear duplex (RFII) DNA was prepared from RFI DNA by digestion with PstI restriction endonuclease.

radA_Da cloning procedure. Two degenerative oligonucleotide PCR primers, 5'-TACATCGACAC(GT)GA(AG)GGIAC-3' and 5'-CT(GT)GCCAT(GT)ACTTGGTTIGT-3', were used in a PCR with highly purified DNA of the Z533 strain of D. amylolyticus, which resulted in synthesis of a 345-bp DNA fragment. Sequencing of this fragment showed that it was a suitable probe in Southern hybridization of the DNA Z533 fragments digested by different restriction endonucleases to detect those which carried the radA_Da gene. By Southern hybridization, a 2.8-kb XhoI fragment containing the radA_Da gene was selected and cloned into the pUC119 vector, and both strands of the insert were sequenced by a standard dideoxy procedure (19).

Protein overproduction and purification. The RadA_Da protein was purified from E. coli BL21(DE3) [F ompT hsdS_B (r_B m_B) dcm gal recA306] carrying the plasmids pLysS and pET21b-radA_Da in the pET expression system developed by Novagen. The pET21b-radA_Da plasmid was constructed from the pET21b vector by replacement of a NdeI-BamHI fragment with an entire radA_Da gene, flanked by NdeI and BglII sites. The inserted radA_Da gene was created by PCR with the primers 5'-GACATATGAGTGAGGAGAAG-3' and 5'-ACAGATCTCACCTACTCTTA-3'. The validity of the sequence was confirmed by a standard dideoxy sequencing procedure.

ssDNA-dependent ATP hydrolysis. The amount of ATP hydrolyzed was determined by thin-layer chromatography with polyethyleneimine cellulose sheets as described earlier (27). Reactions were carried out in the TMD buffer (pH 7.6) (25 mM Tris-HCl, 10 mM MgCl₂, 0.1 mM DTT) containing 0.5 mM [-³²P]ATP, 8 µM RadA_Da, and M13 ssDNA as indicated. The mixture was covered with mineral oil, incubated for a period of 5 to 15 min at the temperature designated, and arrested by stirring on ice.

DNA strand exchange. For DNA strand exchange, the agarose gel assay (8) was used. This assay was conducted and results were visualized as described previously (15). The standard reaction was carried out in TAcMD buffer (pH 7.9) (20 mM Tris-acetate, 10 mM Mg-acetate, 50 mM K-acetate, 0.1 mM DTT) containing 2.5 mM ATP, 8 µM M13mp18 ssDNA, and 8 µM RadA_Da. The mixture was covered with mineral oil and preincubated for 5 min, and then the reaction was initiated by addition of 16 µM M13mp18 dsDNA linearized by PstI digestion. Agarose gels stained with ethidium bromide were documented with the EDAS 120 system (Amersham Pharmacia Biotech). The efficiency of each strand transfer was calculated by making use of the program 1D Software (Kodak Digital Science) via the amount of dsDNA converted to the final product.

CD measurements. The circular dichroism (CD) spectra of 1 µM RadA_Da protein were measured with a Jasco spectropolarimeter, model J-720W, by using a 0.1-cm cuvette in the far-UV region between 200 and 250 nm in a solution containing 20 mM Tris-HCl and 200 mM NaCl. The residue molar ellipticity, [], was defined as 100 []_obs/(lc), where []_obs is the observed molar ellipticity, l is the length of the light path in centimeters, and c is the residue molar concentration of the protein.

Nucleotide sequence accession number. The sequence data have been submitted to the DDBJ, EMBL, and GenBank databases under accession no. AF145465.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

Cloning and sequencing of radA_Da. The recA/RAD51-like gene of D. amylolyticus was cloned as follows. Two degenerative oligonucleotide PCR primers, complementary to the regions corresponding, respectively, to the 2 and 5 strands of the E. coli RecA three-dimensional structure (24), were used in the PCR with highly purified DNA of D. amylolyticus that resulted in synthesis of a 345-bp DNA fragment. The latter was used as a probe in Southern hybridization of the D. amylolyticus DNA fragmented by different restriction endonucleases. Thus, a 2.8-kb XhoI fragment containing the complete radA_Da gene was selected and cloned into the pUC119 vector, and both strands of the insert were sequenced.

View larger version (38K): [in this window] [in a new window]   FIG. 1.   Amino acid sequence of two archaeal RadA proteins (D. amylolyticus RadA [DaRadA] and S. solfataricus RadA [SsRadA]), human Rad51 (Rad51 [HsRad51]), and bacterial RecA (E. coli RecA [EcRecA]). A dash indicates a gap introduced in a sequence to optimize the alignment with the other sequences compared. A period represents a residue identical to that from RadADa. ins1, -2, and -3 and asterisks mark, respectively, the positions of three taxonomic significant insertions relative to RecAEc and nine phylogenetic significant residues described earlier (20). The accession numbers of RadADa, RadASs, Rad51Hs, and RecAEc, respectively, are as follows: AF145465, U45310, D13804, and V00328.

Expression of the radA_Da gene in E. coli and purification of RadA_Da protein. In order to overproduce the RadA_Da protein, an entire radA_Da gene was subcloned into the pET21b expression vector (for details, see Materials and Methods). The degree of RadA_Da protein homogeneity was estimated as more than 98% (Fig. 2). The protein preparations were free from detectable exonucleases, as determined at both 37 and 80°C. The N-terminal amino acid sequence of the RadA_Da protein was determined as follows: SEEKETIK. This sequence was in accordance with that predicted from the nucleotide sequence.

View larger version (18K): [in this window] [in a new window]   FIG. 2.   Purity of RadADa. A 10-µg portion of purified RadADa was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis (12% polyacrylamide gel), and the gel was stained with Coomassie blue. The molecular mass markers, labeled on the left, are in kilodaltons.

ssDNA-dependent ATPase activity. The RadA_Da ATP hydrolysis activity was tested at a wide range of temperatures. The enzyme required two cofactorsmagnesium (data not shown) and ssDNAand was active exclusively at temperatures above 65°C (Fig. 3a). The temperature restriction is in good accordance with that observed for the activity of ATP-dependent topoisomerase purified from D. amylolyticus earlier (23). The RadA_Da ATP hydrolysis showed monomer k_cat values of 1.2 to 2.5 min¹ at the temperature interval 80 to 95°C, which is more closely related to the ATP hydrolysis catalyzed by the S. cerevisiae Rad51 protein at 37°C than that catalyzed by E. coli RecA. The analysis was stopped at 95°C because of a significant decrease of an accuracy of measurements performed at more higher temperatures. Relative to RadA_Sc, showing a significant ATPase activity at 65°C and only 50% more at 75°C (22), the RadA_Da protein had negligible ATPase at 65°C and demonstrated a practically linear increase of this activity up to 95°C.

View larger version (21K): [in this window] [in a new window]   FIG. 3.   ssDNA-dependent ATP hydrolysis. (a) Temperature dependence. (b) ssDNA concentration dependence. The error bars indicate the standard deviations for three sets of experiments. The reaction mixture (20 µl) contained TMD buffer (pH 7.6), 0.5 mM [-32P]ATP, 50 µM M13 ssDNA (ssM13) (unless indicated otherwise in the figure), and 8 µM RadADa. The mixture was incubated for period of 5 to 15 min at the temperatures indicated. , ssDNA-dependent ATP hydrolysis after subtraction of both the spontaneous hydrolysis of ATP () and the ssDNA-independent ATP hydrolysis ().

DNA strand exchange activity. Figure 4 shows characteristics of the strand exchange reaction (schematically presented in Fig. 4a) promoted by the RadA_Da protein. As can be clearly seen, the reaction was very efficient at temperatures close to 90°C (Fig. 4b). At 85°C, the reaction progressed so rapidly that 90% of the dsDNA was already converted to final products after 30 min (Fig. 4c). However, a certain regression of the reaction was also observed, because after an additional 30 min, the amount of final products decreased slightly, whereas the amount of dsDNA increased. The explanation of the latter effect seems to be obvious. In the experiments described, the RadA_Da protein catalyzed the reaction in the absence of a thermoresistant SSB protein or its archaeal analogue, RPA protein (6). As mentioned above, the SSB protein is an essential component of the reaction at both early and late phases to remove, respectively, ssDNA secondary structures and the replaced linear ssDNA. Because the activity of RadA_Da showed a temperature optimum near to the melting temperature of duplex DNA, where ssDNA lacks secondary structures, it appears that the SSB protein is not required for the early phase, but it is still necessary for the late phase in order to arrest the observed regression.

View larger version (30K): [in this window] [in a new window]   FIG. 4.   DNA strand exchange reaction. A scheme of the reaction (a), the temperature (b) and kinetic (c) characteristics, and control reactions (d) are shown. In panels b and c, the reaction mixture (20 µl) contained TAcMD buffer (pH 7.9), 2.5 mM ATP, 8 µM RadADa, 8 µM M13mp18 ssDNA, and 16 µM linearized M13mp18 dsDNA. The latter was added to initiate the reaction after 5 min of preincubation at the temperature indicated. In panel d (homologous controls, lanes 1 to 5), the reaction mixture (20 µl) was the same as described above, but either without RadADa (lane 1) or with RadADa degraded by proteinase K (PrK) (lane 2); a complete mixture (lane 3); the same mixture as in lane 3, but without ATP (lane 4); or the same mixture as in lane 3, but without Mg+2 (lane 5). In panel d (heterologous controls, lanes 6 to 8), the reaction mixture (20 µl) contained TAcMD buffer (pH 7.9), 2.5 mM ATP, 8 µM RadADa, 8 µM M13mp18 ssDNA, and 16 µM linearized X174 dsDNA (lane 6) or 8 µM X174 ssDNA and 16 µM linearized M13mp18 dsDNA (lane 7), or 8 µM X174 ssDNA and 16 µM linearized X174 dsDNA (lane 8, showing the strand exchange reaction with X174 DNA substrates).

View larger version (29K): [in this window] [in a new window]   FIG. 5.   Far-UV CD spectra of RadADa protein. Measurements were performed in solution containing 20 mM Tris-HCl (pH 7.5), 200 mM NaCl, and 5 µM RadADa at the temperatures indicated.