BACKGROUND
the pd-xk superfamily of mg2+-dependent nucleases groups together protein domains initially identified in structurally characterized type ii restriction enzymes   and later found in diverse enzymes involved in dna replication, repair, and recombination, including phage λ exonuclease  <cit> , bacterial enzymes exerting ssdna nicking in the context of methyl-directed and very-short-patch dna repair: muth  <cit>  and vsr  <cit> , tn <dig> transposase tnsa  <cit> , archaeal holliday junction resolvases  hjc/hje  <cit> , phage t <dig> endonuclease i  <cit> , the xpf/rad1/mus <dig> family of nucleases that cleaves branched structures generated during dna repair, replication, and recombination  <cit> , and recb nuclease  <cit> .
all members of the pd-xk superfamily share a common structural core, comprising a mixed β-sheet of  <dig> strands flanked on both sides by α-helices  <cit> .
it serves as a scaffold for a weakly conserved active site, typically including two or three acidic residues  and one lys residue, which together form the hallmark bipartite catalytic motif d...xn.x k .
it was found that some members of the pd-xk superfamily developed different variants of the active site, in which the acidic residues or the lysine have been replaced by asn or gln   <cit> , or in which their side-chains have "migrated" to another region of the polypeptide in a way that only the spatial orientation of functional groups in the active site is maintained, but their sequence is not conserved  <cit>  .
this variability makes it difficult to identify the active site in pd-xk nucleases solely based on sequence comparisons and usually requires analysis of the three-dimensional structures .
it should be mentioned that the mechanistic/catalytic role of the active site sidechains is not fully elucidated even in the well-characterized members with known structure.
moreover, many of pd-xk nucleases contain elaborations of the common fold in the form of large insertions and terminal extensions that form additional subdomains, usually involved in oligomerization or dna-binding.
these regions often contain regular elements of secondary structure and sometimes constitute the majority of the protein, making the detection of the true core a challenging task for protein structure prediction methods .
we have previously used the comparisons of sequence profiles to detect novel members of the pd-xk superfamily  <cit> , as well as applied the sequence-structure threading to map the sequences of reases onto the crystal structures of known members  <cit> .
however, both approaches have shortcomings: the profile-profile analysis is unable to find very remote relationships when families include only a few members xk superfamily) or if the compared proteins contain large unrelated insertions, and the threading analysis works only if an experimentally solved structure is available for one of the potential homologs.
therefore, we decided to use an intermediate approach, in which the compared sequence profiles are combined with the structural information, which can be obtained either from the crystal structure  or predicted by bioinformatics methods, thereby allowing protein fold recognition without the explicit reference to an experimentally solved protein structure.
we used the recently developed method hhsearch that allows comparison of sequence alignments together with secondary structures represented as hidden markov models   <cit>  to compare the library of pd-xk nuclease families obtained previously  <cit>  with the set of domains in the clusters of orthologous groups  <cit>  and pfam  <cit>  databases.
our aim was to identify the previously unknown pd-xk domains in families of proteins that are either completely uncharacterized, or whose function is known , but the structure and phylogenetic relationships are unclear.
RESULTS
identification of new candidate pd-xk nucleases with profile hhm searches
in order to carry out a systematic search for new pd-xk nucleases, we prepared a set of multiple sequence alignments corresponding to previously identified families  <cit> .
for each family we generated a profile hmm that included the sequences and predicted secondary structure .
these profile hmms were compared with hhsearch  <cit>  to a database of profile hmms corresponding to multiple sequence alignments obtained from the cog, kog, and pfam databases, also with predicted secondary structures .
it is noteworthy, that searches initiated with most of the known pd-xk nucleases in the query dataset identified families containing sequences of other known pd-xk nucleases, thereby validating our approach and providing useful threshold values for confident identification of true members of pd-xk superfamily.
for instance, atf16a <dig>  profile detected herpesvirus alkaline exonuclease  with the e-value of 10- <dig>  pfu-hjr detected mrr with the e-value of 10- <dig>  ycjd detected vsr with the e-value of 10- <dig>  etc.
it must be noted, that some of the "best hits" to true positives exhibited poor scores, e.g.
mrr detected hjr  with the e-value of  <dig> .
as expected, our hhm-hhm comparisons revealed also numerous other potential homologs, not included in the query dataset.
based on these results, we generated a preliminary list of candidate new pd-xk subfamilies.
for further analysis, we retained only such proteins, which have not been reported as members of the pd-xk superfamily in earlier analyses carried out by ourselves or other groups .
the preliminary candidates for novel pd-xk nucleases were initially validated by reciprocal hhsearches against the database comprising both the initial query profile hmms as well as all the other cog, kog, and pfam profile hmms.
this search confirmed that for most of the candidate families, the closest homologs are either among the bona fide pd-xk enzymes or among other preliminary candidates.
it also revealed additional families, which were similar to the first set of candidates, but not to the original pd-xk queries, suggesting that they may be more remote members of the superfamily.
in reciprocal searches, a limited number of profile hmms showed significant relationship to other families, unrelated to pd-xk enzymes – such candidates were regarded as false positives and were not further analyzed.
further, each candidate family was analyzed by fold-recognition methods to evaluate its compatibility with the known pd-xk structures .
finally, preliminary comparative models were built for the parts of the sequence aligned to the template structures and the sequence conservation was analyzed in the structural context to detect potential active site residues.
altogether, our analysis revealed previously unknown membership of  <dig> families and one "orphan" protein  in the pd-xk superfamily of nucleases .
discussion
cog1322/pfam <dig>
cog1322/pfam <dig> is represented by the rmuc protein from escherichia coli, which has been implicated in limiting inversions at short-inverted repeats  <cit> .
it has been speculated that rmuc exhibits "limited homology" to human rad <dig> protein, centrosome protein pericentrin, nuclear mitotic apparatus proteins and the sbcc proteins, and therefore it may be a structural protein that protects dna against nuclease action or be itself involved in dna cleavage at the regions of dna secondary structure  <cit> .
our secondary structure predictions revealed that rmuc contains an n-terminal transmembrane helix , coiled-coil structures , a globular α/β domain , and a disordered c-terminus .
the profile hmm analysis confidently identified a relationship of the central globular α/β domain of rmuc and its homologs to the catalytic domain of the mcrc nuclease  <cit> .
this prediction was also confirmed by the fold-recognition analysis, which identified the catalytic domain of rease foki as the best modeling template, albeit with relatively low scores .
analysis of the multiple sequence alignment  reveals that proteins from the rmuc/cog1322/pfam <dig> family exhibit a hallmark pd-xk motif associated with the characteristic pattern of predicted secondary structures, which strongly suggests that rmuc is a nuclease that may cleave dna structures arising during the recombination of short-inverted repeats and thereby thwart the inversion of the internal sequence.
we suspect that the initial predictions of homology between rmuc and proteins involved in the structural maintenance of chromosome   <cit>  was due to a spurious similarity in the regions of low sequence complexity, e.g.
coiled coils.
cog4469/pfam <dig>
cog4469/pfam <dig> is represented by the coia protein from streptococcus pneumoniae, which has been implicated in the dna uptake process and recombination, but without any clues as to the molecular mechanism of its action  <cit> .
the profile hmm analysis identified a relationship  between the central region of coia  and cog <dig>  a family of predicted nucleases abundant in cyanobacteria  <cit> .
this prediction was also confirmed by the fold-recognition analysis, which aligned this domain to the recently solved structure of the cog <dig> member   and the structure of the hef nuclease  <cit> , which both belong to the pd-xk superfamily.
while the template structures exhibit unorthodox configurations of catalytic residues , members of the coia/cog4469/pfam <dig> family exhibit a more typical pd-exq variant of the active site, which has been observed in rease bglii  <cit> .
additionally, coia and its homologs possess a conserved arg residue at the same position as the swapped lys residue in cog <dig>  it will be interesting to determine if this residue is involved in substrate binding or catalysis.
we hypothesize that in competent pneumococcus cells, the coia nuclease degrades one strand of the double-stranded dna, while the other strand is imported inside the cell.
alternatively, coia may be involved in the incorporation of the foreign dna into the chromosome, but this process is more likely to be carried out by the general homologous recombination machinery, such as reca.
cog1489/pfam <dig>
cog1489/pfam <dig> is represented by the sfsa protein from e. coli, which has been implicated in sugar fermentation stimulation by the so far unknown mechanism and was shown to non-specifically bind to dna  <cit> .
the profile hmm analysis confidently identified a relationship  between the c-terminal domain of sfsa  and the yran family of previously predicted endonucleases  <cit> .
this finding was confirmed by the fold-recognition analysis, which confidently identified the vsr nuclease, a member of the pd-xk superfamily  as the best template for the sequence of the sfsa c-terminal domain .
interestingly, while the vsr nuclease exhibits a highly unorthodox active site, with the xk half-motif replaced by fxh, sfsa and its homologs  exhibit a typical version of the nuclease catalytic motif, id-evk.
the fold-recognition analysis revealed also that the n-terminal domain of sfsa  is a member of the ob-fold superfamily of nucleic acid binding proteins  <cit> .
thus, both domains of sfsa are likely to be involved in dna binding, while the c-terminal domain is likely to possess an unforeseen nuclease activity.
it will be interesting to determine whether the n-terminal or c-terminal domains or both are important for the activity of sugar fermentation stimulation and how does this activity relate to the predicted nuclease function.
or perhaps sfsa could be a hydrolase involved in another type of reaction than its homologs from the pd-xk superfamily?
deletion mutagenesis based on our prediction and site-directed mutagenesis of the pd-xk active site may help to address these questions.
kog <dig>
kog <dig> groups together  <dig> large  eukaryotic proteins, including the kia <dig> protein from h. sapiens , which has been annotated as a dna binding protein engaged in dna repair, probably due to a presence of an easily identified rad18-like cchc zinc finger in the n-terminus  <cit> .
however, no suggestions concerning the mechanism of its action have been made.
psi-blast searches revealed that kog <dig> has homologs in other eukaryota  as well as in several bacteria .
the c-terminal domain  shows significant similarity to hjr families  and exhibits the orthodox version of the pd-xk motif, perfectly conserved in all members of kog <dig> as well as in their prokaryotic relatives.
the fold-recognition analysis identified ahjr  as one of the possible templates, but with low confidence .
the analysis of predicted secondary structures and the preliminary tertiary model of kia <dig> suggest that the putative nuclease domain in kog <dig> and related proteins corresponds to the minimal core of the pd-xk fold, i.e.
only  <dig> β-strands and  <dig> α-helices  <cit>  .
combination of reciprocal hhsearches and fold-recognition carried out for the remaining part of the kia <dig> sequence revealed that kog <dig> members possess a long  α-helical domain, most probably comprising 5– <dig> tetratricopeptide-like repeats , an unknown domain  with no clear relationship to any protein families and no confident prediction from fr, and the afore-mentioned rad18-like cchc zinc finger.
the unknown domain and the zinc finger are missing from the shorter prokaryotic members.
such composition of domains suggests that kia <dig> is a dna binding protein with endonuclease activity, possibly engaged in dna repair, with a potential to bind other proteins via tpr domains.
the analysis of genomic neighborhood did not reveal any obvious associations with other conserved proteins, which could shed more light on the possible function of kia <dig> and its homologs.
cog4804/pfam <dig>
cog4804/pfam <dig> is represented by the uncharacterized and functionally not annotated protein yhcg from e. coli, whose homologs can be found in prokaryota – mostly in bacteria, but also in archaea  and bacteriophages.
the profile hmm analysis confidently identified a relationship  between the c-terminal domain of yhcg  and pfam <dig> /cog <dig>  a family of predicted recb-like nucleases  <cit> .
both yhcg and duf <dig> families exhibit the same orthodox endonuclease motif: e-d-ek.
nonetheless, our profile hmm analysis indicates that the endonuclease domain of duf <dig> is more similar to yran-like hjrs rather than to the recb-like nucleases .
this is also confirmed by the fold-recognition analysis for yhcg, which indicates the ahjr structures 1hh <dig> and 1gef/1ipi as possible templates, however with low scores .
cog4804/pfam <dig> members possess an additional n-terminal domain , predicted to be mainly α-helical, but lacking any confident matches to known structures, according to fold-recognition servers.
the analysis of genomic neighborhood did not reveal any obvious associations of the yhcg family with conserved genes.
in rickettsiae  <dig> yhcg paralogs could be found in two gene clusters.
only in methanosarcina acetivorans an yhcg homolog was found associated with a type i restriction-modification gene cluster, also with another predicted pd-xk superfamily member from cog <dig>
pfam <dig>
pfam <dig>  groups together functionally uncharacterized and not annotated proteins from tailed bacteriophages and bacteria , and is represented by the hypothetical product encoded by orf <dig> from s. aureus phage φh.
the profile hmm analysis identified this family as a relative  of ycjd-like proteins – putative members of the pd-xk superfamily identified previously  <cit> .
the fold-recognition analysis identified tnsa  as one of the possible structural templates .
the structure of phage t <dig> endonuclease i, 1fzr, had slightly better fr scores, but exhibited numerous insertions and deletions in the alignment.
nonetheless, proteins from pfam <dig> exhibit a more typical version of the catalytic motif, namely ad-dxk .
psi-blast searches using pfam <dig> members as queries revealed also a low similarity to the pd-xk domain in the res subunit of type iii restriction-modification systems  <cit>   and to cog3372/pfam <dig> , an uncharacterized family predicted to belong to the pd-xk superfamily in this work, see below.
the genomic analysis reveals that pfam <dig> members are typically found only in phage genomes or in prophages in bacterial genomes, however, without any strongly conserved neighbors.
in only one case, s. aureus subsp.
aureus col prophage l54a, the gene coding a putative dna:m <dig> a mtase  was found in a putative operon with a pfam <dig> member – hypothetical protein sacol <dig>  suggesting that together they may form a novel rm system.
we speculate that most members of this family may serve a purpose similar to that of phylogenetically unrelated endonuclease ii of phage t <dig>  namely the degradation of the host dna upon lytic infection as well as production of recombinogenic fragments  <cit>
cog5482
cog <dig> includes a few functionally uncharacterized and not annotated proteins  mainly from proteobacteria .
the profile hmm analysis suggested that they are remotely related to ahjrs .
the fold-recognition analysis has also identified the ahjr structures  as the best templates for modeling of cog <dig> sequences, with very good agreement of alignments reported by different servers despite moderate scores .
the catalytic domain exhibits the consensus motif: e-cd-elk.
interestingly, the c-terminal  <dig> aa was predicted to form a separate domain of the winged helix-turn-helix  fold, typically involved in dna binding  <cit> , which may therefore dictate the target specificity of this putative nuclease.
the precise role of these proteins remains to be determined experimentally.
pfam <dig>
pfam <dig>  groups together very small , functionally uncharacterized and not annotated proteins, exemplified by the gp <dig> protein from the listeria monocytogenes phage psa.
homologs of this protein can be found mainly in other phages and in a few bacteria from different taxa, frequently within putative prophages.
the profile hmm comparison revealed a remote relationship of pfam <dig> members to ahjrs , while sequence analysis revealed an orthodox version of the catalytic motif, d-exk.
all fold-recognition servers identified pd-xk enzymes as the best templates with high scores  identified by inbgu, score  <dig> , recu/prfa recombinase  identified by ffas, score - <dig> ).
structure prediction suggests that the members of the gp <dig> family exhibit a minimal form of the central β-sheet, with only four strands, flanked by three α-helices.
however, they seem to possess an additional β-hairpin similar to the element, which in recu forms a dimerization interface .
interestingly, the analysis of genomic neighborhood reveals that gp <dig> and its homologs are frequently associated with primases or helicases, which suggests they may be involved in dna replication.
we predict that gp <dig> and its relatives are recombinases involved in resolution of branched intermediates of phage dna undergoing replication and/or recombination, similarly to t <dig> endonuclease i.
cog <dig>
cog <dig> groups together archaeal sequences of functionally uncharacterized proteins represented by the pab <dig> protein from pyrococcus abyssi, annotated as putative transcription regulators.
as might have been expected based on the database annotation, the profile hmm analysis identified with high confidence  the helix-turn-helix motif  family like proteins: pfam <dig>  smart <dig>  cd00093) in the central part of the cog <dig> sequences.
however, using hhsearch we found that the n – terminal domain is related to the family of ahjrs  .
the fold-recognition analysis confidently confirmed ahjr  as the best template for modeling of the n-terminal domain.
preliminary modeling of the n-terminal domain  suggests that in pab <dig> and its homologs the catalytic d/e residue from the orthodox xk half-motif migrated to the α-helix following the β-strand in which that half-motif was transformed into the "kl" form.
interestingly, the profile hmm analysis predicted that the c-terminal domain in cog <dig> members may be homologous to the pd-xk nucleases as well.
this prediction was also supported by the fold recognition analysis, which found ahjr 1hh <dig>  as the potentially best template structure.
however, despite the apparent structural conservation and the presence of hydrophobic residues that confer protein stability, the typical active site is not conserved in this domain .
this suggests that the c-terminal domain was generated by intragenic duplication xk domain from another source) and then underwent degeneration.
variants of the pd-xk domains that lost the active site but presumably retained the ability to bind nucleic acids have been already described .
thus, cog <dig> represents a new family of archaeal putative nucleases with a novel domain architecture: pd-xk-hth-pd-xk.
it is noteworthy that a related architecture, namely whth-pd-xk, was observed in the type iie rease naei, in which the pd-xk domain and the whth domain, despite the unrelated folds, bind two copies of the same sequence, but only the copy bound to the nuclease domain is cleaved  <cit> .
a pd-xk-whth fusion  was also observed in the tnsa transposase  <cit> .
on the other hand, a variant of a tandemly duplicated pd-xk domain with a degenerated c-terminal repeat was found in the sau3ai enzyme  <cit> , another type iie rease, which also binds as a dimer two copies of the same sequence, but cleaves only the one bound to the pair of "active" n-terminal domains.
tandemly duplicated pd-xk domains have been also found in type iis restriction enzymes that act as monomers, bind only one asymmetrical dna target, and use different domains for the cleavage of each strand of the substrate .
it will be interesting to study the mode of action of cog <dig> members, i.e.
whether they act as monomers and dimers, how many copies of the target sequence they bind and whether they act similarly to type iie enzymes  or as nickases, which cleave only one strand of the substrate using the c-terminal active domain.
cog4127
cog <dig> groups together  <dig> uncharacterized proteins, of which only one, stm <dig> from s. typhimurium, has been annotated as a predicted restriction endonuclease.
psi-blast searches revealed that cog <dig> members have numerous homologs in the non-redundant database and are related  to the mrr family of reases  <cit> .
the relationship between the "extended" cog <dig> family and the mrr family was confirmed by the profile hmm analysis .
the fold-recognition analysis identified ahjrs  as the best templates for modeling of the c-terminal part of the protein, but failed to suggest a good structural match for the n-terminal domain of a similar size.
it is noteworthy that some members of cog <dig> contain only the n-terminal domain, but either lack the c-terminal mrr-like domain or possess another, apparently unrelated domain.
analysis of sequence conservation and predicted secondary structures suggested that the n-terminal domain also resembles the pd-xk nucleases, but without the hallmark active site, e.g.
like in the afore-mentioned cog <dig>  however, we were unable to confirm the relationship of the n-terminal domain of cog <dig> to known pd-xk nucleases either by fold-recognition or by hhsearches.
thus, the structure and function of the n-terminal domain remains to be determined experimentally; the availability of members of cog <dig> that already lack the c-terminal domain may be particularly useful.
interestingly, analyses of genomic context revealed that a few members of cog <dig> are associated with putative type i rm systems  or with a type iii rm system .
additionally, another gene cluster from d. ethenogenes contains two representatives of cog <dig>  and a member of the sfsa family .
another cog <dig> member from azotobacter vinelandii was found in the neighborhood of the putative mod subunit and a putative protein annotated as a "virulence protein" .
cog4741
cog <dig> consists of functionally uncharacterized proteins mostly from archaea, of which only one, the hypothetical product of the locus tvn <dig> from t. volcanium is annotated as "a predicted secreted endonuclease distantly related to ahjrs".
the profile hmm analysis confidently  identified the relationship of this family to the yran subfamily of pd-xk enzymes  <cit> .
the predicted nuclease domain encompasses  <dig> c-terminal residues and corresponds to the absolutely minimal core, with only  <dig> β-strands and  <dig> α-helices that serve as a scaffold for a well-conserved e-d-ek motif.
the n-terminus reveals a strongly hydrophobic stretch of residues predicted to form a transmembrane helix, which could be either used as a leader peptide to guide the translocation of the nuclease domains through the membrane or it could anchor it to the membrane.
programs for the prediction of transmembrane protein topology hmmtop  <cit> , tmap  <cit> , and tmpred  <cit>  predicted that the nuclease domain has a cytosolic orientation.
we speculate that cog <dig> members could be released to the environment as toxic agents against other cells, like endonuclease colicins , or be used to guard the cell against the uptake of foreign dna and/or to cleave the encountered nucleic acids to produce nucleotides that can be used by the host.
cog5321/pfam06319
cog5321/pfam <dig>  comprises functionally uncharacterized and not annotated short  proteins found almost exclusively in proteobacteria.
the profile hmm analysis confidently identified their relationship to ahjrs .
the fold-recognition analysis confirmed the ahjr structures  as the best templates, however with low scores .
the reason for this low confidence of fold-recognition could be due to the strong divergence of the c-terminal part of the domain and poor consensus of secondary structure prediction.
nonetheless, cog5321/pfam <dig> members exhibit the conserved orthodox ad-ek motif associated with the characteristic pattern of predicted secondary structures in the n-terminal part of the common fold, which is a strong indication that they are genuine members of the pd-xk superfamily, active as nucleases.
however, the analysis of genomic neighborhood did not provide any specific clues as to their specific function.
cog3372/pfam5626
cog3372/pfam <dig>  is represented by the uncharacterized product of pab <dig> gene from pyrococcus abyssi.
members of this family are found almost exclusively in archaea and cyanobacteria.
the profile hmm analysis identified their relationship  to pfam <dig>  family of putative endonucleases  and a more remote similarity to ahjrs .
all fold-recognition servers with the exception of fugue identified with high scores the structure of bacteriophage t <dig> endonuclease i  as the best templates for modeling of the c-terminal domain of cog <dig> .
however, the fold-recognition failed to identify any confident structural matches for the n-terminal domain .
analysis of the genomic neighborhood revealed that cog <dig> members are usually associated with hypothetical proteins from cog <dig> .
this suggests they may be involved in dna repair or recombination, but does not exclude their potential selfish character.
gp <dig> family
the gp <dig> family is represented by a small  protein gp <dig> from the t <dig> bacteriophage, annotated as a head completion protein important for the final stages of bacteriophage head assembly process and predicted to be important for the dna-mediated attachment of independently assembled head and fibers  <cit> .
to our knowledge, only a general functional prediction has been made for this protein and sequences homologous to gp <dig> have not been grouped into a pfam or cog family.
we identified them by psi-blast, using a pfam <dig>  representative as a query, with a very low e-value of  <dig> .
the hhsearch analysis carried out for the gp <dig> family confirmed their relationship to pfam <dig>   as well as to cog3372/pfam <dig>  and gp <dig> families .
exhaustive psi-blast searches  confidently reported structures of bacteriophage t <dig> endonuclease i  as best templates for the gp <dig> tertiary structure .
the consensus of fold-recognition analysis confirmed the prediction pf the pd-xk fold, with tnsa structures  as well as t <dig> endonuclease i  reported as the best structural matches.
gp <dig> is positioned in a cluster of genes encoding structural proteins important for the formation of infectious phage particles and to our knowledge has not been reported to interact with the dna, but to be necessary for the assembly of protein components of the viral particles.
the attachment of independently assembled head and fibers is however dna-dependent  .
thus, it is possible that gp <dig> may be involved in determination if the dna is packed properly or in some yet unknown process involving the dna cleavage upon the attachment of the tail and fibers to the head, for instance when one end of the packaged dna descends into the tail.
pae1662
pae <dig> from pyrobaculum aerophilum str.
im <dig> is an uncharacterized, functionally not annotated protein.
we found it during exhaustive psi-blast searches using pfam <dig>  representative as a query, with e-value only about  <dig> , but also with a hallmark motif e-ad-elk.
we found a characteristic pattern of predicted secondary structure elements associated with the putative nuclease motif, while the fold-recognition servers reported that pae <dig> is similar to several structures of pd-xk superfamily members , score:  <dig> ; 1hh <dig>  score  <dig> ; 1ob <dig>  score:  <dig> ; sam: 1hh <dig>  score:  <dig> ; 1t0f, score:  <dig> ; 1f1z, score  <dig> ; 3dpssm: score: 1hh <dig>  score:  <dig> ).
the consensus predictor pcons selected 1hh <dig>  as a preferred template.
interestingly, pae <dig> is associated with a gene encoding a putative dna:m5c mtase .
in this context it is noteworthy that a reciprocal psi-blast search revealed no homologs of pae <dig>  altogether, the association with a dna mtase, the lack of homologs detectable by database searches and a remote relationship to the pd-xk nucleases detected by fold-recognition suggest that pae <dig> is a novel putative type ii rease, which to date has not been annotated as such in rebase.
CONCLUSIONS
we have carried out a sequence/structure profile hmm search using a new method  <cit>  to identify new members of the pd-xk superfamily.
our results revealed the presence of this highly diverged nuclease domain in families of proteins implicated in dna metabolism but with unknown structure , and in proteins for which no function or structure was assigned to date .
the initial predictions were validated by protein fold-recognition, leading to preliminary structural models, which were used as platforms for identification of the potential active sites.
thus far, all known members of the pd-xk fold were found to be nucleases, mostly acting on the dna, or at least have been implicated in nucleic acid metabolism.
it cannot be excluded that some of the newly reported members may be hydrolases acting on other substrates , but we speculate that most of them would cleave dna.
the predictions reported in this article will facilitate the search for the possible substrates.
our predictions contribute to the reduction of "white spaces" on the sequence-structure-function map of the protein universe and will help to jump-start the experimental characterization of the cellular function of these putative nucleases, as well as the molecular mechanisms of their interactions with the dna.
that we identified several members of the pd-xk superfamily with very low scores suggests that more strongly diverged members still await discovery.
our analysis has provided a set of new sequence profiles that may be used to search for even more members of this important group of enzymes and will help to select targets for experimental analyses.
for instance, it would be interesting to determine high-resolution structures of the presumably "minimal" members of the superfamily  or proteins with interesting combinations of domains .
the availability of the complete catalog of nucleases and the knowledge of their mechanisms of action  in the cell under different conditions is essential for the complete understanding of mechanisms that govern the evolution and stability of the genome.
our analysis provides a small, but important step towards this aim.

 the pd-xk superfamily of mg2+-dependent nucleases groups together protein domains initially identified in structurally characterized type ii restriction enzymes and later found in diverse enzymes involved in dna replication, repair, and recombination, including phage λ exonuclease, bacterial enzymes exerting ssdna nicking in the context of methyl-directed and very-short-patch dna repair: muth and vsr, tn transposase tnsa, archaeal holliday junction resolvases hjc/hje, phage t endonuclease i, the xpf/rad1/mus family of nucleases that cleaves branched structures generated during dna repair, replication, and recombination, and recb nuclease. all members of the pd-xk superfamily share a common structural core, comprising a mixed β-sheet of strands flanked on both sides by α-helices. it serves as a scaffold for a weakly conserved active site, typically including two or three acidic residues and one lys residue, which together form the hallmark bipartite catalytic motif d...xn.x k. it was found that some members of the pd-xk superfamily developed different variants of the active site, in which the acidic residues or the lysine have been replaced by asn or gln, or in which their side-chains have "migrated" to another region of the polypeptide in a way that only the spatial orientation of functional groups in the active site is maintained, but their sequence is not conserved. this variability makes it difficult to identify the active site in pd-xk nucleases solely based on sequence comparisons and usually requires analysis of the three-dimensional structures. it should be mentioned that the mechanistic/catalytic role of the active site sidechains is not fully elucidated even in the well-characterized members with known structure. moreover, many of pd-xk nucleases contain elaborations of the common fold in the form of large insertions and terminal extensions that form additional subdomains, usually involved in oligomerization or dna-binding. these regions often contain regular elements of secondary structure and sometimes constitute the majority of the protein, making the detection of the true core a challenging task for protein structure prediction methods. we have previously used the comparisons of sequence profiles to detect novel members of the pd-xk superfamily, as well as applied the sequence-structure threading to map the sequences of reases onto the crystal structures of known members. however, both approaches have shortcomings: the profile-profile analysis is unable to find very remote relationships when families include only a few members xk superfamily) or if the compared proteins contain large unrelated insertions, and the threading analysis works only if an experimentally solved structure is available for one of the potential homologs. therefore, we decided to use an intermediate approach, in which the compared sequence profiles are combined with the structural information, which can be obtained either from the crystal structure or predicted by bioinformatics methods, thereby allowing protein fold recognition without the explicit reference to an experimentally solved protein structure. we used the recently developed method hhsearch that allows comparison of sequence alignments together with secondary structures represented as hidden markov models to compare the library of pd-xk nuclease families obtained previously with the set of domains in the clusters of orthologous groups and pfam databases. our aim was to identify the previously unknown pd-xk domains in families of proteins that are either completely uncharacterized, or whose function is known, but the structure and phylogenetic relationships are unclear.  identification of new candidate pd-xk nucleases with profile hhm searches in order to carry out a systematic search for new pd-xk nucleases, we prepared a set of multiple sequence alignments corresponding to previously identified families. for each family we generated a profile hmm that included the sequences and predicted secondary structure. these profile hmms were compared with hhsearch to a database of profile hmms corresponding to multiple sequence alignments obtained from the cog, kog, and pfam databases, also with predicted secondary structures. it is noteworthy, that searches initiated with most of the known pd-xk nucleases in the query dataset identified families containing sequences of other known pd-xk nucleases, thereby validating our approach and providing useful threshold values for confident identification of true members of pd-xk superfamily. for instance, atf16a profile detected herpesvirus alkaline exonuclease with the e-value of 10- pfu-hjr detected mrr with the e-value of 10- ycjd detected vsr with the e-value of 10- etc. it must be noted, that some of the "best hits" to true positives exhibited poor scores, e.g. mrr detected hjr with the e-value of. as expected, our hhm-hhm comparisons revealed also numerous other potential homologs, not included in the query dataset. based on these results, we generated a preliminary list of candidate new pd-xk subfamilies. for further analysis, we retained only such proteins, which have not been reported as members of the pd-xk superfamily in earlier analyses carried out by ourselves or other groups. the preliminary candidates for novel pd-xk nucleases were initially validated by reciprocal hhsearches against the database comprising both the initial query profile hmms as well as all the other cog, kog, and pfam profile hmms. this search confirmed that for most of the candidate families, the closest homologs are either among the bona fide pd-xk enzymes or among other preliminary candidates. it also revealed additional families, which were similar to the first set of candidates, but not to the original pd-xk queries, suggesting that they may be more remote members of the superfamily. in reciprocal searches, a limited number of profile hmms showed significant relationship to other families, unrelated to pd-xk enzymes – such candidates were regarded as false positives and were not further analyzed. further, each candidate family was analyzed by fold-recognition methods to evaluate its compatibility with the known pd-xk structures. finally, preliminary comparative models were built for the parts of the sequence aligned to the template structures and the sequence conservation was analyzed in the structural context to detect potential active site residues. altogether, our analysis revealed previously unknown membership of families and one "orphan" protein in the pd-xk superfamily of nucleases. discussion cog1322/pfam cog1322/pfam is represented by the rmuc protein from escherichia coli, which has been implicated in limiting inversions at short-inverted repeats. it has been speculated that rmuc exhibits "limited homology" to human rad protein, centrosome protein pericentrin, nuclear mitotic apparatus proteins and the sbcc proteins, and therefore it may be a structural protein that protects dna against nuclease action or be itself involved in dna cleavage at the regions of dna secondary structure. our secondary structure predictions revealed that rmuc contains an n-terminal transmembrane helix, coiled-coil structures, a globular α/β domain, and a disordered c-terminus. the profile hmm analysis confidently identified a relationship of the central globular α/β domain of rmuc and its homologs to the catalytic domain of the mcrc nuclease. this prediction was also confirmed by the fold-recognition analysis, which identified the catalytic domain of rease foki as the best modeling template, albeit with relatively low scores. analysis of the multiple sequence alignment reveals that proteins from the rmuc/cog1322/pfam family exhibit a hallmark pd-xk motif associated with the characteristic pattern of predicted secondary structures, which strongly suggests that rmuc is a nuclease that may cleave dna structures arising during the recombination of short-inverted repeats and thereby thwart the inversion of the internal sequence. we suspect that the initial predictions of homology between rmuc and proteins involved in the structural maintenance of chromosome was due to a spurious similarity in the regions of low sequence complexity, e.g. coiled coils. cog4469/pfam cog4469/pfam is represented by the coia protein from streptococcus pneumoniae, which has been implicated in the dna uptake process and recombination, but without any clues as to the molecular mechanism of its action. the profile hmm analysis identified a relationship between the central region of coia and cog a family of predicted nucleases abundant in cyanobacteria. this prediction was also confirmed by the fold-recognition analysis, which aligned this domain to the recently solved structure of the cog member and the structure of the hef nuclease, which both belong to the pd-xk superfamily. while the template structures exhibit unorthodox configurations of catalytic residues, members of the coia/cog4469/pfam family exhibit a more typical pd-exq variant of the active site, which has been observed in rease bglii. additionally, coia and its homologs possess a conserved arg residue at the same position as the swapped lys residue in cog it will be interesting to determine if this residue is involved in substrate binding or catalysis. we hypothesize that in competent pneumococcus cells, the coia nuclease degrades one strand of the double-stranded dna, while the other strand is imported inside the cell. alternatively, coia may be involved in the incorporation of the foreign dna into the chromosome, but this process is more likely to be carried out by the general homologous recombination machinery, such as reca. cog1489/pfam cog1489/pfam is represented by the sfsa protein from e. coli, which has been implicated in sugar fermentation stimulation by the so far unknown mechanism and was shown to non-specifically bind to dna. the profile hmm analysis confidently identified a relationship between the c-terminal domain of sfsa and the yran family of previously predicted endonucleases. this finding was confirmed by the fold-recognition analysis, which confidently identified the vsr nuclease, a member of the pd-xk superfamily as the best template for the sequence of the sfsa c-terminal domain. interestingly, while the vsr nuclease exhibits a highly unorthodox active site, with the xk half-motif replaced by fxh, sfsa and its homologs exhibit a typical version of the nuclease catalytic motif, id-evk. the fold-recognition analysis revealed also that the n-terminal domain of sfsa is a member of the ob-fold superfamily of nucleic acid binding proteins. thus, both domains of sfsa are likely to be involved in dna binding, while the c-terminal domain is likely to possess an unforeseen nuclease activity. it will be interesting to determine whether the n-terminal or c-terminal domains or both are important for the activity of sugar fermentation stimulation and how does this activity relate to the predicted nuclease function. or perhaps sfsa could be a hydrolase involved in another type of reaction than its homologs from the pd-xk superfamily? deletion mutagenesis based on our prediction and site-directed mutagenesis of the pd-xk active site may help to address these questions. kog kog groups together large eukaryotic proteins, including the kia protein from h. sapiens, which has been annotated as a dna binding protein engaged in dna repair, probably due to a presence of an easily identified rad18-like cchc zinc finger in the n-terminus. however, no suggestions concerning the mechanism of its action have been made. psi-blast searches revealed that kog has homologs in other eukaryota as well as in several bacteria. the c-terminal domain shows significant similarity to hjr families and exhibits the orthodox version of the pd-xk motif, perfectly conserved in all members of kog as well as in their prokaryotic relatives. the fold-recognition analysis identified ahjr as one of the possible templates, but with low confidence. the analysis of predicted secondary structures and the preliminary tertiary model of kia suggest that the putative nuclease domain in kog and related proteins corresponds to the minimal core of the pd-xk fold, i.e. only β-strands and α-helices. combination of reciprocal hhsearches and fold-recognition carried out for the remaining part of the kia sequence revealed that kog members possess a long α-helical domain, most probably comprising 5– tetratricopeptide-like repeats, an unknown domain with no clear relationship to any protein families and no confident prediction from fr, and the afore-mentioned rad18-like cchc zinc finger. the unknown domain and the zinc finger are missing from the shorter prokaryotic members. such composition of domains suggests that kia is a dna binding protein with endonuclease activity, possibly engaged in dna repair, with a potential to bind other proteins via tpr domains. the analysis of genomic neighborhood did not reveal any obvious associations with other conserved proteins, which could shed more light on the possible function of kia and its homologs. cog4804/pfam cog4804/pfam is represented by the uncharacterized and functionally not annotated protein yhcg from e. coli, whose homologs can be found in prokaryota – mostly in bacteria, but also in archaea and bacteriophages. the profile hmm analysis confidently identified a relationship between the c-terminal domain of yhcg and pfam /cog a family of predicted recb-like nucleases. both yhcg and duf families exhibit the same orthodox endonuclease motif: e-d-ek. nonetheless, our profile hmm analysis indicates that the endonuclease domain of duf is more similar to yran-like hjrs rather than to the recb-like nucleases. this is also confirmed by the fold-recognition analysis for yhcg, which indicates the ahjr structures 1hh and 1gef/1ipi as possible templates, however with low scores. cog4804/pfam members possess an additional n-terminal domain, predicted to be mainly α-helical, but lacking any confident matches to known structures, according to fold-recognition servers. the analysis of genomic neighborhood did not reveal any obvious associations of the yhcg family with conserved genes. in rickettsiae yhcg paralogs could be found in two gene clusters. only in methanosarcina acetivorans an yhcg homolog was found associated with a type i restriction-modification gene cluster, also with another predicted pd-xk superfamily member from cog pfam pfam groups together functionally uncharacterized and not annotated proteins from tailed bacteriophages and bacteria, and is represented by the hypothetical product encoded by orf from s. aureus phage φh. the profile hmm analysis identified this family as a relative of ycjd-like proteins – putative members of the pd-xk superfamily identified previously. the fold-recognition analysis identified tnsa as one of the possible structural templates. the structure of phage t endonuclease i, 1fzr, had slightly better fr scores, but exhibited numerous insertions and deletions in the alignment. nonetheless, proteins from pfam exhibit a more typical version of the catalytic motif, namely ad-dxk. psi-blast searches using pfam members as queries revealed also a low similarity to the pd-xk domain in the res subunit of type iii restriction-modification systems and to cog3372/pfam, an uncharacterized family predicted to belong to the pd-xk superfamily in this work, see below. the genomic analysis reveals that pfam members are typically found only in phage genomes or in prophages in bacterial genomes, however, without any strongly conserved neighbors. in only one case, s. aureus subsp. aureus col prophage l54a, the gene coding a putative dna:m a mtase was found in a putative operon with a pfam member – hypothetical protein sacol suggesting that together they may form a novel rm system. we speculate that most members of this family may serve a purpose similar to that of phylogenetically unrelated endonuclease ii of phage t namely the degradation of the host dna upon lytic infection as well as production of recombinogenic fragments cog5482 cog includes a few functionally uncharacterized and not annotated proteins mainly from proteobacteria. the profile hmm analysis suggested that they are remotely related to ahjrs. the fold-recognition analysis has also identified the ahjr structures as the best templates for modeling of cog sequences, with very good agreement of alignments reported by different servers despite moderate scores. the catalytic domain exhibits the consensus motif: e-cd-elk. interestingly, the c-terminal aa was predicted to form a separate domain of the winged helix-turn-helix fold, typically involved in dna binding, which may therefore dictate the target specificity of this putative nuclease. the precise role of these proteins remains to be determined experimentally. pfam pfam groups together very small, functionally uncharacterized and not annotated proteins, exemplified by the gp protein from the listeria monocytogenes phage psa. homologs of this protein can be found mainly in other phages and in a few bacteria from different taxa, frequently within putative prophages. the profile hmm comparison revealed a remote relationship of pfam members to ahjrs, while sequence analysis revealed an orthodox version of the catalytic motif, d-exk. all fold-recognition servers identified pd-xk enzymes as the best templates with high scores identified by inbgu, score, recu/prfa recombinase identified by ffas, score - ). structure prediction suggests that the members of the gp family exhibit a minimal form of the central β-sheet, with only four strands, flanked by three α-helices. however, they seem to possess an additional β-hairpin similar to the element, which in recu forms a dimerization interface. interestingly, the analysis of genomic neighborhood reveals that gp and its homologs are frequently associated with primases or helicases, which suggests they may be involved in dna replication. we predict that gp and its relatives are recombinases involved in resolution of branched intermediates of phage dna undergoing replication and/or recombination, similarly to t endonuclease i. cog cog groups together archaeal sequences of functionally uncharacterized proteins represented by the pab protein from pyrococcus abyssi, annotated as putative transcription regulators. as might have been expected based on the database annotation, the profile hmm analysis identified with high confidence the helix-turn-helix motif family like proteins: pfam smart cd00093) in the central part of the cog sequences. however, using hhsearch we found that the n – terminal domain is related to the family of ahjrs. the fold-recognition analysis confidently confirmed ahjr as the best template for modeling of the n-terminal domain. preliminary modeling of the n-terminal domain suggests that in pab and its homologs the catalytic d/e residue from the orthodox xk half-motif migrated to the α-helix following the β-strand in which that half-motif was transformed into the "kl" form. interestingly, the profile hmm analysis predicted that the c-terminal domain in cog members may be homologous to the pd-xk nucleases as well. this prediction was also supported by the fold recognition analysis, which found ahjr 1hh as the potentially best template structure. however, despite the apparent structural conservation and the presence of hydrophobic residues that confer protein stability, the typical active site is not conserved in this domain. this suggests that the c-terminal domain was generated by intragenic duplication xk domain from another source) and then underwent degeneration. variants of the pd-xk domains that lost the active site but presumably retained the ability to bind nucleic acids have been already described. thus, cog represents a new family of archaeal putative nucleases with a novel domain architecture: pd-xk-hth-pd-xk. it is noteworthy that a related architecture, namely whth-pd-xk, was observed in the type iie rease naei, in which the pd-xk domain and the whth domain, despite the unrelated folds, bind two copies of the same sequence, but only the copy bound to the nuclease domain is cleaved. a pd-xk-whth fusion was also observed in the tnsa transposase. on the other hand, a variant of a tandemly duplicated pd-xk domain with a degenerated c-terminal repeat was found in the sau3ai enzyme, another type iie rease, which also binds as a dimer two copies of the same sequence, but cleaves only the one bound to the pair of "active" n-terminal domains. tandemly duplicated pd-xk domains have been also found in type iis restriction enzymes that act as monomers, bind only one asymmetrical dna target, and use different domains for the cleavage of each strand of the substrate. it will be interesting to study the mode of action of cog members, i.e. whether they act as monomers and dimers, how many copies of the target sequence they bind and whether they act similarly to type iie enzymes or as nickases, which cleave only one strand of the substrate using the c-terminal active domain. cog4127 cog groups together uncharacterized proteins, of which only one, stm from s. typhimurium, has been annotated as a predicted restriction endonuclease. psi-blast searches revealed that cog members have numerous homologs in the non-redundant database and are related to the mrr family of reases. the relationship between the "extended" cog family and the mrr family was confirmed by the profile hmm analysis. the fold-recognition analysis identified ahjrs as the best templates for modeling of the c-terminal part of the protein, but failed to suggest a good structural match for the n-terminal domain of a similar size. it is noteworthy that some members of cog contain only the n-terminal domain, but either lack the c-terminal mrr-like domain or possess another, apparently unrelated domain. analysis of sequence conservation and predicted secondary structures suggested that the n-terminal domain also resembles the pd-xk nucleases, but without the hallmark active site, e.g. like in the afore-mentioned cog however, we were unable to confirm the relationship of the n-terminal domain of cog to known pd-xk nucleases either by fold-recognition or by hhsearches. thus, the structure and function of the n-terminal domain remains to be determined experimentally; the availability of members of cog that already lack the c-terminal domain may be particularly useful. interestingly, analyses of genomic context revealed that a few members of cog are associated with putative type i rm systems or with a type iii rm system. additionally, another gene cluster from d. ethenogenes contains two representatives of cog and a member of the sfsa family. another cog member from azotobacter vinelandii was found in the neighborhood of the putative mod subunit and a putative protein annotated as a "virulence protein". cog4741 cog consists of functionally uncharacterized proteins mostly from archaea, of which only one, the hypothetical product of the locus tvn from t. volcanium is annotated as "a predicted secreted endonuclease distantly related to ahjrs". the profile hmm analysis confidently identified the relationship of this family to the yran subfamily of pd-xk enzymes. the predicted nuclease domain encompasses c-terminal residues and corresponds to the absolutely minimal core, with only β-strands and α-helices that serve as a scaffold for a well-conserved e-d-ek motif. the n-terminus reveals a strongly hydrophobic stretch of residues predicted to form a transmembrane helix, which could be either used as a leader peptide to guide the translocation of the nuclease domains through the membrane or it could anchor it to the membrane. programs for the prediction of transmembrane protein topology hmmtop, tmap, and tmpred predicted that the nuclease domain has a cytosolic orientation. we speculate that cog members could be released to the environment as toxic agents against other cells, like endonuclease colicins, or be used to guard the cell against the uptake of foreign dna and/or to cleave the encountered nucleic acids to produce nucleotides that can be used by the host. cog5321/pfam06319 cog5321/pfam comprises functionally uncharacterized and not annotated short proteins found almost exclusively in proteobacteria. the profile hmm analysis confidently identified their relationship to ahjrs. the fold-recognition analysis confirmed the ahjr structures as the best templates, however with low scores. the reason for this low confidence of fold-recognition could be due to the strong divergence of the c-terminal part of the domain and poor consensus of secondary structure prediction. nonetheless, cog5321/pfam members exhibit the conserved orthodox ad-ek motif associated with the characteristic pattern of predicted secondary structures in the n-terminal part of the common fold, which is a strong indication that they are genuine members of the pd-xk superfamily, active as nucleases. however, the analysis of genomic neighborhood did not provide any specific clues as to their specific function. cog3372/pfam5626 cog3372/pfam is represented by the uncharacterized product of pab gene from pyrococcus abyssi. members of this family are found almost exclusively in archaea and cyanobacteria. the profile hmm analysis identified their relationship to pfam family of putative endonucleases and a more remote similarity to ahjrs. all fold-recognition servers with the exception of fugue identified with high scores the structure of bacteriophage t endonuclease i as the best templates for modeling of the c-terminal domain of cog. however, the fold-recognition failed to identify any confident structural matches for the n-terminal domain. analysis of the genomic neighborhood revealed that cog members are usually associated with hypothetical proteins from cog. this suggests they may be involved in dna repair or recombination, but does not exclude their potential selfish character. gp family the gp family is represented by a small protein gp from the t bacteriophage, annotated as a head completion protein important for the final stages of bacteriophage head assembly process and predicted to be important for the dna-mediated attachment of independently assembled head and fibers. to our knowledge, only a general functional prediction has been made for this protein and sequences homologous to gp have not been grouped into a pfam or cog family. we identified them by psi-blast, using a pfam representative as a query, with a very low e-value of. the hhsearch analysis carried out for the gp family confirmed their relationship to pfam as well as to cog3372/pfam and gp families. exhaustive psi-blast searches confidently reported structures of bacteriophage t endonuclease i as best templates for the gp tertiary structure. the consensus of fold-recognition analysis confirmed the prediction pf the pd-xk fold, with tnsa structures as well as t endonuclease i reported as the best structural matches. gp is positioned in a cluster of genes encoding structural proteins important for the formation of infectious phage particles and to our knowledge has not been reported to interact with the dna, but to be necessary for the assembly of protein components of the viral particles. the attachment of independently assembled head and fibers is however dna-dependent. thus, it is possible that gp may be involved in determination if the dna is packed properly or in some yet unknown process involving the dna cleavage upon the attachment of the tail and fibers to the head, for instance when one end of the packaged dna descends into the tail. pae1662 pae from pyrobaculum aerophilum str. im is an uncharacterized, functionally not annotated protein. we found it during exhaustive psi-blast searches using pfam representative as a query, with e-value only about, but also with a hallmark motif e-ad-elk. we found a characteristic pattern of predicted secondary structure elements associated with the putative nuclease motif, while the fold-recognition servers reported that pae is similar to several structures of pd-xk superfamily members, score:; 1hh score; 1ob score:; sam: 1hh score:; 1t0f, score:; 1f1z, score; 3dpssm: score: 1hh score: ). the consensus predictor pcons selected 1hh as a preferred template. interestingly, pae is associated with a gene encoding a putative dna:m5c mtase. in this context it is noteworthy that a reciprocal psi-blast search revealed no homologs of pae altogether, the association with a dna mtase, the lack of homologs detectable by database searches and a remote relationship to the pd-xk nucleases detected by fold-recognition suggest that pae is a novel putative type ii rease, which to date has not been annotated as such in rebase.  we have carried out a sequence/structure profile hmm search using a new method to identify new members of the pd-xk superfamily. our results revealed the presence of this highly diverged nuclease domain in families of proteins implicated in dna metabolism but with unknown structure, and in proteins for which no function or structure was assigned to date. the initial predictions were validated by protein fold-recognition, leading to preliminary structural models, which were used as platforms for identification of the potential active sites. thus far, all known members of the pd-xk fold were found to be nucleases, mostly acting on the dna, or at least have been implicated in nucleic acid metabolism. it cannot be excluded that some of the newly reported members may be hydrolases acting on other substrates, but we speculate that most of them would cleave dna. the predictions reported in this article will facilitate the search for the possible substrates. our predictions contribute to the reduction of "white spaces" on the sequence-structure-function map of the protein universe and will help to jump-start the experimental characterization of the cellular function of these putative nucleases, as well as the molecular mechanisms of their interactions with the dna. that we identified several members of the pd-xk superfamily with very low scores suggests that more strongly diverged members still await discovery. our analysis has provided a set of new sequence profiles that may be used to search for even more members of this important group of enzymes and will help to select targets for experimental analyses. for instance, it would be interesting to determine high-resolution structures of the presumably "minimal" members of the superfamily or proteins with interesting combinations of domains. the availability of the complete catalog of nucleases and the knowledge of their mechanisms of action in the cell under different conditions is essential for the complete understanding of mechanisms that govern the evolution and stability of the genome. our analysis provides a small, but important step towards this aim. 
the low degree of amino acid conservation hampers the possibility of identification of new members of the pd-xk superfamily based solely on sequence comparisons to known members. our results contributes to the reduction of "white spaces" on the sequence-structure-function map of the protein universe and will help to jump-start the experimental characterization of new nucleases, of which many may be of importance for the complete understanding of mechanisms that govern the evolution and stability of the genome. therefore, we used a recently developed method hhsearch for sensitive detection of remote similarities between protein families represented as profile hidden markov models enhanced by secondary structure. we carried out a comparison of known families of pd-xk nucleases to the database comprising the cog and pfam profiles corresponding to both functionally characterized as well as uncharacterized protein families to detect significant similarities. thus, it is tempting to speculate that among the uncharacterized protein families, there are potential nucleases that remain to be discovered, but their identification requires more sensitive tools than traditional psi-blast searches. in this article, we report identification of the pd-xk nuclease domain in numerous proteins implicated in interactions with dna but with unknown structure and mechanism of action and in proteins for which no function was assigned to date. the initial candidates for new nucleases were subsequently verified by sequence-structure threading, comparative modeling, and identification of potential active site residues.
 the pd-xk nuclease superfamily, initially identified in type ii restriction endonucleases and later in many enzymes involved in dna recombination and repair, is one of the most challenging targets for protein sequence analysis and structure prediction. typically, the sequence similarity between these proteins is so low, that most of the relationships between known members of the pd-xk superfamily were identified only after the corresponding structures were determined experimentally. thus, it is tempting to speculate that among the uncharacterized protein families, there are potential nucleases that remain to be discovered, but their identification requires more sensitive tools than traditional psi-blast searches.  the low degree of amino acid conservation hampers the possibility of identification of new members of the pd-xk superfamily based solely on sequence comparisons to known members. therefore, we used a recently developed method hhsearch for sensitive detection of remote similarities between protein families represented as profile hidden markov models enhanced by secondary structure. we carried out a comparison of known families of pd-xk nucleases to the database comprising the cog and pfam profiles corresponding to both functionally characterized as well as uncharacterized protein families to detect significant similarities. the initial candidates for new nucleases were subsequently verified by sequence-structure threading, comparative modeling, and identification of potential active site residues.  in this article, we report identification of the pd-xk nuclease domain in numerous proteins implicated in interactions with dna but with unknown structure and mechanism of action and in proteins for which no function was assigned to date. our results contributes to the reduction of "white spaces" on the sequence-structure-function map of the protein universe and will help to jump-start the experimental characterization of new nucleases, of which many may be of importance for the complete understanding of mechanisms that govern the evolution and stability of the genome. 