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J. Biol. Chem., Vol. 275, Issue 43, 33782-33790, October 27, 2000

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Both RadA and RadB Are Involved in Homologous Recombination in Pyrococcus furiosus^*

Kayoko Komori, Tomoko Miyata^§, Jocelyne DiRuggiero^¶, Rhonda Holley-Shanks^¶, Ikuko Hayashi, Isaac K. O. Cann, Kota Mayanagi^§, Hideo Shinagawa, and Yoshizumi Ishino^**

From the Departments of  Molecular Biology and ^§ Structural Biology, Biomolecular Engineering Research Institute, Suita, Osaka 565-0874, Japan, the ^¶ Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, Maryland 21202, and the  Department of Molecular Microbiology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan

Received for publication, May 26, 2000, and in revised form, June 30, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

RecA and Rad51 proteins are essential for homologous recombination in Bacteria and Eukarya, respectively. Homologous proteins, called RadA, have been described for Archaea. Here we present the characterization of two RecA/Rad51 family proteins, RadA and RadB, from Pyrococcus furiosus. The radA and radB genes were not induced by DNA damage resulting from exposure of the cells to  and UV irradiation and heat shock, suggesting that they might be constitutively expressed in this hyperthermophile. RadA had DNA-dependent ATPase, D-loop formation, and strand exchange activities. In contrast, RadB had a very weak ATPase activity that is not stimulated by DNA. This protein had a strong binding affinity for DNA, but little strand exchange activity could be detected. A direct interaction between RadA and RadB was detected by an immunoprecipitation assay. Moreover, RadB, but not RadA, coprecipitated with Hjc, a Holliday junction resolvase found in P. furiosus, in the absence of ATP. This interaction was suppressed in the presence of ATP. The Holliday junction cleavage activity of Hjc was inhibited by RadB in the absence, but not in the presence, of ATP. These results suggest that RadB has important roles in homologous recombination in Archaea and may regulate the cleavage reactions of the branch-structured DNA.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Genetic recombination is important both in generating genetic diversity and in repairing DNA damages. Homologous DNA recombination involves multistep reactions. The molecular mechanisms of the early stage include pairing and strand exchange reactions of two homologous DNA strands. The RecA/Rad51 family proteins have a central role in the initiation step by binding to single-stranded DNA (ssDNA),¹ which results in the formation of a helical nucleoprotein filament (reviewed in Refs. 1 and 2). RecA proteins of eubacteria and Rad51 proteins of eukaryotes have been extensively studied to date (reviewed in Refs. 3-5). RecA/Rad51 structural homologs have also been found in the Archaea and named RadA (6-10). The amino acid sequences of archaeal RadAs are much more similar to those of eukaryotic Rad51 homologs than to those of bacterial RecA homologs. Preliminary characterization shows that the archaeal RadAs found in Sulfolobus solfataricus, Desulfurococcus amylolyticus, and Pyrobaculum islandicum are functionally similar to the RecA/Rad51 family proteins found in the other domains (11-14), and they are now thought to play a critical role in recombination and repair in Archaea.

To understand the detailed mechanism of the DNA recombination in Archaea, we have been investigating the proteins related to this process from the hyperthermophilic archaeon, Pyrococcus furiosus (15). We identified a Rad51-like protein that is encoded in an operon that includes a novel heterodimeric DNA polymerase (polymerase II or D) and an Orc1 (origin recognition complex protein 1)-like protein (9). When we first reported this operon, we called the Rad51-like protein RadA. However, a subsequent report identified two Rad51/Dmc1 homologs in the P. furiosus genome, namely RadA and RadB (16). The protein in the operon described above was renamed RadB. Recently, we found a specific interaction between RadB and DP1, the second subunit of PolD (17). These findings suggest that there is a functional interaction between a recombination protein RadB, and a replication protein, DP1, and that RadB plays a role linking recombination directly with replication. In Escherichia coli, it has been reported that a specific interaction of RecA with DNA polymerase I Klenow fragment enhance the fidelity of DNA synthesis (18). Various lines of evidences suggest that there is a coupling between recombination and replication both in prokaryotes and eukaryotes (19-22).

Many archaeal proteins related to genetic information systems have similar sequences to those found in eukaryotes (23), indicating that research on DNA recombination in archaea would serve as a good model system for understanding the very complex molecular machinery of eukaryotic DNA recombination. In addition to P. furiosus, other archaeal genomes from Methanococcus jannaschii (24), Archaeoglobus fulgidus (25), Methanobacterium thermoautotrophicum (26), and Pyrococcus horikoshii (27) also contain open reading frames homologous to RadA and RadB, respectively. Several eukaryotic proteins with homologous sequences to Rad51, including Saccharomyces cerevisiae Rad55, Rad57, Dmc1, human XRCC2, and XRCC3, are known (reviewed in Ref. 28). Biochemical studies suggest that these proteins do not have redundant function but have distinct roles in the process of homologous recombination in the cells.

In this study, we analyzed the biochemical properties of RadA and RadB from P. furiosus, and the regulation of expression of the radA and radB genes in P. furiosus as the first step to understanding DNA recombination in Archaea. This is also the first report that characterizes two RecA/Rad51-like recombinases from the same archaeon. One remarkable finding is that RadB specifically interacts with Hjc, a Holliday junction resolvase in P. furiosus (29), and regulates its cleavage activity in an ATP-dependent manner. These analyses will contribute to the complete understanding of the molecular mechanism of homologous recombination in Archaea.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Quantification of mRNAs for RadA and RadB-- P. furiosus (DSM 3638) cultures were grown anaerobically in 100-ml serum bottles to approximately 1.0 × 10⁶ cells/ml (30) and subjected to  and UV irradiation and heat shock.  irradiation was performed with a ⁶⁰Co -ray source (at the National Institute of Standards and Technology) from 1500 and 2500 grays at a rate of 45.2 grays/min as described previously (31). Exposure to UV was at a dose of 105 J/m² in salt medium (P. furiosus medium as described earlier (30) without yeast extract and tryptone), on ice and under anaerobic condition, using a Pen-Ray far UV germicidal lamp (254 nm). Heat shock was performed by incubating the cultures at 108 °C for 1 h in an oil bath. After treatment all the cultures were incubated for 1 h at 95 °C before RNA extraction. Total cell counts were performed by acridine orange direct count, and viable cell counts were carried out by the technique of the most probable number as described previously (31). RNA was extracted from 10⁹ to 10¹⁰cells using either a protocol previously described in DiRuggiero et al. (32) or the Bio101 Fastprep RNA extraction kit (Bio101, Vista, CA). All RNAs were submitted to DNase treatment to remove any DNA contamination. Real time quantitative reverse transcriptase-PCR was performed using the ABI Prism 7700 detection system by Perkin-Elmer Biosystems (Foster City, CA), and the TaqMan and SYBR Green I dye kits from Perkin-Elmer (Branchburg, NJ) as recommended by the supplier. The gene for glutamate dehydrogenase (gdh) was used as the housekeeping gene to standardized the reactions. Validation experiments were performed with gdh, radA, and radB primers to validate the calculation. The level of expression of radA and radB mRNA were established as relative expression. Primers for the reverse transcriptase reaction and the PCR were designed using the Primer Express software (Perkin-Elmer Biosystems) and are as follow: gdh primers, 5'-GAATATACAACCCCGATGGTCTTAA-3' and 5'-GAGAACATCAACCTCAAGCTCAAGTA-3'; radA primers, 5'-GTTCGGTAGTGGAAAAACTCAGCTA-3' and 5'-AGGCCTAAATGTGTTCTCTGTGTCT-3'; and radB primers, 5'-GCTTTTAGGTGGTGGAGTTGCT-3' and 5'-CCAACCTGCATTGCAAAAGTT-3'.

Preparation of RadA and RadB Proteins-- The structural genes of radA and radB were amplified by PCR directly from P. furiosus genomic DNA. Pfu DNA polymerase (Stratagene, CA) was used to maintain the accuracy of amplification. To adjust their translational initiation codon, ATG, at the NdeI site (for radA) or NcoI (for RadB) site of an expression vector pET21a (radA) or pET21d (radB) (Novagen, WI), a NdeI (NcoI) recognition sequence was made in each forward primer. In the reverse primers, a XhoI site (radA) or a HindIII site (radB) was made just after the termination codons. The PCR products were digested with NdeI-XhoI or NcoI-BamHI and inserted into the corresponding site of pET21a. The resultant plasmids were designated pPFRADA and pPFRADB, respectively. E. coli JM109 (DE3) carrying pPFRADA was cultivated in 1 liter of LB medium containing 100 µg/ml ampicillin to A₆₀₀ of 0.4, and the expression of radA was induced by adding isopropyl-1-thio--D-galactopyranoside to a final concentration of 1 mM. After incubation for a further 7 h at 37 °C, the cells were harvested by centrifugation. The cells were disrupted by sonication in a sonication buffer (50 mM Tris-HCl, pH 8.0, 0.1 mM EDTA, 0.5 mM DTT, 1 mM phenylmethansulfonyl fluoride) containing 0.4 M NaCl, and the soluble extract was incubated at 80 °C for 20 min. The heat-resistant fraction was taken by centrifugation at 30,000 × g for 20 min and was mixed with polyethylenimine at a final concentration of 0.5%. RadA protein was eluted with 0.3 M ammonium sulfate in buffer A (50 mM Tris-HCl, pH 8.0, 0.1 mM EDTA, 0.5 mM DTT, 10% glycerol) from the precipitate after polyethylenimine treatment. The supernatant was mixed with an equal volume of 2 M ammonium sulfate and applied onto a phenyl-Sepharose column (Amersham Pharmacia Biotech). RadA protein was eluted at 1-0.5 M ammonium sulfate, and the fractions were dialyzed against buffer A containing 0.2 M NaCl. The dialysate was applied onto a heparin column (Amersham Pharmacia Biotech), and the protein was eluted at 0.5-0.7 M NaCl. The fractions were mixed with the same volume of buffer A and were applied onto a Mono Q HR 5/5 column (Amersham Pharmacia Biotech). RadA protein was eluted at 0.35-0.37 M NaCl, and the fraction was stored on ice. E. coli JM109 (DE3) carrying pPFRADB was cultivated in 6 liters of LB-ampicillin medium to A₆₀₀ of 0.4, and isopropyl-1-thio--D-galactopyranoside was added to a final concentration of 0.2 mM for induction of radB gene expression. After incubation for 16 h at 20 °C, the cells were harvested by centrifugation. The cells were suspended in sonication buffer containing 0.2 M NaCl and were disrupted by sonication, and the supernatant collected by centrifugation was incubated at 80 °C for 20 min. The supernatant was treated with 0.5% polyethylenimine, and the precipitate was suspended in buffer A containing 0.8 M of ammonium sulfate to elute RadB protein. The supernatant was mixed with ammonium sulfate at a final concentration of 1.5 M and was applied onto a phenyl-Sepharose column. This step was repeated three times to remove DNA from the RadB fraction. RadB protein eluted at 1-0.5 M ammonium sulfate was dialyzed against buffer B (buffer A containing 0.1 M NaCl) and was applied onto a heparin column. The protein fractions eluted at 0.7-1.2 M NaCl were dialyzed against buffer B and were applied onto a Mono S HR 5/5 column (Amersham Pharmacia Biotech). RadB protein eluted at 0.8-1.4 M NaCl was dialyzed against buffer B, and the fraction was stored on ice. The concentrations of RadA and RadB were determined by using a molar extinction coefficient M of 13,370 M¹·cm¹ and 15930 M¹·cm¹, which were obtained by the method of Gill and von Hippel (33). For the check of nuclease contamination in the purified proteins, deoxyoligonucleotides (both single-stranded and double-stranded) or the linearized pUC118 by PstI digestion were labeled with ³²P and incubated with RadA or RadB at 70 °C for 1 h. Reaction mixtures were subjected to electrophoresis followed by autoradiograpy.

Immunoprecipitation and Western Blot Analysis-- Rabbit polyclonal antibodies were raised against homogenous RadA as previously reported for RadB (17), Hjc (29), protein intron (PI)-PfuI, and PI-PfuII (34). All subsequent steps were carried out at room temperature. RadA, RadB, Hjc, and inteins (PI-PfuI and PI-PfuII, as negative controls) proteins (0.5 µg of each) in various combinations were mixed in 50 µl of TBS-T buffer (20 mM Tris-HCl, pH 7.5, 137 mM NaCl, 0.1% Triton X-100) and treated at 60 °C for 5 min. After cooling down at room temperature, anti-RadA, -RadB, -Hjc, -PI-PfuI, or -PI-PfuII, antiserum was added to the mixtures and incubated for 20 min. The mixtures were added to protein A-Sepharose (Amersham Pharmacia Biotech) and were further incubated for 1 h with rotary shaker. Protein A-Sepharose-bound antigen-antibody complexes were separated from free proteins by centrifugation followed by washing three times in TBS-T buffer. The precipitates were analyzed by Western blotting using an enhanced chemiluminescence system (Amersham Pharmacia Biotech) according to the supplier's protocol. To investigate the effect of ATP on the formation of RadB-Hjc complex, ATP was added to 2.5 mM in the TBS-T buffer.

ATPase Assay-- ATPase activity of RadA and RadB were measured essentially as described previously (35) using [-³²P]ATP or [-³²P]ATP (NEN Life Science Products). RadA or RadB protein (each at 2 µM), [-³²P]ATP (0.1 µ Ci/µl), and 100 µM of ATP were incubated with or without DNA (single-stranded or double-stranded M13 DNA, 0.2 µg) in 20 µl of ATPase buffer (25 mM Tris-HCl, pH 7.5, 6 mM MgCl₂, 0.1 mM DTT) at 70 °C for indicated time, and aliquots of reactions were analyzed by a thin layer chromatography. The amount of ATP and P_i ([-³²P]ATP) or ADP ([-³²P]ATP) were quantified from the autoradiograms using a laser-excited image analyzer (BAS5000, Fuji Film, Tokyo, Japan). In the other experiment, RadA or RadB protein (each at 1 µM) was incubated with 50 or 1 µM of ATP containing [-³²P]ATP in the presence of single-stranded DNA at 70 °C, and aliquots of the reaction were analyzed for ATP hydrolysis and binding activities. Protein-bound nucleotides were separated by a disposable spin column of SUPREC-02 (Takara Shuzo) and were washed with ATPase buffer twice and analyzed by liquid scintillation counting.

Gel Retardation Assay-- Various concentrations of RadA and RadB were incubated with 1 µM (as nucleotide concentration) of a ³²P-labeled single-stranded 83-mer, W16() (its nucleotide sequence was shown earlier (36), or a double-stranded 83-mer, W16()/W16(+), in exchange buffer (20 mM Tris-HCl, pH 7.5, 15 mM MgCl₂, 2 mM DTT, 50 µg/ml bovine serum albumin, 2.5 mM ATP) at 60 °C for 10 min. Then reaction products were analyzed by 1% agarose gel electrophoresis in 0.1× TAE buffer, and the bands were detected by autoradiography. The amounts of protein-DNA complex were quantified from the autoradiograms using a laser-excited image analyzer.

D-loop Formation Assay-- RadA or RadB (at 1 µM) were mixed with 1 µM (as nucleotide) of ³²P-labeled 83-mer oligonucleotide, W16(), at various timing and incubated in 19 µl of exchange buffer at 65 °C for 10 min. Then M13 double-stranded DNA (0.1 µg) was added to the mixtures and further incubated for 2 or 5 min. The reaction using E. coli RecA, which was prepared as described earlier (37), was performed by the same condition except at 37 °C. The reaction mixtures were deproteinized at 37 °C for 20 min in proteinase K (0.1 mg/ml) and SDS (0.5%) and separated by 1% agarose gel electrophoresis in 0.1× TAE buffer. The gels were dried and autoradiographed.

Strand Exchange Reaction-- RadA (7.5 µM) and RadB (3.7 or 7.4 µM) were mixed with single-stranded pUC118 (18 µM nucleotide) at various timing and incubated in exchange buffer at 70 °C for 20 min. Then, PstI-digested and 3'-³²P-labeled double-stranded pUC118 (14.5 µM nucleotide) was added to the mixtures and further incubated for 1 h. RecA (6 µM) was preincubated with single-stranded DNA for 10 min at 37 °C, and then E. coli single-stranded DNA binding protein (Stratagene, CA) was added in the mixture at a concentration of 1 µM. After 10 min of incubation, the strand exchange reaction was initiated by adding 3'-³²P-labeled double-stranded DNA, and the mixture was incubated for 7 min at 37 °C. The reaction mixtures were deproteinized by proteinase K and separated by 1.2% agarose gel electrophoresis in TAE buffer. The bands were detected by autoradiography.

Electron Microscopy-- RadA protein (5.6 µM) was incubated with 21 µM (nucleotides) single-stranded pUC118 in a buffer (20 mM Tris-HCl, pH 7.5, 15 mM MgCl₂, 2 mM DTT) containing 1 mM ATPS at 90 °C for 15 min. RadB protein (7.8 µM) was incubated in the same buffer with or without 1 mM ATPS at 60 °C for 10 min. The reaction mixtures were applied to glow-discharged grids and stained with 2% (w/v) uranyl acetate. Images were recorded at nominally 48,000 in a JEOL 100cx electron microscope at an acceleration voltage of 100 kV. The magnification was calibrated from 23 Å pitch helix of tabacco mosaic virus. Electron micrographs were digitized in a CCD scanner (DSS-1010: DS Scanner Co.) using a step size of 7.2 × 7.2 µm. Contour length of RadA single-stranded pUC118 complexes were measured using SPIDER and Web.

Holliday Junction Cleavage Assay-- Hjc protein (5 nM) was premixed with RadA (3 µM) or RadB (3 µM) proteins and then incubated with 14 µM (as nucleotide concentration) of ³²P-labeled four-way junction, 4Jh in the exchange buffer (29) with or without 2.5 mM of ATP at 60 °C for 1 h. The reaction mixtures were deproteinized by proteinase K and phenol treatment and separated by 6% polyacrylamide gel electrophoresis in 1× TAE buffer. The bands were detected by autoradiography. The amounts of substrate and product were quantified from the autoradiograms using a laser-excited image analyzer.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Structure Comparison of RecA/Rad51 Family Proteins-- Amino acid sequence comparisons showed that RadA and RadB have 54 and 29% similarity, respectively, to the yeast Rad51. In contrast, they exhibited only 29 and 18% similarity to E. coli RecA. The sequence identity between the two Rad proteins is 21% (33% similarity) when they are compared using their entire regions. Fig. 1A shows a diagram of representative RecA/Rad51 family proteins. RadA is about 100 amino acids longer than RadB and has an N-terminal domain (Domain I) that shares conserved sequence with the N-terminal domains of eukaryotic Rad51 proteins. RadB consists solely of the central core domain (Domain II) of this protein family. The amino acid sequence identity of the Domain II in RadA and RadB is about 28% (45% similarity). Both RadA and RadB present the 9-signature amino acids characteristic of archaeal recombinases as described by Sandler et al. (9). In addition, RadA has two insertions and one deletion relative to the E. coli RecA sequence as reported for other RadAs from Euryarchaeotes.

View larger version (39K): [in this window] [in a new window]   Fig. 1.   RadA and RadB proteins from P. furiosus. A, domain structures of RecA/Rad51 family proteins. The black and shaded boxes indicate the Walker A and B motifs, respectively. RadA and RadB, P. furiosus; Rad51 and Dmc1, S. cerevisiae; RecA, E. coli. B, purification of RadA and RadB proteins. RadA and RadB proteins were produced in E. coli cells and were purified by heat treatment, polymin precipitation, and three column chromatographies. 1 µg of protein was analyzed by 15% SDS-PAGE followed by Coomassie Brilliant Blue staining. Protein bands correspond to each size (RadA, 38.4 kDa; RadB, 25.3 kDa). C, detection of RadA and RadB in P. furiosus cells by Western blot analysis. P. furiosus cell extract (Pfu) and recombinant protein (R) were separated by 15% SDS-PAGE, transferred onto polyvinylidene difluoride membranes, and treated with anti-RadA or RadB serum. The arrowhead indicates the RadB band.

Purification of RadA and RadB Proteins-- The structural genes for RadA and RadB (radA and radB) were amplified from P. furiosus genomic DNA by PCR and the products were inserted into the expression vector, pET21a. The recombinant E. coli JM109 (DE3) strains carrying one of the resultant plasmids, pPFRADA or pPFRADB, were cultivated, and the target proteins were successfully overproduced by isopropyl-1-thio--D-galactopyranoside induction. However, the purification of these proteins was difficult because of their high affinity for DNA. Additional chromatographic steps had to be performed to remove the DNA and to purify the two proteins to near homogeneity (Fig. 1B). The bands of RadA and RadB were detected on the SDS-PAGE at positions corresponding to their molecular weights, 38396.8 and 25337.9, respectively, which were calculated from the deduced amino acid sequences. From 1 liter of E. coli culture, about 3.4 mg of RadA and 0.17 mg of RadB were purified. No DNase activity was detected in either RadA or RadB from the assays using ³²P-labeled DNA substrates (data not shown). We prepared polyclonal antibodies using purified RadA and RadB proteins. Western blot analysis showed that proteins reacting specifically with either anti-RadA or anti-RadB antiserum were present in the P. furiosus cell extract (Fig. 1C). By comparisons of the band intensities from the cell extract and from serial dilution of purified RadA and RadB, we calculated that the cellular amounts of native proteins were 70 µg (1.8 nmols) and 0.2 µg (7.9 pmols) for RadA and RadB, respectively, per 1 g (wet weight) of the cells. The amount of RadB was 200 times less than that of RadA in P. furiosus cells. This is in contrast to the RNA data described below.

Expression of radA and radB Genes in P. furiosus-- Using quantitative reverse transcriptase-PCR and the ABI Prism 7700 detection system, we investigated the level of expression of the radA and radB genes in P. furiosus cells exposed to sublethal doses of  and UV irradiation and heat. Cells treated with  irradiation showed no loss of viability at 1500 grays and 75% survival when the culture was exposed to a dose of 2500 grays (31). No loss of viability was observed for the cells exposed to 35 J of UV light at 254 nm when compared with the unirradiated control. Cells subjected to 108 °C for 1 h showed 80% survival compared with the control cells incubated at 95 °C for 1 h. Fig. 2 shows that for all the treatments, the level of radA and radB mRNA did not increase in the treated cells compared with untreated control cells. We obtained the same pattern of expression for the radA and radB genes using nuclease protection assay.² These results suggest that the expressions of radA and radB might be constitutive. This is in contrast to the transcription of the recA (38) and RAD51 genes, which are inducible by UV irradiation and DNA damage (39). We only found severalfold difference in the mRNA levels of the radA and radB genes, whereas the amounts of the two proteins in P. furiosus cells differed by more than 200-fold as described in the above section. Some differential regulation may exist at the translational or post-translational level for the two proteins. Rapid turnover of RadB proteins could also explain those results.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Expression levels of the radA and radB genes in P. furiosus cells exposed to UV (A) or  irradiation (B) or heat shock (C). Cells were grown to mid exponential phase, exposed to challenges, and incubated at 95 °C for 1 h. RNA was extracted and subjected to DNase treatment. Real time quantitative reverse transcriptase-PCR was performed using specific gdh and rad primers, the ABI Prism 7700 detection system (Perkin-Elmer), and the SYBR Green I dye (Perkin-Elmer). The gdh gene, although highly expressed, is constitutive (72) and was used to standardize reactions and to allow relative quantitation. Open and shaded bars indicate the expression levels of radA and radB, respectively.

DNA Binding and ATPase Activity-- Fig. 3A shows that both RadA and RadB have ATPase activity at 70 °C. The ATPase activity of RadA was drastically stimulated by ssDNA and to a lesser extent by dsDNA. The k_cat value of RadA ATPase activity, 0.10 ± 0.01, increased 20 and 10 times when ssDNA (2.07 ± 0.12) and dsDNA (1.01 ± 0.04), respectively, were added to the reaction. The activity increased linearly with ssDNA concentration until a saturation plateau, at which the apparent binding stoichiometry is 3 nucleotides for 1 RadA (data not shown) as observed for RecA (40), Rad51 (41, 42), and S. solfataricus RadA (12). In contrast, RadB ATPase activity is significantly lower than that of RadA. This hydrolysis activity does not appear to be DNA-dependent and stopped at around 1 pmol of ATP/1 pmol protein RadB, suggesting the lack of enzymatic turn over for RadB. To confirm this property, binding affinities of the reaction product (ADP) to RadA and RadB were compared. As shown in Fig. 3B, ADP bound to RadB after ATP hydrolysis, which might explain that only 1 ATP was hydrolyzed per RadB molecule. Gel retardation assay showed that both RadA and RadB bound to deoxyoligonucleotides in a concentration-dependent manner. The binding affinity of RadB to the DNA was about 5 times higher than that of RadA in the gel shift assay condition (Fig. 4). No preference for dsDNA and ssDNA was observed for either RadA or RadB in our assay conditions. The binding of RadA to the DNA was not ATP-dependent (data not shown). However, some difference was observed in the binding patterns of RadB with and without ATP. Sizes of the shifted bands were constant in the presence of ATP, whereas the sizes increased with increasing amounts of RadB without ATP, especially in the case of ssDNA (Fig. 4B). These results suggest that conformational changes of RadB molecules may occur following ATP binding.

View larger version (27K): [in this window] [in a new window]   Fig. 3.   ATPase activity of RadA and RadB. A, RadA or RadB protein (each at 2 µM), [-32P]ATP (0.1 µ Ci/µl), and 100 µM of ATP were incubated with or without DNA at 70 °C for the indicated time, and aliquots of the reaction were analyzed by thin layer chromatography. The graphs show the values after subtraction of the background (radioactivities from the reaction without protein at each time point). , without DNA; , with single-stranded DNA; , with double-stranded DNA. B, RadA or RadB protein (each at 1 µM) was incubated with 50 or 1 µM of ATP containing [-32P]ATP in the presence of single-stranded DNA at 70 °C, and aliquots of the reaction were analyzed for ATP hydrolysis (shaded bars) and binding activities (black bars).

View larger version (35K): [in this window] [in a new window]   Fig. 4.   DNA binding activity of RadA and RadB. Various concentrations of RadA (A) and RadB (B) were incubated with a 32P-labeled single-stranded 83-mer, W16(), indicated by ss or a double-stranded 83 mer, W16(-)/W16(+), indicated by ds in the presence or absence of ATP at 60 °C for 10 min. Reaction products were analyzed by 1% agarose gel electrophoresis in 0.1× TAE buffer, and the bands were detected by autoradiography. C, the gels shown in A and B were subjected to image analysis to obtain data points for a graphical representation of the results. , RadA with ssDNA; , RadA with dsDNA; , RadB with ssDNA; , RadB with dsDNA.

DNA Pairing and Strand Exchange Activities-- To test whether RadA and RadB have strand exchange properties, two different assays were performed. First, as the prototype of strand invasion, D-loop formation ability was investigated. As shown in Fig. 5A, RadA, but not RadB, promoted the invasion of deoxyoligonucleotide (83-mer) into the supercoiled M13 replicative form. RadA-promoted D-loop formation was inhibited when RadB was added, before, after, or simultaneously with the addition of RadA to the reaction mixture. DNA strand exchange activity of RadA and RadB was investigated by three-strand exchange assays using two types of substrates. When using plasmid DNAs (the single-stranded circular form and the double-stranded linear form of pUC118) as substrate, RadA promoted the pairing of homologous strands but was much less active than the E. coli RecA protein. A distinct band corresponding to the joint molecule was observed with RadA (Fig. 5B). However, the nicked circular, which should be produced by complete strand exchange between the two molecules, could not be detected. The strand exchange activity of RadA seems likely to be ATP-dependent, although a faint band for joint molecule was detected in the reaction without ATP or with ATPS (Fig. 5B). This property is the same as that of human Rad51 (36, 43) and is in contrast with the properties of RecA (44, 45) and S. cerevisiae Rad51 (46), for which ATP binding but not hydrolysis is sufficient. However, RadA from D. amylolyticus has no strand exchange activity with ATPS (13). ATPS might not be a suitable co-factor for thermostable RadAs because of its instability at high temperatures. Only a faint band corresponding to joint molecule was observed with RadB and plasmid DNA as substrate (Fig. 5B). To further investigate RadB strand exchange ability, we used oligonucleotides (83-mer) (36) as substrates. RadA and RecA promoted displacement of the ³²P-labeled strand of a DNA duplex by an homologous DNA strand, in an ATP-dependent manner (data not shown). The sequence homology was essential for the exchange reaction. No displaced band was detected in the RadB reaction, suggesting that RadB does not have a strand exchange activity by itself (data not shown), although its amino acid sequence is substantially similar to that of Rad51 proteins. In P. furiosus, RadA appears to be the functional counterpart of bacterial RecA and eukaryotic Rad51. The effects of RadB on RadA strand exchange activity were also investigated. Fig. 5B shows that the reaction efficiency decreased when RadB was added to the reaction with or before RadA. On the other hand, there was no effect when RadB was added after the incubation of RadA with ssDNA. These observations indicate that the binding of RadB to DNA strands interferes with the formation of the RadA-DNA nucleoprotein filaments to proceed to the strand exchange reaction.

View larger version (23K): [in this window] [in a new window]   Fig. 5.   D-loop formation and three-strand exchange activities of RadA and RadB. A, D-loop formation activity of RadA and RadB was examined by using the double-stranded M13 DNA and a oligonucleotide having the homologous sequence as substrates. Asterisks indicate the substrate labeled with 32P (A, upper panel). RadA and/or RadB (each at 1 µM) were mixed with 1 µM of 32P-labeled 83-mer oligonucleotide, W16(), at various times and incubated at 65 °C for 10 min. Then M13 double-stranded DNA (0.1 µg) was added to the mixtures and further incubated for 2 or 5 min. The reaction mixtures were deproteinized and separated by 1% agarose gel electrophoresis in 0.1× TAE buffer. Lanes , no protein; lanes A, RadA; lanes B, RadB; lanes A+B, RadA and RadB (total 2 µM); lanes AB, RadB was added after 5 min of preincubation of ssDNA with RadA; lanes BA, RadA was added after 5 min of preincubation of ssDNA with RadB; lanes RecA, E. coli RecA (1 µM). B, three-strand exchange activity of RadA and RadB was examined by using plasmid DNAs as substrates. Time course and ATP dependence of RadA-promoted strand exchange reactions are examined in left panel. Effects of RadB on RadA-promoted reaction were analyzed by adding RadB in the reaction mixture at various timing (B, right panel). RadA and/or RadB was preincubated with ssDNA for 20 min at 70 °C, and then 32P-labeled dsDNA was added to the mixtures and further incubated for further 1 h. The reaction mixtures were deproteinized and separated by 1.2% agarose gel electrophoresis in TAE buffer. The bands were detected by autoradiography. Lane , no protein; lane A, 7.5 µM RadA; lane A+B, 7.5 µM RadA and 3.7 µM RadB; lane AB, RadB was added (3.7 µM) after 10 min of preincubation of ssDNA with 7.5 µM RadA; lane BA, RadA was added (7.5 µM) after 10 min of preincubation of ssDNA with 3.7 µM RadB; lane AB, RadB was added (3.7 µM) 15 min after starting exchange reaction (addition of dsDNA to the mixture of ssDNA and RadA); lane B, 7.4 µM RadB. E. coli RecA and single-stranded DNA binding protein (SSB) were used as a control (lane RecA).

Visualization of RadA and RadB Proteins-- One of the common features of strand exchange proteins is that they form nucleoprotein filaments with DNA in presence of ATPS. Complexes of RadA protein and M13 ssDNA were observed by electron microscopy (Fig. 6A). This is consistent with the observation that RadA is a strand exchange protein. However, the observed nucleoprotein filaments were different from those with other proteins in the RecA/Rad51 family (discussed below). In contrast, imaging of RadB-DNA complex never succeeded despite the fact that RadB has strong affinity to DNA as shown above. Images of each protein without DNA were also recorded. RadA protein formed ring-like structures with and without ATPS (Fig. 6A). At about the same concentration (7.8 µM), RadB aggregated, forming bulky structures that look like helical filaments (Fig. 6A, lower panels). The conformations of RadB filaments in the presence and absence of ATPS were different, which may reflect the dissimilarity of DNA binding mode for RadB as shown in the gel shift assay (Fig. 4B). The filament structure of RadB decreased in proportion to the amount of DNA added to the mixture (data not shown). Many reports show that the protein-DNA complexes of this family of proteins have extended structure with similarities in helicity, pitch, diameter, and extension of the axial rise per base pair of DNA (1.5 times the length of B-form duplex DNA). The filaments of RadA-ssDNA were not extended in our observation. Instead, the contour length of the nucleoprotein rings were shortened to less than half the length of the naked DNA. The length of the pUC118 DNA ring is about 1,100 nm. However, the average length of the 46 observed nucleoprotein filaments of RadA and ssDNA was 380 ± 28 nm (Fig. 6B). The nucleoprotein complex structure of human Dmc1-ssDNA looks like beads on a string rather than filaments, even though Dmc1 has a sequence very similar to other Rad51 family proteins and has strand transfer activity (47). The biological significance of the different structures of RecA/Rad51 family proteins remains to be elucidated.

View larger version (74K): [in this window] [in a new window]   Fig. 6.   Electron micrographs of RadA and RadB proteins. A, RadA (5.6 µM) and RadB protein (7.8 µM) were incubated at 90 °C for 15 min or 60 °C for 10 min with or without pUC118 ssDNA (21 µM) in the presence and absence of ATPS. B, the contour lengths of pUC118 ssDNA ring covered by RadA protein in the presence of ATPS were calculated from 46 independent images and shown by a histogram.

Physical Interactions of RadA-RadB and RadB-Hjc-- In yeast, it has been shown that Rad51 specifically interacts with Rad55-Rad57 heterodimer to promote strand exchange (48, 49). Furthermore, direct interactions between Rad51 and Xrcc3 (50), Rad51C and Rad51B, and Rad51C and Xrcc3 (51) in human cells have been reported. We investigated the possibility of interaction between RadA and RadB in P. furiosus cells using immunoprecipitation assay. RadB was co-precipitated with RadA by anti-RadA antibodies (Fig. 7A). Neither of the two truncated mutants, RadAn with the N-terminal one-third (Met¹-Arg¹⁰⁷) or RadAc with the C-terminal two-thirds (Als¹⁰⁸-Asp³⁴⁹), interacted with RadB (Fig. 7B). Furthermore, we investigated the interactions of RadB with other recombinational proteins. As shown in Fig. 7 (C and D), RadB, but not RadA, was co-precipitated with Hjc, a Holliday junction resolvase in P. furiosus, by anti-Hjc antibody. The amount of RadB precipitated with Hjc decreased in the presence of ATP (Fig. 7D). When anti-RadB antibodies were used for precipitation in presence of RadA or Hjc, only the RadB band was detected in the antibody-bound fraction. The anti-RadB antibody used for this experiment may interfere with the binding of RadB by competing with these proteins.

View larger version (46K): [in this window] [in a new window]   Fig. 7.   Immunoprecipitation of RadA, RadB, and Hjc proteins by anti-RadA, anti-RadB, and anti-Hjc antibodies in vitro. RadB and the wild-type (A) or the truncated (B) RadA proteins in indicated combinations were immunoprecipitated by anti-RadA or anti-RadB antiserum. The precipitates were analyzed by Western blot using each specific antibody indicated on the left side. Interaction of Hjc with RadA (C) or RadB (D) proteins were analyzed by the same procedure. The panels with (+ATP) in D show the precipitates prepared from the reaction in the presence of 2.5 mM of ATP in the binding and washing buffer. PI-PfuI, PI-PfuII, and their antisera were used as negative controls.

RadB Regulates Holliday Junction Cleavage Activity of Hjc-- We investigated the biological meaning of RadB-Hjc interaction by observing the effect of RadB on the Holliday junction cleavage by Hjc. The standard in vitro cleavage assay was used with a synthetic four-way junction as a substrate, and the cleavage efficiency of Hjc was compared in the presence and absence of RadB. Fig. 8 shows that the RadB suppressed the Holliday junction cleavage of Hjc to one-fifth in the absence, but not in the presence, of ATP. In contrast, RadA suppressed the activity in the presence of ATP, probably because RadA protects from Hjc cleavage by binding to the DNA substrate. These results suggest that RadB has some regulatory role on the Holliday junction cleavage activity of Hjc in the recombination process.

View larger version (48K): [in this window] [in a new window]   Fig. 8.   Effects of RadA and RadB on the Holliday junction DNA cleavage activity of Hjc. Hjc protein was mixed with RadA or RadB and reacted with 32P-labeled 4Jh at 60 °C for 1 h. The products were deproteinized and separated on 6% polyacrylamide gel. The bands were visualized by autoradiography.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Two different genes named radA and radB encoding Rad51-like proteins were found in the genomes of several archaea, raising the question of their biological functions. We presented here the regulation of expression of the radA and radB genes in P. furiosus cells and the biochemical properties of the corresponding proteins.

As suggested by this study, and in contrast to RecA in bacteria and Rad51 in eukaryotes, both RadA and RadB appeared to be constitutively expressed. This might be necessary to repair the constant damage inflicted to the DNA by exposure to high temperatures. Homologs of the bacterial DinF, DinG, UvrA, and UvrB proteins have been found in Archaea. All of these repair proteins are under the control of recA and lexA regulation in E. coli. Therefore, it is of great interest to study whether an adaptive DNA repair system similar to the bacterial SOS system (52) also exists in Archaea. Adaptive responses to heat shock have been reported for several Archaea (53-55), and stress response genes are known to be involved in DNA repair, heat shock, transcription, and cell cycle regulation in eukaryotes (56). Therefore, both constitutive and damage-inducible DNA repair functions may coexist in Archaea. An alternative to the constitutive expression hypothesis is that at 95 °C, the growth temperature of P. furiosus in the laboratory, both RadA and RadB might be already fully induced. Experiments with cells grown at the lowest temperature might bring an answer.

RadA possesses DNA-dependent ATPase, DNA pairing, and strand exchange activities, suggesting that it might be the counterpart of bacterial RecA and eukaryotic Rad51 proteins in P. furiosus. In contrast, our experiments show that the biochemical properties of RadB differ greatly from those of RadA. RadB consists of only Domain II of the RecA/Rad51 family proteins and has a weak DNA-independent ATPase activity. Little strand pairing or strand exchange activity was detected with RadB. These results suggest that the archaeal RadB is not a strand exchange protein. Stoichiometric rather than catalytic nature of RadB-mediated hydrolysis of ATP (Fig. 3) suggests that ATP may work as a molecular switch, and interconversion of active and inactive conformation of RadB may be regulated by the ATP hydrolysis. The same manner of interconversion is well understood in the case of E. coli DnaA, which regulates the initiation of DNA replication (57). There is a report that the RadB homolog from Pyrococcus sp. KOD (Pk-REC) can complement the UV sensitivity of a recA-deficient strain of E. coli (7). However, in contrast to this report, our experiments using RadB did not complemented the UV sensitivity of E. coli DM2569 (recA and lexA) at all, although the production of RadB protein in the cells was confirmed by Western blot analysis (data not shown). Pk-REC is 84% identical to RadB at the amino acid level, suggesting that it is the functional homolog of RadB in Pyrococcus sp. KOD (recently named Pyrococcus kodakaraensis KOD). The second RecA/Rad51 family protein has not yet been identified in P. kodakaraensis KOD. Further studies are necessary to understand the difference of the two Pyrococcal proteins.

Sandler et al. (9) suggested that the archaeal RadB might be a functional counterpart of the eukaryotic Rad55-Rad57 heterodimer. Rad55 and Rad57 interact with each other, and the interactions of Rad51, Rad55, and Rad57 are important for recombinational DNA repair (48). More recently, it was reported that the Rad55-Rad57 complex promoted the replication protein A-associated strand exchange reaction by Rad51 (49). The complex possesses an ATPase activity, which is not stimulated by ssDNA or dsDNA. Furthermore, an immunological analysis showed that the molar cellular abundance of the Rad55-Rad57 heterodimer is one-tenth of that of Rad51. Present studies showed that the ATPase activity of RadB is not stimulated by DNA and that RadB is present in much lower amounts than RadA in P. furiosus cells. Immunoprecipitation experiments showed a specific interaction between RadA and RadB, suggesting that RadB might be the functional homolog of the eukaryotic Rad55-Rad57 heterodimer in Archaea. Analogy to the Rad55/Rad57 system could be studied further by examining whether RadB enhances the strand exchange activity of RadA in the presence of P. furiosus replication protein A.

In addition to Rad55-Rad57, Rad51 protein is known to interact with other proteins in RAD52 epistasis group (58-62) and tumor suppressors (63-70). Our immunoprecipitation experiment shows that there is a specific interaction between RadB and Hjc. This is a new finding, and cleavage assays of Hjc with or without RadB showed that RadB regulates the Hjc activity. RadB might bind as an inhibitor to Hjc, and ATP might change the conformation of RadB to promote the dissociation of the protein from the complex with Hjc. As a consequence, Hjc becomes active to cleave the Holliday junction. The exact binding ratio of RadB to Hjc has to be determined to examine this possibility. The cellular amount of Hjc is low from our previous work (29). In E. coli, it has been proposed that RuvABC proteins form a complex, which promotes branch migration and dissociates at DNA sequences that are suitable for cleavage by RuvC resolvase (71). One possible speculation from our observation is that RadB and Hjc may form a complex with unidentified proteins, which promotes branch migration. When the complex with the junction reaches some suitable site for cleavage, RadB dissociates from the complex to activate Hjc for cleavage. Scouting of the functional homolog of RuvAB in Archaea is essential to prove this hypothesis.

Genetic analyses are now necessary to understand the function in vivo of RadB. However, no reliable technique for genetic engineering is currently amendable to P. furiosus. A radA deletion mutation of Haloferax volcanii, a halophilic euryarchaeote, resulted in an UV-hypersensitive phenotype (11). This microorganism could be employed to study the in vivo function of radB gene and its product. In addition, studies on functional and structural interactions among archaeal proteins related to RadA and RadB will contribute immensely to the understanding of the more complex eukaryotic mechanisms of DNA recombination.

	ACKNOWLEDGEMENTS

We thank Drs. H. Iwasaki, E. Akaboshi, T. Morita, A. Shinohara, T. Ogawa, and F. Robb for discussions and S. Kowalczykowski for critical reading of the manuscript. We also thank N. Fujita and S. Ishino for some experiments in the early stage of this study. We are grateful to Dr. Y. Shimura, the director of BERI, for continuous encouragement.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence should be addressed: Dept. of Molecular Biology, Biomolecular Engineering Research Institute, 6-2-3 Furuedai, Suita, Osaka 565-0874, Japan. Tel.: 81-6-6872-8203; Fax: 81-6-6872-8219; E-mail: ishino@beri.co.jp.

Published, JBC Papers in Press, July 19, 2000, DOI 10.1074/jbc.M004557200

² J. DiRuggiero and F. Chaussard, unpublished results.

	ABBREVIATIONS

The abbreviations used are: ssDNA, single-stranded DNA; PCR, polymerase chain reaction; DTT, dithiothreitol; ATPS, adenosine 5'-O-(thiotriphosphate); PAGE, polyacrylamide gel electrophoresis.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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