Identification of a Novel Helicase Activity Unwinding Branched DNAs from the Hyperthermophilic Archaeon, Pyrococcus furiosus*

To identify the branch migration activity in archaea, we fractionated Pyrococcus furiosus cell extracts by several chromatography and assayed for ATP-dependent resolution of synthetic Holliday junctions. The target activity was identified in the column fractions, and the optimal reaction conditions for the branch migration activity were determined using the partially purified fraction. We successfully cloned the corresponding gene by screening a heat-stable protein library made by P. furiosus genomic DNA. The gene, hjm (Holliday junction migration), encodes a protein composed of 720 amino acids. The Hjm protein is conserved in Archaea and belongs to the helicase superfamily 2. A homology search revealed that Hjm shares sequence similarity with the human PolΘ, HEL308, and Drosophila Mus308 proteins, which are involved in a DNA repair, whereas no similar sequences were found in bacteria and yeast. The Hjm helicase may play a central role in the repair systems of organisms living in extreme environments.

Homologous recombination plays important roles in DNA metabolisms, including several DNA repair processes and the generation of genetic diversity in living cells. The Holliday junction (HJ) 1 is an important intermediate during the homologous recombination process (1). The molecular mechanism of the early stage of homologous recombination has been extensively investigated, and the proteins involved in this process are conserved in the three biological domains: Bacteria, Eukarya, and Archaea (2,3). On the other hand, the protein factors involved in the late stage of the process have been identified only in Bacteria, in which the RuvABC-Holliday junction complex processes the recombinational intermediates (4,5). In Eukarya, the activities for branch migration and resolution of the HJ have been reported (6 -9); however, the corresponding proteins have yet to be identified. A recent report showed that RAD51C, a RAD51 paralog in human cells, is involved in HJ processing (10).
Archaea, the third domain of life (11), is distinct from both Bacteria and Eukarya. Archaea share many similarities with Eukarya in their genetic information processing pathways, including DNA replication, transcription, and translation (12), although cellular structure is prokaryotic. Therefore, the archaeal processes provide a useful model systems to understand the much more complex mechanisms of their eukaryotic equivalents. Proteins involved in homologous recombination are also conserved between Archaea and Eukarya. We characterized the Pyrococcus furiosus RadA and RadB proteins (13)(14)(15), which play a central role in the initiation of homologous recombination. These archaeal recombinases have sequences more similar to that of eukaryotic Rad51 than bacterial RecA (16,17). In addition, we showed that the P. furiosus RPA, which is composed of three subunits, RPA41, RPA14, and RPA32, like the eukaryotic RPA (p70-p14-p32), but different from bacterial SSB (homotetramer), clearly stimulated a RadA-mediated strand exchange reaction (18). The homologs of the eukaryotic Rad50 and Mre11 proteins, which may work in the very early steps of homologous recombination, after double-stranded break repair, are also conserved in Archaea (19,20). Regarding the late stage, in which the HJ intermediates are processed, we identified an archaeal HJ resolvase and named it Hjc (21). Contrary to our expectations, Hjc is an archaea-specific protein, and neither its sequence nor its three-dimensional structure is similar to other known HJ resolvases (22,23). Therefore, the HJ resolvase is quite interesting, from an evolutional point of view (24).
In our series of experiments, we fractionated P. furiosus cell extracts by several chromatography steps and assayed for the ATP-dependent resolution of synthetic Holliday junctions to identify the activity for HJ branch migration in archaea. The target activity was identified in the column fractions, and the optimal reaction conditions for the activity were determined, using the partially purified fraction. Then we succeeded in cloning the corresponding gene by screening a heat-stable protein library of P. furiosus. The gene, hjm (Holliday junction migration), encodes Hjm, which is composed of 720 amino acids. The Hjm protein is conserved in Archaea, and there are some eukaryotic proteins sharing sequence similarity to Hjm. The physiological functions of Hjm in archaeal cells and the evolution of the Hjm protein are very interesting themes to consider.

EXPERIMENTAL PROCEDURES
Synthetic DNA Substrates-Oligonucleotides with the following sequences were synthesized: sequence 1, 5Ј-GTGACCGTCTCCGGGAGC-TGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAG-ACGAAAGG-3Ј; sequence 2, 5Ј-CCTTTCGTCTCGCGCGTTTCGGTGA-TGACGGTGAAAACCTCTGACACATGGCCAGCCCCGACACCCGCC-A-3Ј; sequence 3, 5Ј-TGGCGGGTGTCGGGGCTGGCCATGTGTCAGA-* This work was supported in part by the Japan New Energy and Industrial Technology Development Organization. This work was also supported in part by a Grant-in Aid from the Ministry of Education, Science, and Sports (to Y. I. and H. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Partial Purification of the Branch Migration Activity from P. furiosus Cells-P. furiosus cells (DSM 3838 strain) were cultivated as described earlier (27), and 52 g of cells were obtained. The cells were disrupted by sonication in 500 ml of buffer A (50 mM Tris-HCl, pH 8.0, 0.5 mM dithiothreitol, 0.1 mM EDTA and 10% glycerol) containing 1 mM phenylmethylsulfonyl fluoride. After centrifugation at 100,000 ϫ g for 60 min at 4°C, the cell-free extract was treated with polyethylenimine (final concentration, 0.15%) and centrifuged at 30,000 ϫ g for 15 min at 4°C. After centrifugation, the precipitate was resuspended in 300 ml of buffer A containing 1 M ammonium sulfate, and the proteins were precipitated with ammonium sulfate (80% saturation). The precipitated proteins were resuspended in 60 ml of buffer A containing 2 M NaCl, and the eluted proteins were dialyzed against buffer A containing 0.5 M NaCl. After dialysis, the supernatant was diluted with 2.5 volumes of buffer A, and the dialysate was applied to a heparin column (HiTrap heparin; Amersham Biosciences), which was developed with a 0.2-1.5 M NaCl gradient in buffer A. The active fractions, which eluted at 0.46 -0.70 M NaCl, were dialyzed against buffer B (50 mM Tris-HCl, pH 7.0, 0.5 mM dithiothreitol, 0.1 mM EDTA, and 10% glycerol) containing 0.1 M NaCl. The dialysate was applied to a HiTrap SP column (Amersham Biosciences), which was developed with a 0.10 -0.60 M NaCl gradient in buffer B. The activity eluted at 0.18 -0.38 M NaCl and was dialyzed against buffer A. The dialysate was applied to a HiTrap Q column, which was developed with a 0 -1 M NaCl gradient. The activity eluted at 0.30 -0.55 M NaCl and was dialyzed against buffer C (10 mM potassium phosphate, pH 6.8, 7 mM 2-mercaptoethanol, 0.01 mM CaCl 2 , and 10% glycerol). The dialysate was applied to a hydroxylapatite column (CHT-II; Bio-Rad), which was developed with a 0.01-1 M potassium phosphate gradient. The activity eluted at 0.10 -0.30 M potassium phosphate and was dialyzed against buffer D (50 mM Tris-HCl, pH 7.0, 0.5 mM DTT, and 0.1 mM EDTA). The dialysate was applied to a phosphocellulose column (Whatman P-11), which was developed with 0 -1.5 M NaCl. The activity eluted at 0.25-0.43 M NaCl and was dialyzed against buffer D. The dialysate was applied to a MonoS 5/5 column (Amersham Biosciences), which was developed with 0 -0.6 M NaCl. The activity eluted at 0.40 -0.45 M NaCl. The active fractions were stored at 4°C. The protein concentrations of the active fractions were quantified by using a protein assay kit (Advanced protein assay kit; Cytoskelton Inc.).
Branch Migration Assay-Aliquots (2 l) of each column fraction or purified Hjm protein were incubated with 5 nM of 32 P-labeled synthetic HJ (HSL) in an 18-l reaction mixture, containing 10 mM Tris-HCl, pH 8.8, 2 mM ATP, 5 mM MgCl 2 , and 0.1 mM DTT at 55°C for 30 -60 min. The reactions were stopped by phenol, and the products were analyzed by PAGE using TAE buffer (40 mM Tris acetate, pH 7.8, 1 mM EDTA) followed by autoradiography. For the quantitative analysis, the reaction products from each experiment were quantified from the autoradiograms using a laser-excited image analyzer (FLA5000; Fuji Film).
Cloning of the Gene Responsible for the Branch Migration Activity-The cosmid-based genomic library and the heat-resistant fractions (heat-stable protein library) from each E. coli clone were prepared as described previously (21). The branch migration assay using a synthetic Holliday junction, HSL, was performed with the heat-stable protein library from 496 independent clones. Cosmid DNA was prepared from the clone encoding the branch migration activity and was digested by BamHI. The fragments were inserted into the pUC118 vector, and their nucleotide sequences were partially determined. The gene encoding the branch migration activity was identified by searching the prospective region of the P. furiosus genome with the determined sequence for an open reading frame encoding a nucleotide binding motif (P-loop). This gene was amplified by PCR, using the oligonucleotides 5Ј-CGAGCCA-TGGGGGTTGATGAGCTGAGAGTT-3Ј and 5Ј-CGGGTCGACTCAAGA-TTTGAGAAAGTAATC-3Ј as primers, from the P. furiosus genome. The amplified gene was cloned into the pGEM-T easy vector (Promega). The cloned gene was digested by NcoI-SalI and was inserted into the corresponding sites of pET21d (Novagen). The constructed plasmid was designated as pHJM100.
Overexpression and Purification of the Hjm Protein-To obtain the recombinant Hjm protein, E. coli BL21 codonPlus TM (DE3)-RIL (Stratagene) carrying pHJM100 was grown in 1 liter of LB medium, containing 50 g/ml ampicillin and 34 g/ml chloramphenicol, at 37°C. The cells were cultured to an A 600 of 0.35, and then the expression of the hjm gene was induced by adding isopropyl ␤-D-thiogalactopyranoside to a final concentration of 1 mM and continuing the culture for 5 h at 37°C. After cultivation, the cells were harvested and disrupted by sonication in buffer A containing 50 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 0.5 mM EDTA, 1 mM DTT, and 10% glycerol. The soluble fraction, obtained by centrifugation (12,000 ϫ g, 15 min), was heated at 80°C for 20 min. The heat-resistant fraction was treated with 0.15% polyethylenimine. The soluble proteins were precipitated by 80% saturated ammonium sulfate precipitation. The precipitate was resuspended in buffer B containing 50 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 1.25 M (NH 4 ) 2 SO 4 , 0.5 mM EDTA, 1 mM DTT, and 10% glycerol and was subjected to chromatography on a HiTrap Butyl (Amersham Biosciences). The proteins were eluted at 0 M ammonium sulfate, and the eluted proteins were dialyzed against buffer C containing 10 mM potassium phosphate, 7 mM ␤-mercaptoethanol, 0.01 mM CaCl 2 , and 10% glycerol. The dialysate was loaded onto a CHT-II column, and the proteins were eluted at 0.25-0.35 M potassium phosphate. The eluted proteins were dialyzed against buffer D containing 10% glycerol), and the dialysate was subjected to chromatography on a MonoQ 5/5 column (Amersham Biosciences). The proteins were eluted at 0.32-0.37 M NaCl. The eluted protein was pooled and stored at 4°C.
Western Blot Analysis-P. furiosus cells (1 g) were disrupted by sonication in 15 ml of buffer B containing proteinase inhibitor (Complete TM ), and the extract was obtained by centrifugation. The P. furiosus cell extract (800 ng) and the purified Hjm proteins (1 ng) were separated by 12% SDS-PAGE, blotted onto polyvinylidene difluoride membranes, and reacted with anti-Hjm antiserum. The bands were detected by using an enhanced chemiluminescence system (Amersham Biosciences) according to the supplier's recommendations.
Gel Filtration and Glycerol Gradient Centrifugation-Gel filtration chromatography was performed with the SMART system (Amersham Biosciences). The MonoS fraction (fraction number 16) or purified recombinant Hjm protein was applied to a Superdex 200 3.2/30 column (Amersham Biosciences), pre-equilibrated with buffer E (50 mM Tris-HCl, pH 8.0, 0.5 mM DTT, 0.1 mM EDTA, and 0.3 M NaCl). The eluted fractions were subjected to a branch migration assay, as described above. The molecular mass of the protein with the branch migration activity was estimated from the elution profiles of standard marker proteins, including thyroglobulin (670,000), ␥-globulin (158,000), ovalbumin (44,000), and myoglobin (17,000). Glycerol gradient centrifugation was performed as described earlier (28). The purified Hjm protein (100 g) was sedimented through a 10-ml continuous 10 -35% (v/v) glycerol gradient by 40,000 rpm at 4°C for 24 h in a Beckman SW41 rotor. The standard marker proteins as described above were sedimented under identical conditions.
Branch Migration of Recombination Intermediates-The recombination intermediates were produced by in vitro strand exchange reaction using plasmid DNAs, as described previously (21,29). The singlestranded circular pUC118 DNA and the KasI-PstI-digested doublestranded pUC118 were mixed to produce a gapped DNA, and the gapped DNA (6 M as nucleotide concentration) was preincubated with the E. coli RecA protein (2.7 M) in a buffer containing 20 mM Tris-HCl, pH 7.5, 15 mM MgCl 2 , 2 mM DTT, 2 mM ATP, and 100 g/ml bovine serum albumin at 37°C for 5 min for the strand exchange reaction. To produce the ␣-structured DNA, recombination intermediates, 3Ј-32 Plabeled linear DNA (PstI-digested) was added to 3 M. After 15 min of incubation at 37°C, the reaction mixture was deproteinized by phenol extraction. Purified Hjm protein was added to 100 nM to the solution containing ␣-structured DNA in 10 mM Tris-HCl, pH 8.0, 2 mM ATP, 5 mM MgCl 2 , and 0.1 mM DTT and was incubated at 55°C for 0 -60 min. The reaction products were deproteinized by incubation with proteinase K followed by phenol extraction and were separated by 12% agarose gel electrophoresis in TAE buffer. Detection of the products was performed by autoradiography.
Computer Analysis of the Amino Acid Sequences-Search for the homologous sequences in the databases with BLAST was carried out at a website (www.ncbi.nlm.nih.gov/cgi-bin/BLAST/). The gene corresponding to hjm has been registered as the gene number of PF0677 in the P. furiosus genome data base. The hjm homologs in the other archaeal genomes correspond to the gene numbers, PAB0592 (Pyrococues abyssi), AF2245m (Archaeoglobus fulgidus), HaloRad24b (Halobacterium salinarium), MTH810 (Methanothermobacter thermoautotrophicus), PAE0894 (Pyrobaculum aerophilum), SSO02462 (Sulfolobus solfataricus), STO 0590 (Sulfolobus tokodaii), and APE0191 (Aeropyrum pernix) in each genome data base. The sequences of human DNA polymerase , Hel308, and Drosophila melanogaster Mus308 were derived from the genes with accession numbers AAK39635, NP_598375, and AAB67306, respectively.

RESULTS
Identification and Partial Purification of the Branch Migration Activity-We tried to identify the branch migration activity corresponding to that from the bacterial RuvB protein in P. furiosus cells. The total cell extracts of P. furiosus were fractionated by anion exchange (HiTrap SP) or cation exchange (HiTrap Q) chromatography, and the target activity was assayed using a 32 P-labeled synthetic HJ. However, the branch migration activity to produce splayed arm DNAs was not detected in any fraction. Then polyethylenimine precipitation was done to concentrate the nucleic acid-binding proteins before the ammonium sulfate precipitation and the column chromatography as described under "Experimental Procedures." These procedures were effective, and the branch migration activity eluted at 0.46 -0.6 M NaCl during the elution of the heparin-Sepharose column with a linear NaCl gradient (Fig.  1A). As shown in the figure, the HJ resolvase activity by Hjc was separated from the branch migration activity in this chromatography step. The active fractions were further purified by sequential chromatography. Using the active fractions (fractions 15 and 16) from the cation exchange chromatography, as shown in Fig. 1B, the substrate specificity was examined. Several kinds of DNA substrates, including four-way junction, half-cruciform, looped-out, and normal duplex DNAs, were prepared by the combination of synthetic oligonucleotides as described under "Experimental Procedures." Among these substrates, the four-way junction was preferentially dissolved (data not shown). As shown in Fig. 1C, the fraction sample dissociated oligonucleotide annealed to M13 mp18 singlestranded DNA, in addition to the two HJs, HSL with large homologous core, and JY with small homologous core. This new helicase activity may have wider substrate preference when compared with that of RuvB helicase.
Cloning and Expression of the Gene Encoding the Branch Migration Helicase-Based on the reaction conditions determined from the above section, we screened for the branch migration activity from the heat-stable P. furiosus protein libraries, as described under "Experimental Procedures." Among the 496 independent heat extracts of E. coli transformants, we found a clone producing a protein that dissolved the four-way junction DNA. Then the cosmid DNA was recovered from the E. coli clone, and the region containing the gene encoding the target activity was subcloned into the plasmid vector. A certain open reading frame with an ATP-binding motif (P-loop) was found after sequencing the cloned DNA. The gene for the open reading frame was cloned into the expression vector, pET21d for overexpression in E. coli. The recombinant E. coli cells carrying the resultant plasmid, pHJM100, were cultivated, and the encoded protein was successfully overproduced by isopropyl ␤-D-thiogalactopyranoside induction. The protein was purified to homogeneity by the three sequential chromatography steps as shown in Fig. 2A. The highly purified protein was used for the following characterization. Based on its activity to dissolve four-way junction DNA, we named the protein Hjm (Holliday junction migration). Using the highly purified Hjm protein, a polyclonal antibody was prepared. Western blotting analysis showed that a protein the same size as the recombinant Hjm protein, which also specifically reacted with the antibody, was present in the total extract from P. furiosus cells (Fig. 2B). The active fractions obtained during the purification procedure, as described above, also had a protein that reacted with the antibody (Fig. 2C). To investigate the oligomeric state of Hjm in solution, gel filtration chromatography was done (Fig. 2D). The elution profile showed that the estimated molecular weight of Hjm is 73,500, which is slightly smaller than that calculated from the deduced amino acid sequence (82, 631). This inconsistency may be explained by some interaction of Hjm with Sephadex resin, because chromatographic profile showed that the elution peak of Hjm was not symmetric but had some tailing. Glycerol gradient separation experiment showed that Hjm was sedimented to the position corresponding to the molecular weight of 75,800 (Fig. 2E). These results suggest that Hjm exists as a monomeric protein in solution. Further analyses including analytical ultracentrifugation will provide more detailed information.
Biochemical Characterization of Hjm-Using purified Hjm protein, its HJ unwinding activity was characterized in more detail. Hjm dissociated the synthetic HJ, HSL, in a concentrationdependent manner in vitro as shown in Fig. 3A. Thermus thermophilus RuvB and RuvA-RuvB complex were used for the same assay to compare the efficiencies of the unwinding reaction. The Hjm dissociated the synthetic HJ, HSL DNA, to two splayed arms at lower concentrations (3-10 nM). In the higher concentrations (Ͼ10 nM), Hjm dissociated HSL DNA very efficiently, and in these cases, approximately half of the splayed arms DNA was dissociated to single-stranded DNAs (Fig. 3). In the same unwinding assay, T. thermophilus RuvAB, with 200 nM as RuvB monomer concentration, dissociated 34% of HLS to two splayed arms in 30 min. With the same concentration of the proteins, Hjm showed more efficient dissociation of HSL DNA (Fig. 3B). This dissolving activity was ATP-dependent (Fig.  4A). However, some reaction products were observed in the reactions with ADP and ATP␥S. Further investigations should be done to confirm that these nucleotides actually work for the reaction. The reaction was most efficient with an ATP concentration of at ϳ5-10 mM (Fig. 4A). Interestingly, the HJ were dissociated to single-stranded DNA with increasing concentrations of ATP. A divalent cation was also essential for the junction dissolving reaction, and Mg 2ϩ worked most efficiently among five kinds of metal cations (Fig. 4B). In the case of MgCl 2 , the reaction progressed well with less than 10 mM MgCl 2 (Fig. 4C). This junction dissolving activity was quite sensitive to the salt concentration, because the reaction was inhibited with increasing concentrations of NaCl (Fig. 4D). In addition to these properties, Hjm is very heat-stable. No activity was lost even after an incubation of the protein at 98°C for 90 min (data not shown). The optimal temperature of the junction dissolving reaction was difficult to determine, because the synthetic four-way junction substrates were disrupted at higher temperatures. However, the dissolving reaction was observed at least at 80°C (data not shown).
Hjm-induced Branch Migration of RecA-mediated Recombination Intermediates-To investigate whether the Hjm protein has branch migration activity with a natural recombinational intermediate, we prepared the Holliday junction by using a RecA-mediated strand exchange reaction between gapped circular pUC18 and homologous linear duplex DNA labeled at its 3Ј-termini with 32 P, as shown in Fig. 5A. The recombination intermediate, called an ␣-structured DNA, can be dissolved to nicked circular and linear duplex DNA by a branch migration reaction. The RecA-mediated ␣-structured DNA was deproteinized by phenol extraction and then was reacted with purified Hjm. As shown in Fig. 5B, the amount of the ␣-structured DNA decreased, and the processed products increased with the reaction time. This result suggests that Hjm may function like bacterial RuvB in the homologous recombination process. Hjm Homologs in Archaea and Eukarya-The deduced amino acid sequence revealed that the N-terminal region of Hjm has the seven characteristic motifs found in the helicase family (Fig. 6), and therefore, Hjm belongs to superfamily 2 in the helicase classification (30). The C-terminal region may be responsible for recognizing a specific structure of DNA or for interacting with other protein for its physiological function. The sequence of Hjm is not similar to that of the bacterial RuvB protein. Moreover, no other protein with a similar sequence to Hjm has been found in eubacteria and yeast. However, the sequence of Hjm is highly conserved in Archaea. One open reading frame sharing more than 30% amino acid sequence homology to Hjm was found in each of the genomes from both euryarchaeal and crenarchaeal organisms (Table I). Our homology search using the Hjm sequence as a query identified the human Pol⌰ and Hel308 proteins and the Drosophila Mus308 protein (Fig. 6). The mus308 gene was identified in D. melanogaster as a gene required for resistance to DNA cross-linking reagents (31). The Hel308 protein (a helicase similar to Mus308) is a human homolog of the helicase domain of D. melanogaster Mus308, and it has a single-stranded DNA-dependent ATPase activity with a dissociation activity for duplex DNA (32). Hel308 does not have a DNA polymerase-like sequence in the C-terminal region. Pol⌰, with both a helicase motif and a polymerase-like sequence, is more similar to Mus308, and the POLQ gene seems to be the mus308 ortholog in the human genome (33). The DNA-dependent ATPase activity and the DNA polymerase activity of Pol⌰ have also been characterized (33). It will be very interesting to further characterize and classify these Mus308 family helicases in terms of their structures and functions. DISCUSSION We previously identified the Holliday junction resolvase, Hjc, in archaea and proposed that the formation and resolution of the Holliday junction represent a common mechanism of homologous recombination in the three domains of life (21). During our continuing efforts to identify the proteins responsible for the processing of the Holliday junction intermediate in archaea, in this study, we identified a novel helicase, which dissolves the four-way junction in an ATP-dependent manner. To isolate the target activity in the P. furiosus cell extracts, we used the same strategy as used for Hjc. However, we could not find the branch migration activity by using the total cell extract directly with a synthetic Holliday junction. The purification procedure to concentrate nucleic acid-binding proteins was critical to identify the target activity in this case, and the fractionation by polyethylenimine treatment followed by ammonium sulfate precipitation was very effective. Actually, the purified Hjm protein has very high affinity for DNA, and its branch migration activity was inhibited by increasing the amount of nonspecific DNA in the reaction mixture (data not shown).
It has been proposed that RuvA, RuvB, and RuvC can form a resolvasome complex and work cooperatively to process the Holliday junction (34 -37). The formation of a resolvasome has also been suggested by the fact that the branch migration and junction cleavage activities in a mammalian cell-free extract fractionated together after chromatography on phosphocellulose, Butyl-Sepharose, and heparin-Sepharose columns (8). In this archaeal case, Hjm and Hjc were separated even by affinity chromatography (heparin-Sepharose). Hjc has strong affinity for heparin (21), and this interaction may be stronger than that between Hjc and Hjm if these proteins actually form the resolvasome in archaeal cells.
It seems likely that Hjm processes the homologous recombination intermediate in archaeal cells, from the result that Hjm processed the RecA-mediated ␣-structure in vitro, as shown in Fig. 5. However, it remains to be elucidated whether Hjm is the real functional counterpart of the bacterial RuvB in the Archaeal domain. Some genetic studies to analyze the phenotype of hjm mutants are necessary to determine the function of Hjm in cells. In terms of the early steps of homologous recombination, the homologs of the eukaryotic Mre11, Rad50, Rad51, and RPA have been found in Archaea. However, the Holliday junction resolvase, Hjc, is completely unique in Archaea, and no protein with sequence homology to Hjc has been found in Eukarya. The Holliday junction resolvases have been recognized as a very interesting issue from evolutional aspects (24). It is possible that the proteins responsible for the processing of the Holliday junction in Archaea are totally diversified, and neither the bacterial nor eukaryotic proteins involved in this process have similarity. FIG. 4. Requirements for Hjm branch migration activity. A, ATP dependence. Reactions were carried out at 55°C for 30 min in a buffer containing ATP at the indicated concentrations. B, cation preference: Mg 2ϩ , Mn 2ϩ , Zn 2ϩ , Ca 2ϩ , or Co 2ϩ , each at 5 mM, was used. C, Mg 2ϩ requirement: Reactions were performed in the presence of MgCl 2 at the indicated concentrations. D, effect of salt concentrations: The NaCl concentration in the reaction was varied as indicated. The reaction products from each experiment were quantified from the autoradiograms using a laser-excited image analyzer.
A sequence homology search revealed some similarity between Hjm and eukaryotic Mus308, Hel308, and Pol⌰ (Fig. 6). The D. melanogaster mus308 mutant is sensitive to crosslinking agents, such as psoralen, diepoxybutane, and nitrogen mustard, and therefore, the gene product probably functions in interstrand cross-link repair, from genetic studies (31). The Mus308 protein has a family A DNA polymerase (bacterial DNA polymerase I)-like sequence at the C-terminal region, in addition to the N-terminal SF2 helicase sequence (31). Moreover, this protein probably has DNA polymerase activity (38). The human Hel308 has been purified from recombinant insect cells, and activities including a single-stranded DNA-dependent ATPase and a DNA helicase, which translocates on DNA with 3Ј to 5Ј polarity, were demonstrated using the purified protein (32). Human Pol⌰, which has a bacterial Pol I-like sequence (39), has been identified recently to have a helicaselike sequence at the N-terminal region (33). Therefore, Pol⌰ is now entirely similar to Mus308 (Fig. 6), and these two genes seem to be orthogonous. In the bacterial interstrand cross-link repair system, the excision and synthesis events involve UvrABC and UvrD, as well as DNA polymerase I (40). It would be very interesting to investigate whether the helicase and polymerase activities in Mus308 and Pol⌰ are involved in the interstrand cross-link repair system in eukaryotes. We investigated the DNA polymerase activity of Hjm by a nucleotide incorporation assay, but no activity was detected (data not Asterisks show the 3Ј-32 P-labeled ends. B, the Hjm protein was incubated with deproteinized ␣-structured DNA. The reaction products were separated by 1.2% agarose gel electrophoresis and detected by autoradiography. As a positive control, T. thermophilus RuvAB was used for the reaction. ATP␥S was used for ATP to investigate the effect of ATP hydrolysis on the branch migration activity of Hjm. FIG. 6. Structure comparison of Hjm and the eukaryotic Mus308, Pol⌰, and Hel308 proteins. The N-terminal regions of the P. furiosus Hjm, Drosophila Mus308, and human Pol⌰ and Hel308 proteins contain sequences (motifs I, Ia, II, III, IV, and VI) similar to those within superfamily 2 helicases (indicated as Helicase). The sequences of each motif in Hjm protein are also indicated at the top. In addition, the C-terminal region of Hjm shares some similarity to these eukaryotic proteins. Pol⌰ and Mus308 have a family A DNA polymerase (bacterial Pol I)-like sequence at their C-terminal regions. The source of the sequences used for comparison is indicated under "Experimental Procedures." shown). It remains to be determined whether the helicase activity of Hjm is related to interstrand cross-link repair in Archaea. More detailed characterizations of Hjm, in terms of its structural specificities for the DNA binding, helicase, and ATPase activities, in addition to genetic studies, will provide important information about its functions in living cells.