BACKGROUND
restriction-modification systems  usually consist of at least two enzymatic activities: a restriction endonuclease , which recognises and cleaves a specific dna sequence, and a cognate dna methyltransferase  that can methylate the same target site, thereby preventing the cleavage of host dna by the rease  <cit> .
on the basis of subunit composition, cofactor requirements, and the mode of recognition and cleavage, rm systems have been traditionally classified into three major types: i, ii and iii.
type iv has been re-introduced to describe a heterogeneous group of enzymes cleaving only modified dna, with ill-defined sequence specificity  <cit> .
type i and type iii reases are complex multisubunit molecular machines that utilize atp and perform either dna modification or cleavage  <cit> .
type ii reases are simpler: they recognise short, 4– <dig> bp, nucleotide sequences and cleave phosphodiester bonds in dna within their recognition sites or at up to a distance of  <dig> bp from them  <cit> .
in most cases, methylation and cleavage are divided between two separate enzymes.
more than  <dig> various reases that recognise more than  <dig> different nucleotide sequences have been described as members of this large and divergent class of enzymes  <cit> .
most of them have been isolated from mesophilic microorganisms and only a few from hyperthermophiles  <cit>  and thermophiles, in particular from the genus thermus  <cit> .
in order to account for their structural and functional heterogeneity type ii reases have been divided into multiple subtypes, often overlapping with each other  <cit> .
for example, a growing group of atypical type ii reases, represented by bifunctional enzymes with rease and mtase activities within a single polypeptide , is a subclass of subtype iis enzymes that recognise a specific dna sequence and cleave outside it at a defined distance, within any sequence.
examples of this group include eco57i  <cit> , haeiv  <cit> , aloi  <cit> , bsemii  <cit>  and enzymes from the thermus sp.
family: tspgwi , taqii , tspdti  and tth111ii   <cit> .
in this paper we describe the cloning of the tspgwi gene based on degenerated primers pcr protocol, site-directed mutagenesis, expression and purification of recombinant and mutant proteins followed by comparative biochemical and genetic analysis.
the constructed mutants are more suitable than native tspgwi in gene cloning methodology, as the cleavage and modification functions were separated into distinct, active proteins.
tspgwi-like enzymes  <cit>  are difficult to fit unambiguously into the conventional classification, as their unique structural and functional features can be found in rease sub-types iia, iic, iis, iif, iih and type i.
RESULTS
sequencing, cloning and expression of the tspgwi gene
neither the biochemical selection for methylation phenotype approach nor the "white-blue" screen for dna damage/modification to yield recombinant clones were successful , due to tspgwi low activity and lack of complete cleavage of the plasmid dna.
we devised a cloning protocol for thermophilic rease without accompanying  mtase, which includes two stages:  a gene nucleotide sequence determination starting from internal amino acid sequences of rease proteolytic fragments followed by various pcr methods using combination of degenerated and non-degenerated primers.
the primers are designed using probability of codon usage from other closely related bacteria and  direct in-frame insertion of amplified orf into tightly temperature-regulated expression vector, taking advantage of low temperature cultivation at non-permissive conditions, which both prevent rease expression and suppresses activity of any leaking thermophilic rease.
the tspgwi gene nucleotide sequence determination was initiated from very limited amino acid sequence information about the n-termini of native tspgwi proteolytic internal fragments.
we were unable to determine the amino acid sequence of the intact protein, thus homogeneous tspgwi preparation was subjected to limited proteolysis, yielding  <dig> major bands and more than  <dig> minor bands, as determined by sds/page .
the immobilised tpck-trypsin was readily removed by centrifugation; hence, the resulting bands were derivatives of tspgwi protein only.
two of the predominant bands of approximate sizes  <dig> kda and  <dig> kda were subjected to n-terminal sequencing .
based on the obtained short 19- and 10- amino acid sequences gpiggggspeaqlvplitr and effterlaqe, sets of non-degenerated and degenerated primers, forming alternative pairs, were designed.
the primers were designed arbitrarily as based on back-translated amino acid sequence using codon usage data from orfs of thermus sp.
genes available in genbank.
the high gc content of thermus genes was also considered.
sets of primer pairs used were either with low degeneracy and non-degenerated, containing incorporated codons of the assumed highest probability to exist in thermus sp.
one such pair yielded a pcr fragment containing an internal portion of the tspgwi gene.
combination of pcr with degenerated primers using thermus sp.
gw genomic dna as a template as well as casette-ligation-pcr using pbr <dig> dna as the known sequence donor for non-degenerated primers, yielded a sequence of  <dig> bp contig with the complete tspgwi gene.
each contig strand was sequenced; in addition, regions containing discrepancies were sequenced several times under different conditions.
the tspgwi gene orf coding for the rease-mtase bifunctional protein is  <dig> bp in length coding for the  <dig> amino acid polypeptide .
the calculated molecular weight of the tspgwi is  <dig>  <dig> da, which perfectly matches data from sds/page  and size exclusion chromatography of the native protein  <cit> .
the calculated isoelectric point of recombinant tspgwi is  <dig>  , very similar to calculated isoelectric point of recombinant tspdti of  <dig>  .
no sequence similarity to any mtase or dna-binding protein was found in the orf flanking regions.
the orf begins with the atg start codon and contains  <dig> putative upstream ribosome binding sites : - <dig> 5'-agg and - <dig> 5'-agga.
the orf is extremely gc rich:  <dig> %.
to ensure that no errors are incorpoated during tspgwi gene amplification, all the pcr reactions were performed with a proofreading blend of taq and pfu dna polymerases.
sequencing was repeated three times for each strand, both directly for the independent pcr products and for the cloned the gene fragments.
native tspgwi purification was reported by us previously  <cit> .
since tspgwi activity was hardly noticeable in its natural host thermus gw, the development of an extensive isolation procedure was essential, which included: polyethyleneimine precipitation of nucleic acids, ammonium sulfate fractionations followed by five chromatographic steps and size-exclusion chromatography  <cit> .
to the contrary, recombinant tspgwi and its mutants were purified to homogeneity using much simplified procedure with the tspgwi protein expressed in e. coli.
the protocol included heat-denaturing e. coli proteins, polyethyleneimine precipitation of nucleic acids, ammonium sulfate fractionations, followed by two chromatographic steps.
interestingly, in spite of the cloning in the presence of a strong pr promoter, the protein becomes detectable in induced cells only after  <dig> h of growth under non-permissive conditions, and keeps accumulating until the late stationary phase, even after  <dig> h cultivation at 42°c .
this is probably due to the combination of the following factors:  gc rich orf sequence distant from e. coli optimum codon usage,  slow transcription of the extremely gc-rich tspgwi gene,  the presence of numerous putative hairpin structures within the gene and  the very large size of the protein to be translated.
nevertheless, optimisation of expression culture growth/induction conditions yielded adequate amounts of tspgwi .
it was also observed that the initially homogenous protein, after prolonged storage at -20°c, undergoes minor degradation .
bioinformatic analyses of tspgwi: prediction of domains and functional motifs
isolation and sequencing of the tspgwi gene enabled detailed analysis of its amino acid sequence.
searches of rease sequences deposited in rebase revealed an overall similarity to a number of genuine and putative type iic enzymes, including the previously characterised nuclease taqii  <cit>  .
two other members of the thermus family, i.e.
tspdti  revealed the expected significant similarity of the central region of tspgwi  to the catalytic domains of known dna:m6a mtases.
no significant similarity of the terminal regions  to any protein family was observed in simple sequence searches.
nonetheless, multiple sequence alignment of tspgwi homologues revealed the presence of a candidate pd-xk motif , resembling the active site of many reases and other nucleases  <cit> .
we carried out a protein fold-recognition  analysis  to confirm this preliminary prediction for the n-terminal region, as well as to identify potential homologues and predict the structure for the c-terminal region.
since the fr method is designed to identify remote homology and predict structure for domain-size sequence fragments , the tspgwi sequence was split into a series of overlapping segments and submitted to the genesilico metaserver  <cit> .
the fr analysis of the tspgwi sequence identified the structures of a type i dna:m6a mtase m. ecoki hsdm subunit , a putative hsdm subunit of an experimentally uncharacterised rm system bthvorf4518p , and type ii dna:m6a mtase m. taqi  as the best structural templates for the central region comprising residues 130– <dig> of tspgwi .
interestingly, the fr alignments for this region revealed not only the s-adenosylmethionine -binding/catalytic mtase domain , but also a helical domain characteristic of the n-terminus of the hsdm subunit .
for the n-terminal region of tspgwi  the fr methods also supported the prediction of the pd-xk domain, albeit with scores below the level of significance .
the closest match on the sequence level  by the hhsearch method for profile-profile searches was the n-terminal pd-xk domain of the hsdr subunit from type i rm systems  <cit> .
this prediction made for the n-terminal domain of tspgwi further supports the classification of the catalytic domain of taqii-related enzymes as a member of the pd-xk fold  <cit> .
fr methods failed to report any significant matches to known structures for the c-terminal part of the tspgwi sequence .
interestingly, the c-terminal region was predicted to be almost entirely disordered, with only a short segment comprising residues 960– <dig> predicted to form a stable helical secondary structure.
this segment  shows sequence similarity to the c-terminus of taqii.
we found no similarity of this region to target recognition domains  of any mtase family; nevertheless, we speculate that it might play an analogous role.
altogether, the results of our fr analysis suggest that type ii enzyme tspgwi is a fusion protein comprising a tandem arrangement of type i-like domains: a pd-xk nuclease domain related to the corresponding domains in hsdr subunits, a hsdm-like module comprising a helical domain and a highly conserved adomet-binding/catalytic mtase domain, and a c-terminal extension, presumably responsible for dna sequence recognition .
on the other hand, tspgwi lacks other domains characteristic of type i rm enzymes, including the atp-dependent translocase module of the hsdr subunit and c-terminal domains of either hsdm or hsdr, which are important for interactions between type i subunits  <cit> .
this domain organization resembles the structure of the recently analysed mmei enzyme  <cit> , to which tspgwi exhibits only very limited sequence similarity, restricted primarily to the central mtase domain .
enzymatic properties of tspgwi
purified preparation of native tspgwi protein was used for the detailed study of biochemical properties, reaction conditions and various cofactors influencing either dna cleavage or the methylation activity of the enzyme.
the apparent molecular mass of the wt recombinant protein under denaturing conditions was found to be  <dig> kda, adequately to the molecular mass of tspgwi isolated from thermus sp.
gw  <cit> .
comparative assay of activities of both enzymes revealed no difference .
control mock-purification from e. coli did not show any of the described activities .
wt recombinant protein was also subjected to analytical gel filtration in a buffer with a composition close to the physiological, containing  <dig> mm mgcl <dig> .
the experiment revealed that the native endonuclease molecular mass is 110– <dig> kda, indicating that under physiological conditions the protein exists as a monomer  <cit> .
the temperature activity range extends from 42°c to 89°c , with the maximum observed at 60–70°c.
incubation at 37°c resulted in 5% activity only .
tspgwi is inactivated by  <dig> min incubation at 89°c and ph greatly affects activity, which drops sharply below ph  <dig>  and above  <dig>  , with the maximum at  <dig> – <dig>  .
the optimal ionic strength is below  <dig> mm kcl; at  <dig> mm only 50% of activity remains .
the optimum restriction reaction conditions for tspgwi endonuclease in vitro are in  <dig> mm tris-hcl ph  <dig>  at 65°c,  <dig> mm mgcl <dig>   <dig> mm dtt.
tspgwi maintains the absolute requirement for mg2+ for cleavage activity, with the optimum between  <dig> and  <dig> mm .
mg2+ ions can be effectively substituted with mn2+, fe2+ and partially with co2+ ions .
remarkably, ca2+ ions do not support tspgwi cleavage activity, while in contrast, they stimulate methylation activity of the enzyme .
no difference was observed in response to divalent cations by both recombinant and native wt tspgwi .
unlike most of the other bifunctional type iic reases/mtases  <cit>   <dig> μm adomet slightly inhibits the rease activity of tspgwi enzyme , while s-adenosylhomocysteine  has essentially no effect on lambda dna cleavage .
the restriction activity of tspgwi is neither inhibited nor stimulated by spermidine or atp .
as adomet is a co-substrate for the tspgwi mtase activity, it indirectly promotes dna modification rather than cleavage.
the presence of refractory sites, observed in vitro without adomet in the reaction buffer, could alternatively be explained by partial dna methylation due to certain enzyme-bound adomet carry over during purification.
however, since eight purification steps were used  <cit> , this possibility is rather remote.
in addition, partial protection should be uniform among different recognition sites, instead of observed site preference.
a recombinant tspgwi gene alone can be cloned into e. coli without an additional mtase  and stably maintained at 28°c.
a much reduced restriction activity < 5% at low temperature and the presence of cellular adomet and ca2+ appears to favour methylation in vivo.
taken together, the adomet and ca2+ effects as well as apparent preference for the opposite arrangement of recognition sites may provide an effective mechanism for controlling undesired restriction in vivo, thus removing the necessity for the existence of a separate tspgwi mtase.
another conclusion from the above results is that dna methylation and dna cleavage events occur by different mechanisms, in parallel and essentially independently.
as a result, some of the target sites become protected, even under conditions favouring cleavage.
such concurrent methylation and restriction activities seem to resemble the behaviour of another bifunctional type iic enzyme aloi  <cit> .
to further verify the marked independence of cleavage and methylation as well as to define the functional motifs within tspgwi , a series of amino acid replacements by site-directed mutagenesis was performed on tspgwi.
we targeted three residues in the catalytic motif "pd-xk" of the nuclease domain , and two residues in the mtase domain of tspgwi: in the adomet-binding motif i "dpavgtg"  and in the main mtase catalytic motif iv: "nppy" .
all the mutant proteins used were of high purity, isolated using simplified recombinant tspgwi protocol and their activity in vitro was analysed .
some of the mutants lost their rease activity, while maintaining the wild type  level of methylation; others lost their mtase activity while retaining the ability to cut dna, although at reduced efficiency .
the mtase activities of both the wt  tspgwi and mutant proteins were dependent on the presence of divalent cations .
further experiments are in progress to evaluate complex effect of ca2+ and mg2+.
dna substrate preference of tspgwi
under the variety of conditions initially tested no complete tspgwi digestion was obtained; instead a stable partial cleavage pattern was observed.
it is probably due to the preferential site cleavage in various dna substrates.
among them puc <dig> was found to be the most efficiently cleaved supercoiled dna substrate  <cit> .
however, even in the case of puc <dig> small fraction of molecules remained uncut, as determined by agarose gel electrophoresis of overloaded digestion samples and by transformation assay .
the cleavage depends on the substrate used: it varies from the near complete digestion of two tspgwi sites in puc <dig> dna  to the trace cleavage of a subset of refractive sites in pbr <dig> and pacyc <dig> plasmids  <cit> .
the refractory site phenomenon was preliminary evaluated here using two pcr substrates, containing either single  or double  tspgwi recognition sites.
on a  <dig> bp pcr fragment containing a single recognition site for tspgwi and taqii, no detectable cleavage was observed in the case of tspgwi , while closely related taqii exhibits complete digestion .
however, when the  <dig> bp fragment is cloned into puc <dig> plasmid , the tspgwi site becomes cleavable, as evident from the appearance of predicted  <dig> bp and  <dig> bp bands .
the  <dig> bp pcr fragment contained two divergent tspgwi recognition sites, separated by a  <dig> bp spacer, which may allow two tspgwi molecules to bind simultaneously both recognition sequences located on the same face of dna, without steric interference, while being close enough to allow for protein-protein interaction.
it turned out to be the most completely cleaved substrate among those we tested .
further experiments are in progress to carefully evaluate the observed phenomenon, as the presence of two sites on the same dna molecule may be an intrinsic part of the tspgwi scission mechanism.
the requirement for the presence of two recognition sites of opposite orientation for dna cleavage is a characteristic feature of type iii reases  <cit> .
thus, the tspgwi-like enzymes  show a mixture of features characteristic of type i, ii, or iii rm systems.
type iii enzymes methylate only one strand, regardless of the number and orientation of cognate sites, while still providing adequate protection against cleavage.
within type ii , bspmi is the one rease described to date that strongly prefers a tandem, head-to-tail orientation of double sites  <cit> .
another subtype iis endonuclease foki, has a preference towards a pair of sites with a tandem head-to-tail orientation  <cit> .
the divergent orientations of the recognition sites may ensure cleavage of both sequences, as was proposed for type iis reases bsgi, bpmi and acc36i  <cit> , which are, however, rather remotely related to tspgwi .
nevertheless, these enzymes retain the ability to cut single sites, although with a reduced efficiency compared to the tandem sites.
tspgwi is unable to cut a single site, unlike bsgi, bpmi, acc36i and the closely related taqii rease.
it is intriguing that taqii rease, despite the very high amino acid sequence similarity to tspgwi  and in spite of numerous biochemical similarities  <cit>  cleaves single sites efficiently.
the ability to cleave a single recognition sequence may be correlated with the existence of rease activity stimulation by adomet.
although this cofactor has no effect on tspgwi rease activity, it does stimulate taqii rease approximately 2– <dig> fold .
as we have pointed out previously  <cit> , the new thermus sp.
family including: tspgwi, taqii, tspdti and tth111ii, is internally diversified.
even though these enzymes exhibit striking similarities in protein size, quaternary structure, recognition sequences, cleavage distances and related origin from various thermus strains, they display marked differences in their response to adomet and to the arrangement requirements of different recognition sites.
interestingly, this group of enzymes seems to be also heterogenous at the level of amino acid sequence similarity, as tspgwi and taqii seem to be closely related to each other, but only remotely to the two other enzymes tspdti and tth111ii.
this latter pair of enzymes appear to be closely related to each other, but exhibit a different pattern of similarities compared to the tspgwi-taqii pair.
however, all four enzymes share significant biochemical similarities, including related recognition and cleavage sequences, very large molecular weights of around  <dig> kda, monomeric protein organization  <cit>  and are negatively charged with calculated isoelectric points of  <dig>  ,  <dig>  ,  <dig>   and  <dig>   .
although tspgwi is not particularly closely related to any known subtype iib rease  <cit> , the similarity of the domain structure and the mode of action of these enzymes  suggests that they have evolved from a common ancestor.
thus, type iib enzymes forming tight dimers and taqii-like enzymes that hardly dimerise if at all, may represent two extremes of an evolutionary landscape, where different enzymes have specialised towards either separation or pairing of recognition sites by developing subunit-subunit interactions of different strength.
in this classification, tspgwi would be located "between" type iib enzymes and taqii.
endonucleolytic activity of tspgwi wt and the mutant proteins
site-directed mutagenesis was used to test whether the amino acids predicted by bioinformatics were essential for the mtase and rease catalytic activity of the tspgwi enzyme.
to this end, variants of the tspgwi protein with substitutions in the putative pd-xk domain and in the mtase domain were constructed by saturation site-directed mutagenesis.
the mutant proteins were purified as described for recombinant tspgwi protein and subjected to in vitro biochemical experiments.
to calibrate the protein concentrations, each variant was assayed, then diluted with the storage buffer to achieve equal protein concentration in every assay.
the purity and concentration of the diluted proteins were further assessed by poliacrylamide gel electrophoresis .
the cleavage reaction was performed at 65°c for one hour and overnight.
mutants in the pd-xk domain d58a, d58r, e72a, e72l, e72n, e72r, e72s, k74a, k74g, k74p, k74r did not show any dna cleavage activity under optimal buffer conditions at a concentration of  <dig> μg/ml, the equivalent of  <dig> u wt tspgwi per reaction.
higher enzyme concentrations could not be tested, as the specific activity of wt tspgwi rease is relatively low , and increasing the amount of protein per reaction caused inhibition because of its non-specific binding to the substrate dna.
only one mutant variant e72d retained its rease activity – its cleavage rate was even slightly higher than that of the wt protein.
thus, conservative substitution of aspartic acid for glutamic acid in the  site keeps the pd-xk motif of tspgwi fully functional.
it also confirms the key role of acidic amino acid in position  <dig> in catalysis.
since preferential cleavage of substrate dna by native tspgwi was observed at concentrations as low as  <dig>  μg/ml , the decrease in rease activity of tspgwi mutants was estimated to be more than 200-fold, thus confirming the predicted role of the amino acids d <dig>  e <dig> and k <dig> in catalysis.
surprisingly, the mutants in the mtase domain – v356m  and n473a  – also displayed a significantly reduced cleavage activity in vitro in comparison to the wt tspgwi protein.
the double mutant v356m/n473a exhibited no detectable rease activity in vitro.
taqii, the closest relative of tspgwi, exhibits the "dpamgtg" variant of motif i. this observation, together with the fact that taqii rease activity is strongly stimulated by adomet , points to the complex role of adomet in both enzymatic activities and suggests that some interaction occurs between the nuclease and mtase domains in the course of the cleavage reaction.
the substitution of methionine for valine in the adomet-binding motif of tspgwi  could disturb adomet-affected communication between the nuclease domain and the mtase domain.
the nature of the communication between the domains remains to be defined by further analyses.
mtase activity of tspgwi wt and the mutant proteins
mutant proteins were further analysed for their methylation activity.
in vitro methylation reactions were performed using wt tspgwi enzyme and mutant proteins  in the dna protection assay as described in materials and methods.
two types of substrate dna were used: pbr <dig> plasmid dna and the  <dig> bp pcr fragment.
the results presented in table  <dig> and table  <dig> demonstrate that the mutant n473a  is unable to modify the tspgwi target sequence.
interestingly, the n473a substitution not only completely eliminates the methylation activity, but also decreases the cleavage activity of tspgwi; even so, the resulting protein retains sufficient activity to be considered a functional rease.
thus, both the rease and mtase activities of tspgwi were uncoupled, and rease-only or mtase-only tspgwi variants were obtained.
this is also of high practical methodological value in gene cloning technology, allowing to use tspgwi mutants as traditional class-ii enzymes for dna cleavage or methylation.
mutants with d58a  or v356m  substitutions exhibit mtase activity at the same level as the wt enzyme, despite the total or partial loss of their ability to cleave the substrate dna.
we observed that d58a slightly increased the modification activity  in comparison to the wt protein .
this could have been due to the absence of a competing rease activity in this variant.
interestingly, mg2+ at  <dig> mm concentration tested has a strong stimulatory effect on the mtase activity of the d58a mutant .
in the case of wt tspgwi, mg2+ is utilised for cleavage, which hinders its effect on the mtase activity.
thus, the d58a mutation demonstrates that the competing mtase and rease activities both share the same mg2+ cofactor.
however, it cannot be ruled out that the presence of divalent cations stabilizes not perfectly folded mutant protein tspgwi, thus contributing non-specifically to retaining their enzymatic activities.
taken together with the observed inhibition of the tspgwi rease activity by adomet, our results show that the dna methylation and cleavage activities of tspgwi are closely intertwined, but autonomous enough to have their functions separated.
CONCLUSIONS
the tspgwi gene coding for  <dig> kda tspgwi has been sequenced and cloned.
active bifunctional rease-mtase protein has been expressed in e. coli and purified.
bioinformatics studies predicted rease and mtase binding/catalytic motifs: pd-xk, dpavgtg, nppy and showed modular structure of tspgwi.
series of mutants in the predicted motifs were constructed, yielding rease-deficient and mtase-deficient enzymes.
uncoupled rease and mtase activities exhibited high autonomy.
mutants more suitable than native tspgwi for gene cloning methodology were constructed.
the cleavage and modification functions were separated into distinct proteins, while maintaining enzymatic activity, thus allowing practical use of tspgwi in vitro as a regular class ii enzyme.

 restriction-modification systems usually consist of at least two enzymatic activities: a restriction endonuclease, which recognises and cleaves a specific dna sequence, and a cognate dna methyltransferase that can methylate the same target site, thereby preventing the cleavage of host dna by the rease. on the basis of subunit composition, cofactor requirements, and the mode of recognition and cleavage, rm systems have been traditionally classified into three major types: i, ii and iii. type iv has been re-introduced to describe a heterogeneous group of enzymes cleaving only modified dna, with ill-defined sequence specificity. type i and type iii reases are complex multisubunit molecular machines that utilize atp and perform either dna modification or cleavage. type ii reases are simpler: they recognise short, 4– bp, nucleotide sequences and cleave phosphodiester bonds in dna within their recognition sites or at up to a distance of bp from them. in most cases, methylation and cleavage are divided between two separate enzymes. more than various reases that recognise more than different nucleotide sequences have been described as members of this large and divergent class of enzymes. most of them have been isolated from mesophilic microorganisms and only a few from hyperthermophiles and thermophiles, in particular from the genus thermus. in order to account for their structural and functional heterogeneity type ii reases have been divided into multiple subtypes, often overlapping with each other. for example, a growing group of atypical type ii reases, represented by bifunctional enzymes with rease and mtase activities within a single polypeptide, is a subclass of subtype iis enzymes that recognise a specific dna sequence and cleave outside it at a defined distance, within any sequence. examples of this group include eco57i, haeiv, aloi, bsemii and enzymes from the thermus sp. family: tspgwi, taqii, tspdti and tth111ii. in this paper we describe the cloning of the tspgwi gene based on degenerated primers pcr protocol, site-directed mutagenesis, expression and purification of recombinant and mutant proteins followed by comparative biochemical and genetic analysis. the constructed mutants are more suitable than native tspgwi in gene cloning methodology, as the cleavage and modification functions were separated into distinct, active proteins. tspgwi-like enzymes are difficult to fit unambiguously into the conventional classification, as their unique structural and functional features can be found in rease sub-types iia, iic, iis, iif, iih and type i.  sequencing, cloning and expression of the tspgwi gene neither the biochemical selection for methylation phenotype approach nor the "white-blue" screen for dna damage/modification to yield recombinant clones were successful, due to tspgwi low activity and lack of complete cleavage of the plasmid dna. we devised a cloning protocol for thermophilic rease without accompanying mtase, which includes two stages: a gene nucleotide sequence determination starting from internal amino acid sequences of rease proteolytic fragments followed by various pcr methods using combination of degenerated and non-degenerated primers. the primers are designed using probability of codon usage from other closely related bacteria and direct in-frame insertion of amplified orf into tightly temperature-regulated expression vector, taking advantage of low temperature cultivation at non-permissive conditions, which both prevent rease expression and suppresses activity of any leaking thermophilic rease. the tspgwi gene nucleotide sequence determination was initiated from very limited amino acid sequence information about the n-termini of native tspgwi proteolytic internal fragments. we were unable to determine the amino acid sequence of the intact protein, thus homogeneous tspgwi preparation was subjected to limited proteolysis, yielding major bands and more than minor bands, as determined by sds/page. the immobilised tpck-trypsin was readily removed by centrifugation; hence, the resulting bands were derivatives of tspgwi protein only. two of the predominant bands of approximate sizes kda and kda were subjected to n-terminal sequencing. based on the obtained short 19- and 10- amino acid sequences gpiggggspeaqlvplitr and effterlaqe, sets of non-degenerated and degenerated primers, forming alternative pairs, were designed. the primers were designed arbitrarily as based on back-translated amino acid sequence using codon usage data from orfs of thermus sp. genes available in genbank. the high gc content of thermus genes was also considered. sets of primer pairs used were either with low degeneracy and non-degenerated, containing incorporated codons of the assumed highest probability to exist in thermus sp. one such pair yielded a pcr fragment containing an internal portion of the tspgwi gene. combination of pcr with degenerated primers using thermus sp. gw genomic dna as a template as well as casette-ligation-pcr using pbr dna as the known sequence donor for non-degenerated primers, yielded a sequence of bp contig with the complete tspgwi gene. each contig strand was sequenced; in addition, regions containing discrepancies were sequenced several times under different conditions. the tspgwi gene orf coding for the rease-mtase bifunctional protein is bp in length coding for the amino acid polypeptide. the calculated molecular weight of the tspgwi is da, which perfectly matches data from sds/page and size exclusion chromatography of the native protein. the calculated isoelectric point of recombinant tspgwi is, very similar to calculated isoelectric point of recombinant tspdti of. no sequence similarity to any mtase or dna-binding protein was found in the orf flanking regions. the orf begins with the atg start codon and contains putative upstream ribosome binding sites: - 5'-agg and - 5'-agga. the orf is extremely gc rich: %. to ensure that no errors are incorpoated during tspgwi gene amplification, all the pcr reactions were performed with a proofreading blend of taq and pfu dna polymerases. sequencing was repeated three times for each strand, both directly for the independent pcr products and for the cloned the gene fragments. native tspgwi purification was reported by us previously. since tspgwi activity was hardly noticeable in its natural host thermus gw, the development of an extensive isolation procedure was essential, which included: polyethyleneimine precipitation of nucleic acids, ammonium sulfate fractionations followed by five chromatographic steps and size-exclusion chromatography. to the contrary, recombinant tspgwi and its mutants were purified to homogeneity using much simplified procedure with the tspgwi protein expressed in e. coli. the protocol included heat-denaturing e. coli proteins, polyethyleneimine precipitation of nucleic acids, ammonium sulfate fractionations, followed by two chromatographic steps. interestingly, in spite of the cloning in the presence of a strong pr promoter, the protein becomes detectable in induced cells only after h of growth under non-permissive conditions, and keeps accumulating until the late stationary phase, even after h cultivation at 42°c. this is probably due to the combination of the following factors: gc rich orf sequence distant from e. coli optimum codon usage, slow transcription of the extremely gc-rich tspgwi gene, the presence of numerous putative hairpin structures within the gene and the very large size of the protein to be translated. nevertheless, optimisation of expression culture growth/induction conditions yielded adequate amounts of tspgwi. it was also observed that the initially homogenous protein, after prolonged storage at -20°c, undergoes minor degradation. bioinformatic analyses of tspgwi: prediction of domains and functional motifs isolation and sequencing of the tspgwi gene enabled detailed analysis of its amino acid sequence. searches of rease sequences deposited in rebase revealed an overall similarity to a number of genuine and putative type iic enzymes, including the previously characterised nuclease taqii. two other members of the thermus family, i.e. tspdti revealed the expected significant similarity of the central region of tspgwi to the catalytic domains of known dna:m6a mtases. no significant similarity of the terminal regions to any protein family was observed in simple sequence searches. nonetheless, multiple sequence alignment of tspgwi homologues revealed the presence of a candidate pd-xk motif, resembling the active site of many reases and other nucleases. we carried out a protein fold-recognition analysis to confirm this preliminary prediction for the n-terminal region, as well as to identify potential homologues and predict the structure for the c-terminal region. since the fr method is designed to identify remote homology and predict structure for domain-size sequence fragments, the tspgwi sequence was split into a series of overlapping segments and submitted to the genesilico metaserver. the fr analysis of the tspgwi sequence identified the structures of a type i dna:m6a mtase m. ecoki hsdm subunit, a putative hsdm subunit of an experimentally uncharacterised rm system bthvorf4518p, and type ii dna:m6a mtase m. taqi as the best structural templates for the central region comprising residues 130– of tspgwi. interestingly, the fr alignments for this region revealed not only the s-adenosylmethionine -binding/catalytic mtase domain, but also a helical domain characteristic of the n-terminus of the hsdm subunit. for the n-terminal region of tspgwi the fr methods also supported the prediction of the pd-xk domain, albeit with scores below the level of significance. the closest match on the sequence level by the hhsearch method for profile-profile searches was the n-terminal pd-xk domain of the hsdr subunit from type i rm systems. this prediction made for the n-terminal domain of tspgwi further supports the classification of the catalytic domain of taqii-related enzymes as a member of the pd-xk fold. fr methods failed to report any significant matches to known structures for the c-terminal part of the tspgwi sequence. interestingly, the c-terminal region was predicted to be almost entirely disordered, with only a short segment comprising residues 960– predicted to form a stable helical secondary structure. this segment shows sequence similarity to the c-terminus of taqii. we found no similarity of this region to target recognition domains of any mtase family; nevertheless, we speculate that it might play an analogous role. altogether, the results of our fr analysis suggest that type ii enzyme tspgwi is a fusion protein comprising a tandem arrangement of type i-like domains: a pd-xk nuclease domain related to the corresponding domains in hsdr subunits, a hsdm-like module comprising a helical domain and a highly conserved adomet-binding/catalytic mtase domain, and a c-terminal extension, presumably responsible for dna sequence recognition. on the other hand, tspgwi lacks other domains characteristic of type i rm enzymes, including the atp-dependent translocase module of the hsdr subunit and c-terminal domains of either hsdm or hsdr, which are important for interactions between type i subunits. this domain organization resembles the structure of the recently analysed mmei enzyme, to which tspgwi exhibits only very limited sequence similarity, restricted primarily to the central mtase domain. enzymatic properties of tspgwi purified preparation of native tspgwi protein was used for the detailed study of biochemical properties, reaction conditions and various cofactors influencing either dna cleavage or the methylation activity of the enzyme. the apparent molecular mass of the wt recombinant protein under denaturing conditions was found to be kda, adequately to the molecular mass of tspgwi isolated from thermus sp. gw. comparative assay of activities of both enzymes revealed no difference. control mock-purification from e. coli did not show any of the described activities. wt recombinant protein was also subjected to analytical gel filtration in a buffer with a composition close to the physiological, containing mm mgcl. the experiment revealed that the native endonuclease molecular mass is 110– kda, indicating that under physiological conditions the protein exists as a monomer. the temperature activity range extends from 42°c to 89°c, with the maximum observed at 60–70°c. incubation at 37°c resulted in 5% activity only. tspgwi is inactivated by min incubation at 89°c and ph greatly affects activity, which drops sharply below ph and above, with the maximum at –. the optimal ionic strength is below mm kcl; at mm only 50% of activity remains. the optimum restriction reaction conditions for tspgwi endonuclease in vitro are in mm tris-hcl ph at 65°c, mm mgcl mm dtt. tspgwi maintains the absolute requirement for mg2+ for cleavage activity, with the optimum between and mm. mg2+ ions can be effectively substituted with mn2+, fe2+ and partially with co2+ ions. remarkably, ca2+ ions do not support tspgwi cleavage activity, while in contrast, they stimulate methylation activity of the enzyme. no difference was observed in response to divalent cations by both recombinant and native wt tspgwi. unlike most of the other bifunctional type iic reases/mtases μm adomet slightly inhibits the rease activity of tspgwi enzyme, while s-adenosylhomocysteine has essentially no effect on lambda dna cleavage. the restriction activity of tspgwi is neither inhibited nor stimulated by spermidine or atp. as adomet is a co-substrate for the tspgwi mtase activity, it indirectly promotes dna modification rather than cleavage. the presence of refractory sites, observed in vitro without adomet in the reaction buffer, could alternatively be explained by partial dna methylation due to certain enzyme-bound adomet carry over during purification. however, since eight purification steps were used, this possibility is rather remote. in addition, partial protection should be uniform among different recognition sites, instead of observed site preference. a recombinant tspgwi gene alone can be cloned into e. coli without an additional mtase and stably maintained at 28°c. a much reduced restriction activity < 5% at low temperature and the presence of cellular adomet and ca2+ appears to favour methylation in vivo. taken together, the adomet and ca2+ effects as well as apparent preference for the opposite arrangement of recognition sites may provide an effective mechanism for controlling undesired restriction in vivo, thus removing the necessity for the existence of a separate tspgwi mtase. another conclusion from the above results is that dna methylation and dna cleavage events occur by different mechanisms, in parallel and essentially independently. as a result, some of the target sites become protected, even under conditions favouring cleavage. such concurrent methylation and restriction activities seem to resemble the behaviour of another bifunctional type iic enzyme aloi. to further verify the marked independence of cleavage and methylation as well as to define the functional motifs within tspgwi, a series of amino acid replacements by site-directed mutagenesis was performed on tspgwi. we targeted three residues in the catalytic motif "pd-xk" of the nuclease domain, and two residues in the mtase domain of tspgwi: in the adomet-binding motif i "dpavgtg" and in the main mtase catalytic motif iv: "nppy". all the mutant proteins used were of high purity, isolated using simplified recombinant tspgwi protocol and their activity in vitro was analysed. some of the mutants lost their rease activity, while maintaining the wild type level of methylation; others lost their mtase activity while retaining the ability to cut dna, although at reduced efficiency. the mtase activities of both the wt tspgwi and mutant proteins were dependent on the presence of divalent cations. further experiments are in progress to evaluate complex effect of ca2+ and mg2+. dna substrate preference of tspgwi under the variety of conditions initially tested no complete tspgwi digestion was obtained; instead a stable partial cleavage pattern was observed. it is probably due to the preferential site cleavage in various dna substrates. among them puc was found to be the most efficiently cleaved supercoiled dna substrate. however, even in the case of puc small fraction of molecules remained uncut, as determined by agarose gel electrophoresis of overloaded digestion samples and by transformation assay. the cleavage depends on the substrate used: it varies from the near complete digestion of two tspgwi sites in puc dna to the trace cleavage of a subset of refractive sites in pbr and pacyc plasmids. the refractory site phenomenon was preliminary evaluated here using two pcr substrates, containing either single or double tspgwi recognition sites. on a bp pcr fragment containing a single recognition site for tspgwi and taqii, no detectable cleavage was observed in the case of tspgwi, while closely related taqii exhibits complete digestion. however, when the bp fragment is cloned into puc plasmid, the tspgwi site becomes cleavable, as evident from the appearance of predicted bp and bp bands. the bp pcr fragment contained two divergent tspgwi recognition sites, separated by a bp spacer, which may allow two tspgwi molecules to bind simultaneously both recognition sequences located on the same face of dna, without steric interference, while being close enough to allow for protein-protein interaction. it turned out to be the most completely cleaved substrate among those we tested. further experiments are in progress to carefully evaluate the observed phenomenon, as the presence of two sites on the same dna molecule may be an intrinsic part of the tspgwi scission mechanism. the requirement for the presence of two recognition sites of opposite orientation for dna cleavage is a characteristic feature of type iii reases. thus, the tspgwi-like enzymes show a mixture of features characteristic of type i, ii, or iii rm systems. type iii enzymes methylate only one strand, regardless of the number and orientation of cognate sites, while still providing adequate protection against cleavage. within type ii, bspmi is the one rease described to date that strongly prefers a tandem, head-to-tail orientation of double sites. another subtype iis endonuclease foki, has a preference towards a pair of sites with a tandem head-to-tail orientation. the divergent orientations of the recognition sites may ensure cleavage of both sequences, as was proposed for type iis reases bsgi, bpmi and acc36i, which are, however, rather remotely related to tspgwi. nevertheless, these enzymes retain the ability to cut single sites, although with a reduced efficiency compared to the tandem sites. tspgwi is unable to cut a single site, unlike bsgi, bpmi, acc36i and the closely related taqii rease. it is intriguing that taqii rease, despite the very high amino acid sequence similarity to tspgwi and in spite of numerous biochemical similarities cleaves single sites efficiently. the ability to cleave a single recognition sequence may be correlated with the existence of rease activity stimulation by adomet. although this cofactor has no effect on tspgwi rease activity, it does stimulate taqii rease approximately 2– fold. as we have pointed out previously, the new thermus sp. family including: tspgwi, taqii, tspdti and tth111ii, is internally diversified. even though these enzymes exhibit striking similarities in protein size, quaternary structure, recognition sequences, cleavage distances and related origin from various thermus strains, they display marked differences in their response to adomet and to the arrangement requirements of different recognition sites. interestingly, this group of enzymes seems to be also heterogenous at the level of amino acid sequence similarity, as tspgwi and taqii seem to be closely related to each other, but only remotely to the two other enzymes tspdti and tth111ii. this latter pair of enzymes appear to be closely related to each other, but exhibit a different pattern of similarities compared to the tspgwi-taqii pair. however, all four enzymes share significant biochemical similarities, including related recognition and cleavage sequences, very large molecular weights of around kda, monomeric protein organization and are negatively charged with calculated isoelectric points of,, and. although tspgwi is not particularly closely related to any known subtype iib rease, the similarity of the domain structure and the mode of action of these enzymes suggests that they have evolved from a common ancestor. thus, type iib enzymes forming tight dimers and taqii-like enzymes that hardly dimerise if at all, may represent two extremes of an evolutionary landscape, where different enzymes have specialised towards either separation or pairing of recognition sites by developing subunit-subunit interactions of different strength. in this classification, tspgwi would be located "between" type iib enzymes and taqii. endonucleolytic activity of tspgwi wt and the mutant proteins site-directed mutagenesis was used to test whether the amino acids predicted by bioinformatics were essential for the mtase and rease catalytic activity of the tspgwi enzyme. to this end, variants of the tspgwi protein with substitutions in the putative pd-xk domain and in the mtase domain were constructed by saturation site-directed mutagenesis. the mutant proteins were purified as described for recombinant tspgwi protein and subjected to in vitro biochemical experiments. to calibrate the protein concentrations, each variant was assayed, then diluted with the storage buffer to achieve equal protein concentration in every assay. the purity and concentration of the diluted proteins were further assessed by poliacrylamide gel electrophoresis. the cleavage reaction was performed at 65°c for one hour and overnight. mutants in the pd-xk domain d58a, d58r, e72a, e72l, e72n, e72r, e72s, k74a, k74g, k74p, k74r did not show any dna cleavage activity under optimal buffer conditions at a concentration of μg/ml, the equivalent of u wt tspgwi per reaction. higher enzyme concentrations could not be tested, as the specific activity of wt tspgwi rease is relatively low, and increasing the amount of protein per reaction caused inhibition because of its non-specific binding to the substrate dna. only one mutant variant e72d retained its rease activity – its cleavage rate was even slightly higher than that of the wt protein. thus, conservative substitution of aspartic acid for glutamic acid in the site keeps the pd-xk motif of tspgwi fully functional. it also confirms the key role of acidic amino acid in position in catalysis. since preferential cleavage of substrate dna by native tspgwi was observed at concentrations as low as μg/ml, the decrease in rease activity of tspgwi mutants was estimated to be more than 200-fold, thus confirming the predicted role of the amino acids d e and k in catalysis. surprisingly, the mutants in the mtase domain – v356m and n473a – also displayed a significantly reduced cleavage activity in vitro in comparison to the wt tspgwi protein. the double mutant v356m/n473a exhibited no detectable rease activity in vitro. taqii, the closest relative of tspgwi, exhibits the "dpamgtg" variant of motif i. this observation, together with the fact that taqii rease activity is strongly stimulated by adomet, points to the complex role of adomet in both enzymatic activities and suggests that some interaction occurs between the nuclease and mtase domains in the course of the cleavage reaction. the substitution of methionine for valine in the adomet-binding motif of tspgwi could disturb adomet-affected communication between the nuclease domain and the mtase domain. the nature of the communication between the domains remains to be defined by further analyses. mtase activity of tspgwi wt and the mutant proteins mutant proteins were further analysed for their methylation activity. in vitro methylation reactions were performed using wt tspgwi enzyme and mutant proteins in the dna protection assay as described in materials and methods. two types of substrate dna were used: pbr plasmid dna and the bp pcr fragment. the results presented in table and table demonstrate that the mutant n473a is unable to modify the tspgwi target sequence. interestingly, the n473a substitution not only completely eliminates the methylation activity, but also decreases the cleavage activity of tspgwi; even so, the resulting protein retains sufficient activity to be considered a functional rease. thus, both the rease and mtase activities of tspgwi were uncoupled, and rease-only or mtase-only tspgwi variants were obtained. this is also of high practical methodological value in gene cloning technology, allowing to use tspgwi mutants as traditional class-ii enzymes for dna cleavage or methylation. mutants with d58a or v356m substitutions exhibit mtase activity at the same level as the wt enzyme, despite the total or partial loss of their ability to cleave the substrate dna. we observed that d58a slightly increased the modification activity in comparison to the wt protein. this could have been due to the absence of a competing rease activity in this variant. interestingly, mg2+ at mm concentration tested has a strong stimulatory effect on the mtase activity of the d58a mutant. in the case of wt tspgwi, mg2+ is utilised for cleavage, which hinders its effect on the mtase activity. thus, the d58a mutation demonstrates that the competing mtase and rease activities both share the same mg2+ cofactor. however, it cannot be ruled out that the presence of divalent cations stabilizes not perfectly folded mutant protein tspgwi, thus contributing non-specifically to retaining their enzymatic activities. taken together with the observed inhibition of the tspgwi rease activity by adomet, our results show that the dna methylation and cleavage activities of tspgwi are closely intertwined, but autonomous enough to have their functions separated.  the tspgwi gene coding for kda tspgwi has been sequenced and cloned. active bifunctional rease-mtase protein has been expressed in e. coli and purified. bioinformatics studies predicted rease and mtase binding/catalytic motifs: pd-xk, dpavgtg, nppy and showed modular structure of tspgwi. series of mutants in the predicted motifs were constructed, yielding rease-deficient and mtase-deficient enzymes. uncoupled rease and mtase activities exhibited high autonomy. mutants more suitable than native tspgwi for gene cloning methodology were constructed. the cleavage and modification functions were separated into distinct proteins, while maintaining enzymatic activity, thus allowing practical use of tspgwi in vitro as a regular class ii enzyme. 
tspgwi also possesses an n-terminal pd-xk nuclease domain related to the corresponding domains in hsdr subunits, but lacks the atp-dependent translocase module of the hsdr subunit and the additional domains that are involved in subunit-subunit interactions in type i systems. tspgwi is a bifunctional protein comprising a tandem arrangement of type i-like domains; particularly noticeable is the central hsdm-like module comprising a helical domain and a highly conserved s-adenosylmethionine-binding/catalytic mtase domain, containing dpavgtg and nppy motifs. the deduced amino acid sequence of the enzyme shares significant sequence similarity with another representative of the thermus sp. restriction-modification systems are a diverse class of enzymes. we have previously proposed the existence of a thermus sp. the mtase and rease activities of tspgwi are autonomous and can be uncoupled. enzyme family, which belongs to type ii restriction endonucleases, however, it features also some characteristics of types i and iii. both the restriction endonuclease and methyltransferase activities of wild type tspgwi are dependent on the presence of divalent cations. we cloned, expressed, and mutagenised the tspgwi gene and investigated the properties of its product, the bifunctional tspgwi restriction/modification enzyme. they are classified into four major types: i, ii, iii and iv.
 restriction-modification systems are a diverse class of enzymes. they are classified into four major types: i, ii, iii and iv. we have previously proposed the existence of a thermus sp. enzyme family, which belongs to type ii restriction endonucleases, however, it features also some characteristics of types i and iii. members include related thermophilic endonucleases: tspgwi, taqii, tspdti, and tth111ii.  here we describe cloning, mutagenesis and analysis of the prototype tspgwi enzyme that recognises the 5'-acgga-3' site and cleaves 11/ nt downstream. we cloned, expressed, and mutagenised the tspgwi gene and investigated the properties of its product, the bifunctional tspgwi restriction/modification enzyme. since tspgwi does not cleave dna completely, a cloning method was devised, based on amino acid sequencing of internal proteolytic fragments. the deduced amino acid sequence of the enzyme shares significant sequence similarity with another representative of the thermus sp. family – taqii. interestingly, these enzymes recognise similar, yet different sequences in the dna. both enzymes cleave dna at the same distance, but differ in their ability to cleave single sites and in the requirement of s-adenosylmethionine as an allosteric activator for cleavage. both the restriction endonuclease and methyltransferase activities of wild type tspgwi are dependent on the presence of divalent cations.  tspgwi is a bifunctional protein comprising a tandem arrangement of type i-like domains; particularly noticeable is the central hsdm-like module comprising a helical domain and a highly conserved s-adenosylmethionine-binding/catalytic mtase domain, containing dpavgtg and nppy motifs. tspgwi also possesses an n-terminal pd-xk nuclease domain related to the corresponding domains in hsdr subunits, but lacks the atp-dependent translocase module of the hsdr subunit and the additional domains that are involved in subunit-subunit interactions in type i systems. the mtase and rease activities of tspgwi are autonomous and can be uncoupled. structurally and functionally, the tspgwi protomer appears to be a streamlined'half' of a type i enzyme. 