BACKGROUND
dna helicases are enzymes that translocate along dna and unwind double-stranded regions in an atp-dependent manner  <cit> .
they play crucial and universal roles in dna metabolism, such as dna replication and recombinational repair.
as a consequence of their physiologically important functions, many reports have been published regarding protein characterization and catalytic mechanisms, including the relationships between enzymatic dysfunctions and several human genetic diseases  <cit> .
our on-going structural analysis of the late stage of homologous recombination, such as the ruvabc-holliday junction  complex  <cit> , tempted us to investigate the molecular machinery involved in holliday junction processing in eukaryotes.
we also noticed that the archaeal proteins involved in dna metabolism generally have amino acid sequences and three-dimensional  structures that are highly similar to their eukaryotic homolog.
the proteins from the hyperthermophilic archaea, including pyrococcus furiosus, are more advantageous for structural studies than their eukaryotic counterparts, because of their remarkable thermal stability.
in fact, we were the first group to successfully identify the holliday junction resolvase from archaea, which we designated as hjc  <cit> , and we also determined its crystal structure by x-ray analysis  <cit> .
a subsequent screening study for a new protein factor that stimulates the hj resolving activity by hjc led to the identification of a new protein factor, termed hef  <cit> .
biochemical and sequence analyses revealed that this protein should be classified as an xpf/rad1/mus <dig> nuclease, which bears endonuclease activity specific for flap or fork structures.
interestingly, the full-length hef molecule contains a super family  <dig>  helicase at the amino terminus.
we determined the crystal structures of each region that individually folds into a distinct, rigid architecture, such as the helicase region, the nuclease domain, and the c-terminal domain containing the two repeated hhh motifs  <cit> .
the combined approach of structural and functional analyses of the nuclease regions also revealed the bipartite substrate recognition mode, which is quite likely to be conserved in the xpf/rad1/mus <dig> nuclease family.
intriguingly, the human hef ortholog was found to be an important component of the fanc core complex, which plays a crucial role in the fanconi anemia-related dna repair process responding to cross-link damage  <cit> .
in parallel with these studies, we initiated experiments to identify the branch migration activity of the holliday junction in archaea.
in p. furiosus, we successfully indentified a novel dna helicase, which we designated as hjm , according to its functional activity, holliday junction migration  <cit> .
its primary structure of  <dig> amino acids indicated that the hjm helicase belongs to sf <dig>  and was intriguingly found to share significant similarity to the helicase-like regions of the human dna polymerase Θ , hel <dig>  and drosophila mus <dig> proteins, which are all involved in dna repair.
hjm appears to be unique to archaea, because of the lack of sequence similarity to proteins from bacteria and yeast.
however, it was recently found that this structure-specific helicase preferentially binds to fork-related y-structured dnas and unwinds their double-stranded regions.
additionally, hjm partially complements the recq function in e. coli dnae486recq mutant cells in vivo  <cit> .
similar results were also reported for another archaeal homologous helicase from methanothermobacter thermautotrophicus  <cit> .
these results suggest that hjm may be a functional counterpart of the recq helicases in archaea.
the functional interaction of hjm with pcna also revealed that this helicase could participate in a reconstituted replisome to restart a stalled replication fork  <cit> .
most recently, the crystal structure of the archaeal homolog of hel <dig>  from archaeoglobus fulgidus, was determined in both the dna-free and dna-complexed states  <cit> .
another structure of hel <dig> from sulfolobus solfataricus was also reported, and a unique role for the small c-terminal domain to regulate its unwinding activity was proposed in combination with biochemical studies  <cit> .
despite these intriguing findings, many aspects of the hjm helicase, such as its actual substrates in vivo and its atp-dependent unwinding mechanism of dna duplexes, still remain elusive.
in order to obtain more detailed and clearer insights into the 3d structure and the helicase action at the atomic level, we determined the crystal structure of pfuhjm, in two apo-states at  <dig>  and  <dig>  Å resolution, in the adp-bound form at  <dig>  Å, and in the atp-analog bound form at  <dig>  Å. in combination with single particle electron microscopy of the enzyme complexed with a putative synthetic dna substrate, the atomic structure revealed clearer views of the functional and structural aspects of each domain, such as dna substrate recognition and nucleotide binding, in comparison with the structural data of the previously reported hel <dig> helicases.
RESULTS
overview of the structure
we obtained the two different pfuhjm crystals  in the nucleotide free-state, and determined their structures at  <dig>  Å  and  <dig>  Å  resolutions, respectively .
in the form  <dig> crystal, the c-terminal  <dig> residues, about two thirds of the c-terminal domain, are missing in the final model, presumably because of structural disorder.
on the other hand, in the form  <dig>  we could build the model of almost the entire molecule except for the c-terminal twenty residues.
although we could not obtain cocrystals with nucleotides, we soaked atp analogs into the crystals, and successfully determined the nucleotide complex structures.
the structures of the two apo-forms are quite similar, with a root-mean square deviation  of  <dig>  Å for the corresponding  <dig> cα atoms.
nucleotide binding to the protein also causes no large structural change; the overall rmsd value between the apo- and atpγs-soakd states is  <dig>  Å in form  <dig>  and similarly that between the apo- and amppcp-bound structures is  <dig>  Å in form  <dig>
arfree was calculated using 5% of the total reflections, which were chosen randomly and omitted from the refinement.
pfuhjm folds into five domains  with dimensions of approximately  <dig> ×  <dig> ×  <dig> Å. the protein possesses a concave surface on the front-view side and a hole  at the center of the molecule .
the two n-terminal domains  <dig>  and  <dig>  form typical helicase domains with a cleft between them, as commonly observed in the helicase superfamily.
the seven conserved helicase sequence motifs  <cit>  line the cleft walls in an arrangement similar to that observed in other helicase structures  <cit> .
domain  <dig> contains the walker a and b motifs that are widely conserved in nucleotide triphosphate hydrolases.
the form  <dig> atpγs-soaked crystal exhibited electron density corresponding to the hydrolyzed product adp, rather than the soaked atpγs, in the nucleotide-binding pocket .
on the other hand, clear electron density for the bound triphosphate was observed in the form  <dig> amppcp-soaked crystal .
regardless of the crystal form and the nucleotide binding, the structures of the walker-a motif and the surrounding region are very similar to each other.
the atp-analog amppcp is bound to the binding pocket of domain  <dig>  and it participates in several key interactions with the protein: the adenine moiety is surrounded mainly by four hydrophobic residues, ile <dig>  phe <dig>  tyr <dig>  and leu <dig>  and the two nitrogen atoms hydrogen bond with gln <dig>  in a bidentate manner.
the triphosphate is wrapped up by the walker a motif , which contains the invariant lysine residue .
the γ-phosphate faces the two acidic residues in the walker b motif .
interestingly, in the hjm structures, the conformations around the nucleotide binding sites are almost the same, including the side-chain conformations, independently of the nucleotide-binding states.
figure 1d shows a close-up view around the nucleotide binding sites of the three family members.
a comparison of the nucleotide binding pocket of hjm with those in the two hel <dig> helicases revealed that the pocket of a. fulgidus hel <dig> is partly disrupted: in the superimposed structure, the three amino acids of the a. fulgidus hel <dig> sterically clash with the atp-analog molecule bound to hjm , indicating that the a. fulgidus hel <dig> segments should undergo a structural change upon nucleotide binding.
on the other hand, the s. solfataricus enzyme exhibits a highly similar structure around the nucleotide binding site, and therefore seems to be ready to bind the nucleotide.
the c-terminal region is divided into three domains .
domain  <dig>  has a structural segment similar to the winged-helix  motif.
this motif is often used for the recognition and binding of double-stranded dna   <cit> .
in the case of hjm, however, it is unclear whether this segment is important for dna binding, because the electrostatic potential surface has few notably positive areas in this region.
consistently, in the structure of the a. fulgidus hel308-dna complex, the corresponding segment was not involved in dna binding.
domain  <dig>  folds into a seven α-helix bundle structure.
this fold seems to be unique within this helicase family, as thus far.
the c-terminal domain  <dig>  is the smallest and contains the hhh motif.
the hhh motif is present in many dna metabolizing proteins that recognize ssdna  <cit> .
indeed, the corresponding element in the a. fulgidus hel <dig> helicase interacts with dna  <cit> .
in the case of the s. solfataricus and m. thermautotrophicus hel <dig>  this domain exhibited a regulatory function to tune the processivity of its helicase activity as a molecular brake  <cit> .
pfuhjm possesses a pcna-interacting protein  box at the c-terminus, which is required for the physical interaction with pcna, and the unwinding activity of pfuhjm for the fork-structured dna is enhanced by pcna in vitro  <cit> .
however, the c-terminal segment was invisible in both the form  <dig> and form  <dig> crystals, suggesting that this segment is highly mobile.
the interaction of pfuhjm with dna is similar to that of the archaeal hel <dig> helicase
based on the a. fulgidus hel308-dna crystal structure, a dna unwinding mechanism has been proposed for this helicase  <cit> .
in this mechanism, the central helix of domain  <dig> acts as the "ratchet" formed by two key amino acid residues .
these residues form stacking interactions on base moieties of the dna, thus pushing out 3' tails of unwound dnas from the tunnel, formed by the domains  <dig>   <dig>  and  <dig>  toward an exit near domain  <dig>  an interesting feature is that the ratchet helix is located near the conserved helicase motifs, ia and ib of domain  <dig>  and iv of domain  <dig>  which are associated with atpase activity.
pfuhjm shares 30% and 37% amino acid identity with the a. fulgidus and s. solfataricus hel <dig> helicases, respectively , and the overall folding of pfuhjm is very similar to those of those proteins throughout the molecule.
pfuhjm was fitted to the protein of the a. fulgidus hel308-dna complex, with an rmsd of  <dig>  Å, for the corresponding  <dig> ca atoms, while rmsd values for individual fitting of each domain is  <dig> ,  <dig> ,  <dig> ,  <dig> , and  <dig>  Å, for domains  <dig> to  <dig>  respectively.
this indicates that the spatial arrangements of the five domains significantly differ between pfuhjm and the dna-bound a. fulgidus hel <dig>  and hence each domain of pfuhjm was separately fitted to the corresponding domain of a. fulgidus hel <dig>
first, we superimposed pfuhjm on a. fulgidus hel <dig> only using domain  <dig>  which recognizes the branch points of the substrate dna, and then the other four domains were further moved separately to the best fitted positions.
the shifts of the second fitting could correspond to the movements of each domain upon dna binding.
the second shifts, defined as the center of mass, were  <dig> ,  <dig> ,  <dig> , and  <dig>  Å for domains  <dig>   <dig>   <dig>  and  <dig>  respectively .
therefore, as suggested previously for a. fulgidus hel <dig>  <cit> , the domain rearrangement of pfuhjm should be small upon branched dna processing.
according to the scheme of the helicase-dna recognition revealed from the a. fulgidus hel308-dna complex crystal structure, we visually inspected which amino acids interact with dna in the fitted pfuhjm-dna binding model, and found out that such amino acids and their locations are substantially conserved .
furthermore, the prominent β-hairpin loop in domain  <dig>  which melts the duplex dna in a. fulgidus hel <dig>  is shorter by one residue in pfuhjm.
however, the residues that contact the dna in the a. fulgidus hel308-dna complex are substantially conserved in the sequence, and thus the protein-dna interactions at this β-hairpin loop could be quite similar between the two enzymes.
in the pfuhjm crystal structures, several segments exhibit high temperature factors, and the side chain atoms could not be assigned in the electron density maps.
among these, segments 332– <dig> and 347– <dig> are located on the possible dna interacting surfaces.
we presume that their conformational flexibility would be important for the continuous dna translocating and unwinding reaction, which is coupled with atp binding/hydrolysis.
the form <dig> crystals were obtained using ammonium sulfate as a precipitant, and it was found that five sulfate ions were bound to the protein.
notably, all of the sulfate ions lie on possible dna binding surfaces .
similarly, it was reported that phosphate ions are bound in the a. fulgidus and s. solfataricus hel <dig> structures  <cit> .
collectively, these results indicate that the sulfate/phosphate ions mimic dna backbone phosphates.
for instance, a sulfate ion is strongly bound to arg <dig> and arg <dig> in domain  <dig> of pfuhjm.
two point mutations  in pfuhjm significantly decreased the dna binding ability .
taken together, the pfuhjm structures strongly suggest that this helicase recognize branched dnas in a similar manner to that in the a. fulgidus hel308-dna complex.
therefore, it is also likely that the dna unwinding mechanism is conserved between them.
electron microscopy of pfuhjm complexed with dna
we were not successful in obtaining pfuhjm dna complex crystals.
therefore, we used single particle electron microscopy to analyze the structure of a pfuhjm in complex with a 3' overhang dna, and indeed, a 3d image was obtained at 23Å resolution .
the complex has a main body with a protruded portion.
the main body corresponds to pfuhjm, as the atomic structure of pfuhjm fits well into the electron density isosurface.
consequently, the protruded portion should correspond to the ds dna lying outside of the protein molecule.
it should be noted that the orientation of the ds dna is different between the pfuhjm-dna em structure and the a. fulgidus hel308-dna crystal structure.
the ds dna in our complex is tilted by about  <dig> degrees, as compared to that in the a. fulgidus enzyme complex.
the sequence and the secondary structure of dna used in our study is slightly different from that of the hel <dig> complex.
however, it is unlikely that this caused the difference in dna orientations.
in fact, the double-stranded region of the dna substrate, in both of the protein-dna complexes, weakly interacts with the helicases through minor contacts.
for instance, our previous electrophoresis mobility shift assay  indicated that the apparent dissociation constant of pfuhjm against ds dna was about  <dig> times higher than those against single-stranded or y-shaped dna  <cit> .
thus, the ds dna may have happened to be fixed at the distinct positions, because crystallographic and em analyses target different states of protein or protein-dna complexes.
comparison with other helicases
apart from the hel <dig> helicases, hjm is closest to a bacterial recq helicase   <cit>  in its n-terminal region .
on the other hand, the c-terminal halves of hjm and the archaeal hel308s adopt unique folds.
however, we could detect local fold similarity of domain  <dig> to transcriptional factors .
likewise domain  <dig> shares local similarity to the signal recognition particle protein   <cit> , while the c-terminal domain  <dig> shares similarity to dna excision repair protein   <cit>  and hj dna binding protein   <cit> .
the hjm structure appears to be composed of a unique combination of the domains used for dna/rna-binding or processing.
the overall structural comparison among the sf <dig> helicases is shown in figure 4b.
when these structures are aligned using the well-conserved helicase domains, the configurations of the other domains are quite variable.
this indicates that these enzymes share the two helicase domains that are fundamental for the helicase activity, while the structural and spatial arrangements of the other domains are designed to correspond to their individual dna unwinding mechanisms and substrate specificities.
homology modeling of the human polΘ helicase domain indicates structural and functional similarity to pfuhjm
the dna metabolizing proteins from archaea are both structurally and functionally similar to those from eukaryote, and therefore, the structures of archaeal proteins are useful to understand the complicated dna transaction mechanisms in eukaryotes.
in this study, we showed that the 3d structure of pfuhjm is similar to those of the a. fulgidus and s. solfataricus hel <dig> helicases, implying that these structural features could be extended to this helicase family, which includes the human polΘ and hel <dig> and drosophila mus <dig> proteins.
human polΘ is a-family dna polymerase and works in translesion dna synthesis  <cit> .
this protein is unique because it has both helicase and dna polymerase domains on a single polypeptide chain.
a homology model of the helicase domain of human polΘ, which was built using the program moe , is highly similar to the pfuhjm and hel <dig> helicases .
the model seems to be reasonable in that, as in the case of pfuhjm, the putative dna-interacting segments are both sequentially and spatially conserved in the human polΘ helicase domain.
in this domain, polΘ contains seventeen cysteine residues that are not present in pfuhjm.
the homology model indicates that twelve cysteine residues are exposed to the solvent, and that two of them form a disulfide linkage in a region corresponding to domain  <dig> of pfuhjm.
furthermore, several cysteine residues are conserved in polΘ helicase domains in eukaryotes other than human .
it is tempting to speculate that these cysteines are used for sensing oxidative stress, because a genetic analysis showed that vertebrate polΘ gene-deficient cells exhibited hypersensitivity to oxidative base damage induced by h2o <dig>  <cit> .
CONCLUSIONS
we determined the high-resolution crystal structures of the archaeal sf- <dig> helicase, pfuhjm.
although we could not obtain the protein-dna complex structures, in comparison with the previously reported hel308-dna complex, the 3d em image of the hjm-dna complex suggested that the two helicases unwind dna by essentially the same mechanism.
furthermore, homology modeling of the human dna polymerase Θ helicase domain strongly suggested the structural conservation across the domains of life.
as suggested by the structural study of the a. fulgidus hel308-dna complex, the dna unwinding mechanism itself may differ between the hel <dig> family proteins and e. coli recq and related proteins, because of the lack of the β-hairpin loop.
however, accumulating biochemical evidence suggests that pfuhjm, and probably the closely-related archaeal proteins, are the functional counterparts of the e. coli recq helicase.

 dna helicases are enzymes that translocate along dna and unwind double-stranded regions in an atp-dependent manner. they play crucial and universal roles in dna metabolism, such as dna replication and recombinational repair. as a consequence of their physiologically important functions, many reports have been published regarding protein characterization and catalytic mechanisms, including the relationships between enzymatic dysfunctions and several human genetic diseases. our on-going structural analysis of the late stage of homologous recombination, such as the ruvabc-holliday junction complex, tempted us to investigate the molecular machinery involved in holliday junction processing in eukaryotes. we also noticed that the archaeal proteins involved in dna metabolism generally have amino acid sequences and three-dimensional structures that are highly similar to their eukaryotic homolog. the proteins from the hyperthermophilic archaea, including pyrococcus furiosus, are more advantageous for structural studies than their eukaryotic counterparts, because of their remarkable thermal stability. in fact, we were the first group to successfully identify the holliday junction resolvase from archaea, which we designated as hjc, and we also determined its crystal structure by x-ray analysis. a subsequent screening study for a new protein factor that stimulates the hj resolving activity by hjc led to the identification of a new protein factor, termed hef. biochemical and sequence analyses revealed that this protein should be classified as an xpf/rad1/mus nuclease, which bears endonuclease activity specific for flap or fork structures. interestingly, the full-length hef molecule contains a super family helicase at the amino terminus. we determined the crystal structures of each region that individually folds into a distinct, rigid architecture, such as the helicase region, the nuclease domain, and the c-terminal domain containing the two repeated hhh motifs. the combined approach of structural and functional analyses of the nuclease regions also revealed the bipartite substrate recognition mode, which is quite likely to be conserved in the xpf/rad1/mus nuclease family. intriguingly, the human hef ortholog was found to be an important component of the fanc core complex, which plays a crucial role in the fanconi anemia-related dna repair process responding to cross-link damage. in parallel with these studies, we initiated experiments to identify the branch migration activity of the holliday junction in archaea. in p. furiosus, we successfully indentified a novel dna helicase, which we designated as hjm, according to its functional activity, holliday junction migration. its primary structure of amino acids indicated that the hjm helicase belongs to sf and was intriguingly found to share significant similarity to the helicase-like regions of the human dna polymerase Θ, hel and drosophila mus proteins, which are all involved in dna repair. hjm appears to be unique to archaea, because of the lack of sequence similarity to proteins from bacteria and yeast. however, it was recently found that this structure-specific helicase preferentially binds to fork-related y-structured dnas and unwinds their double-stranded regions. additionally, hjm partially complements the recq function in e. coli dnae486recq mutant cells in vivo. similar results were also reported for another archaeal homologous helicase from methanothermobacter thermautotrophicus. these results suggest that hjm may be a functional counterpart of the recq helicases in archaea. the functional interaction of hjm with pcna also revealed that this helicase could participate in a reconstituted replisome to restart a stalled replication fork. most recently, the crystal structure of the archaeal homolog of hel from archaeoglobus fulgidus, was determined in both the dna-free and dna-complexed states. another structure of hel from sulfolobus solfataricus was also reported, and a unique role for the small c-terminal domain to regulate its unwinding activity was proposed in combination with biochemical studies. despite these intriguing findings, many aspects of the hjm helicase, such as its actual substrates in vivo and its atp-dependent unwinding mechanism of dna duplexes, still remain elusive. in order to obtain more detailed and clearer insights into the 3d structure and the helicase action at the atomic level, we determined the crystal structure of pfuhjm, in two apo-states at and Å resolution, in the adp-bound form at Å, and in the atp-analog bound form at Å. in combination with single particle electron microscopy of the enzyme complexed with a putative synthetic dna substrate, the atomic structure revealed clearer views of the functional and structural aspects of each domain, such as dna substrate recognition and nucleotide binding, in comparison with the structural data of the previously reported hel helicases.  overview of the structure we obtained the two different pfuhjm crystals in the nucleotide free-state, and determined their structures at Å and Å resolutions, respectively. in the form crystal, the c-terminal residues, about two thirds of the c-terminal domain, are missing in the final model, presumably because of structural disorder. on the other hand, in the form we could build the model of almost the entire molecule except for the c-terminal twenty residues. although we could not obtain cocrystals with nucleotides, we soaked atp analogs into the crystals, and successfully determined the nucleotide complex structures. the structures of the two apo-forms are quite similar, with a root-mean square deviation of Å for the corresponding cα atoms. nucleotide binding to the protein also causes no large structural change; the overall rmsd value between the apo- and atpγs-soakd states is Å in form and similarly that between the apo- and amppcp-bound structures is Å in form arfree was calculated using 5% of the total reflections, which were chosen randomly and omitted from the refinement. pfuhjm folds into five domains with dimensions of approximately × × Å. the protein possesses a concave surface on the front-view side and a hole at the center of the molecule. the two n-terminal domains and form typical helicase domains with a cleft between them, as commonly observed in the helicase superfamily. the seven conserved helicase sequence motifs line the cleft walls in an arrangement similar to that observed in other helicase structures. domain contains the walker a and b motifs that are widely conserved in nucleotide triphosphate hydrolases. the form atpγs-soaked crystal exhibited electron density corresponding to the hydrolyzed product adp, rather than the soaked atpγs, in the nucleotide-binding pocket. on the other hand, clear electron density for the bound triphosphate was observed in the form amppcp-soaked crystal. regardless of the crystal form and the nucleotide binding, the structures of the walker-a motif and the surrounding region are very similar to each other. the atp-analog amppcp is bound to the binding pocket of domain and it participates in several key interactions with the protein: the adenine moiety is surrounded mainly by four hydrophobic residues, ile phe tyr and leu and the two nitrogen atoms hydrogen bond with gln in a bidentate manner. the triphosphate is wrapped up by the walker a motif, which contains the invariant lysine residue. the γ-phosphate faces the two acidic residues in the walker b motif. interestingly, in the hjm structures, the conformations around the nucleotide binding sites are almost the same, including the side-chain conformations, independently of the nucleotide-binding states. figure 1d shows a close-up view around the nucleotide binding sites of the three family members. a comparison of the nucleotide binding pocket of hjm with those in the two hel helicases revealed that the pocket of a. fulgidus hel is partly disrupted: in the superimposed structure, the three amino acids of the a. fulgidus hel sterically clash with the atp-analog molecule bound to hjm, indicating that the a. fulgidus hel segments should undergo a structural change upon nucleotide binding. on the other hand, the s. solfataricus enzyme exhibits a highly similar structure around the nucleotide binding site, and therefore seems to be ready to bind the nucleotide. the c-terminal region is divided into three domains. domain has a structural segment similar to the winged-helix motif. this motif is often used for the recognition and binding of double-stranded dna. in the case of hjm, however, it is unclear whether this segment is important for dna binding, because the electrostatic potential surface has few notably positive areas in this region. consistently, in the structure of the a. fulgidus hel308-dna complex, the corresponding segment was not involved in dna binding. domain folds into a seven α-helix bundle structure. this fold seems to be unique within this helicase family, as thus far. the c-terminal domain is the smallest and contains the hhh motif. the hhh motif is present in many dna metabolizing proteins that recognize ssdna. indeed, the corresponding element in the a. fulgidus hel helicase interacts with dna. in the case of the s. solfataricus and m. thermautotrophicus hel this domain exhibited a regulatory function to tune the processivity of its helicase activity as a molecular brake. pfuhjm possesses a pcna-interacting protein box at the c-terminus, which is required for the physical interaction with pcna, and the unwinding activity of pfuhjm for the fork-structured dna is enhanced by pcna in vitro. however, the c-terminal segment was invisible in both the form and form crystals, suggesting that this segment is highly mobile. the interaction of pfuhjm with dna is similar to that of the archaeal hel helicase based on the a. fulgidus hel308-dna crystal structure, a dna unwinding mechanism has been proposed for this helicase. in this mechanism, the central helix of domain acts as the "ratchet" formed by two key amino acid residues. these residues form stacking interactions on base moieties of the dna, thus pushing out 3' tails of unwound dnas from the tunnel, formed by the domains and toward an exit near domain an interesting feature is that the ratchet helix is located near the conserved helicase motifs, ia and ib of domain and iv of domain which are associated with atpase activity. pfuhjm shares 30% and 37% amino acid identity with the a. fulgidus and s. solfataricus hel helicases, respectively, and the overall folding of pfuhjm is very similar to those of those proteins throughout the molecule. pfuhjm was fitted to the protein of the a. fulgidus hel308-dna complex, with an rmsd of Å, for the corresponding ca atoms, while rmsd values for individual fitting of each domain is,,,, and Å, for domains to respectively. this indicates that the spatial arrangements of the five domains significantly differ between pfuhjm and the dna-bound a. fulgidus hel and hence each domain of pfuhjm was separately fitted to the corresponding domain of a. fulgidus hel first, we superimposed pfuhjm on a. fulgidus hel only using domain which recognizes the branch points of the substrate dna, and then the other four domains were further moved separately to the best fitted positions. the shifts of the second fitting could correspond to the movements of each domain upon dna binding. the second shifts, defined as the center of mass, were,,, and Å for domains and respectively. therefore, as suggested previously for a. fulgidus hel, the domain rearrangement of pfuhjm should be small upon branched dna processing. according to the scheme of the helicase-dna recognition revealed from the a. fulgidus hel308-dna complex crystal structure, we visually inspected which amino acids interact with dna in the fitted pfuhjm-dna binding model, and found out that such amino acids and their locations are substantially conserved. furthermore, the prominent β-hairpin loop in domain which melts the duplex dna in a. fulgidus hel is shorter by one residue in pfuhjm. however, the residues that contact the dna in the a. fulgidus hel308-dna complex are substantially conserved in the sequence, and thus the protein-dna interactions at this β-hairpin loop could be quite similar between the two enzymes. in the pfuhjm crystal structures, several segments exhibit high temperature factors, and the side chain atoms could not be assigned in the electron density maps. among these, segments 332– and 347– are located on the possible dna interacting surfaces. we presume that their conformational flexibility would be important for the continuous dna translocating and unwinding reaction, which is coupled with atp binding/hydrolysis. the form crystals were obtained using ammonium sulfate as a precipitant, and it was found that five sulfate ions were bound to the protein. notably, all of the sulfate ions lie on possible dna binding surfaces. similarly, it was reported that phosphate ions are bound in the a. fulgidus and s. solfataricus hel structures. collectively, these results indicate that the sulfate/phosphate ions mimic dna backbone phosphates. for instance, a sulfate ion is strongly bound to arg and arg in domain of pfuhjm. two point mutations in pfuhjm significantly decreased the dna binding ability. taken together, the pfuhjm structures strongly suggest that this helicase recognize branched dnas in a similar manner to that in the a. fulgidus hel308-dna complex. therefore, it is also likely that the dna unwinding mechanism is conserved between them. electron microscopy of pfuhjm complexed with dna we were not successful in obtaining pfuhjm dna complex crystals. therefore, we used single particle electron microscopy to analyze the structure of a pfuhjm in complex with a 3' overhang dna, and indeed, a 3d image was obtained at 23Å resolution. the complex has a main body with a protruded portion. the main body corresponds to pfuhjm, as the atomic structure of pfuhjm fits well into the electron density isosurface. consequently, the protruded portion should correspond to the ds dna lying outside of the protein molecule. it should be noted that the orientation of the ds dna is different between the pfuhjm-dna em structure and the a. fulgidus hel308-dna crystal structure. the ds dna in our complex is tilted by about degrees, as compared to that in the a. fulgidus enzyme complex. the sequence and the secondary structure of dna used in our study is slightly different from that of the hel complex. however, it is unlikely that this caused the difference in dna orientations. in fact, the double-stranded region of the dna substrate, in both of the protein-dna complexes, weakly interacts with the helicases through minor contacts. for instance, our previous electrophoresis mobility shift assay indicated that the apparent dissociation constant of pfuhjm against ds dna was about times higher than those against single-stranded or y-shaped dna. thus, the ds dna may have happened to be fixed at the distinct positions, because crystallographic and em analyses target different states of protein or protein-dna complexes. comparison with other helicases apart from the hel helicases, hjm is closest to a bacterial recq helicase in its n-terminal region. on the other hand, the c-terminal halves of hjm and the archaeal hel308s adopt unique folds. however, we could detect local fold similarity of domain to transcriptional factors. likewise domain shares local similarity to the signal recognition particle protein, while the c-terminal domain shares similarity to dna excision repair protein and hj dna binding protein. the hjm structure appears to be composed of a unique combination of the domains used for dna/rna-binding or processing. the overall structural comparison among the sf helicases is shown in figure 4b. when these structures are aligned using the well-conserved helicase domains, the configurations of the other domains are quite variable. this indicates that these enzymes share the two helicase domains that are fundamental for the helicase activity, while the structural and spatial arrangements of the other domains are designed to correspond to their individual dna unwinding mechanisms and substrate specificities. homology modeling of the human polΘ helicase domain indicates structural and functional similarity to pfuhjm the dna metabolizing proteins from archaea are both structurally and functionally similar to those from eukaryote, and therefore, the structures of archaeal proteins are useful to understand the complicated dna transaction mechanisms in eukaryotes. in this study, we showed that the 3d structure of pfuhjm is similar to those of the a. fulgidus and s. solfataricus hel helicases, implying that these structural features could be extended to this helicase family, which includes the human polΘ and hel and drosophila mus proteins. human polΘ is a-family dna polymerase and works in translesion dna synthesis. this protein is unique because it has both helicase and dna polymerase domains on a single polypeptide chain. a homology model of the helicase domain of human polΘ, which was built using the program moe, is highly similar to the pfuhjm and hel helicases. the model seems to be reasonable in that, as in the case of pfuhjm, the putative dna-interacting segments are both sequentially and spatially conserved in the human polΘ helicase domain. in this domain, polΘ contains seventeen cysteine residues that are not present in pfuhjm. the homology model indicates that twelve cysteine residues are exposed to the solvent, and that two of them form a disulfide linkage in a region corresponding to domain of pfuhjm. furthermore, several cysteine residues are conserved in polΘ helicase domains in eukaryotes other than human. it is tempting to speculate that these cysteines are used for sensing oxidative stress, because a genetic analysis showed that vertebrate polΘ gene-deficient cells exhibited hypersensitivity to oxidative base damage induced by h2o.  we determined the high-resolution crystal structures of the archaeal sf- helicase, pfuhjm. although we could not obtain the protein-dna complex structures, in comparison with the previously reported hel308-dna complex, the 3d em image of the hjm-dna complex suggested that the two helicases unwind dna by essentially the same mechanism. furthermore, homology modeling of the human dna polymerase Θ helicase domain strongly suggested the structural conservation across the domains of life. as suggested by the structural study of the a. fulgidus hel308-dna complex, the dna unwinding mechanism itself may differ between the hel family proteins and e. coli recq and related proteins, because of the lack of the β-hairpin loop. however, accumulating biochemical evidence suggests that pfuhjm, and probably the closely-related archaeal proteins, are the functional counterparts of the e. coli recq helicase. 
we determined the crystal structures of pfuhjm, in two apo-states and two nucleotide bound forms, at resolutions of – Å. the overall structures and the local conformations around the nucleotide binding sites are almost the same, including the side-chain conformations, irrespective of the nucleotide-binding states. previous biochemical and genetic analyses revealed that pfuhjm preferentially binds to fork-related y-structured dnas and unwinds their double-stranded regions, suggesting that this helicase is a functional counterpart of the bacterial recq helicase, which is essential for genome maintenance. it belongs to helicase superfamily and shares homology with the human dna polymerase Θ, hel and drosophila mus proteins, which are involved in dna repair. our present structures allowed reasonable homology model building of the helicase region of human polΘ, indicating the strong conformational conservation between archaea and eukarya.
 pyrococcus furiosus hjm is a structure-specific dna helicase that was originally identified by in vitro screening for holliday junction migration activity. it belongs to helicase superfamily and shares homology with the human dna polymerase Θ, hel and drosophila mus proteins, which are involved in dna repair. previous biochemical and genetic analyses revealed that pfuhjm preferentially binds to fork-related y-structured dnas and unwinds their double-stranded regions, suggesting that this helicase is a functional counterpart of the bacterial recq helicase, which is essential for genome maintenance. elucidation of the dna unwinding and translocation mechanisms by pfuhjm will require its three-dimensional structure at atomic resolution.  we determined the crystal structures of pfuhjm, in two apo-states and two nucleotide bound forms, at resolutions of – Å. the overall structures and the local conformations around the nucleotide binding sites are almost the same, including the side-chain conformations, irrespective of the nucleotide-binding states. the architecture of hjm was similar to that of archaeoglobus fulgidus hel complexed with dna. an hjm-dna complex model, constructed by fitting the five domains of hjm onto the corresponding hel domains, indicated that the interaction of hjm with dna is similar to that of hel notably, sulphate ions bound to hjm lie on the putative dna binding surfaces. electron microscopic analysis of an hjm-dna complex revealed substantial flexibility of the double stranded region of dna, presumably due to particularly weak protein-dna interactions. our present structures allowed reasonable homology model building of the helicase region of human polΘ, indicating the strong conformational conservation between archaea and eukarya.  the detailed comparison between our dna-free pfuhjm structure and the structure of hel complexed with dna suggests similar dna unwinding and translocation mechanisms, which could be generalized to all of the members in the same family. structural comparison also implied a minor rearrangement of the five domains during dna unwinding reaction. the unexpected small contact between the dna duplex region and the enzyme appears to be advantageous for processive helicase activity. 