BACKGROUND
nucleases of the s <dig> family are widely used as tools for probing single-stranded regions of dna and rna  <cit>  as well as for the removal of single-stranded regions from dsdna  <cit> .
one class of plant homologs of s <dig>  represented by cel i from celery, are particularly capable of efficient cutting at single base substitutions and loops  <cit> .
several cel i-based mutation detection techniques have been developed  <cit> .
they are relatively simple yet highly reliable and capable of detecting a mutation in pools of several dna samples.
adaptation of this approach to the tilling method of recovering chemically derived mutations at target regions  <cit>  has allowed cel i to contribute to many plant genetics programs  <cit> , as well as zebrafish  <cit> , drosophila  <cit> , and mouse es cells research  <cit> .
moreover, it is beginning to be successfully applied to programs of disease mutation detection  <cit> .
a cel i ortholog, cel ii nuclease, is the principal component of the surveyor mutation detection kits   <cit> .
the p <dig> nuclease of penicillium citrinum is a close ortholog of the s <dig> nuclease.
although its crystal structure has provided important clues to the mechanism of phosphodiester bond cleavage and single-stranded oligonucleotide binding  <cit> , the applicability of this model for cel i orthologs has not been tested.
several important questions remain.
how is the wide range of mismatch substrates recognized by cel i?
what determines the ph optima for rnase and dnase activities  <cit> ?
how can these enzymes be engineered into even better mutation-detection tools?
clearly, a better mechanistic understanding of single-strand specific nucleases is needed to answer these questions.
development of an expression system for this class of enzymes will be an important step in this direction.
most single-strand specific nucleases are extracellular glycoproteins containing one or more disulfide bridges per monomer to confer high enzyme stability  <cit> .
such proteins are hard to express in an active form in a heterologous system which often fails to provide the correct pattern of posttranslational modifications.
indeed, our attempts to express active cel i in various prokaryotic hosts have not succeeded.
in the present study we employed a baculovirus system for expression and site-directed mutagenesis studies of enzymes of the cel i family.
active cel i nuclease was purified from the cell culture media and used for detection of single-base substitutions in patient-derived dna.
we also report the cloning, expression and site-directed mutagenesis of the cdna of a close homolog of cel i nuclease from spinach, which we termed sp i. we show that the properties of recombinant cel i and sp i nucleases may be different from their native counterparts purified from plant tissues.
RESULTS
cloning of the sp i nuclease cdna
a nuclease isolated from spinach, called sp, is a particularly intriguing cel i ortholog, showing properties intermediate between cel i and s <dig>  it has a strong preference for at-rich regions, yet is able to cut single-base mismatches and has a neutral ph optimum.
unlike cel i, it is unable to recognize mismatches with guanine bases at the mismatched site  <cit> .
we cloned this cel i ortholog from spinach mrna.
we called this putative nuclease sp i to distinguish the properties of this clonally purified form from the native sp nuclease preparations that may contain more than one homolog that are very difficult to separate during native enzyme purification.
the sp i mrna sequence was deposited to genbank under accession no.
.
sp i and cel i amino acid sequences are  <dig> % identical.
amino acid sequence alignment of sp i with other known s1-like nucleases reveals that all residues identified by structural studies  <cit>  as crucial for binding of the three zn2+ atoms and for catalysis are preserved in sp i. interestingly, the nucleotide binding site shows less sequence conservation .
a significant divergence was observed in a fragment within a loop located close to the ligand, comprising residues 134– <dig> in sp i and 127– <dig> in p <dig>  respectively.
the h <dig> residue is one of the most prominent sequence features within this fragment of the sp i nuclease, compared with cel i and p <dig>  to test if h <dig> is important for the sp i mismatch-specific nuclease activity, we produced a recombinant virus expressing a h135a mutant of the sp i nuclease.
recombinant expression of cel i and sp i
infection of a sf <dig> cell culture with recombinant viruses containing the cel i or sp i genes under control of a constitutive promoter resulted in accumulation of a single-strand specific nuclease activity both in the culture media and cell extract .
this activity adhered to a ni2+ affinity column and was eluted with  <dig> mm imidazole.
a single major nuclease band was observed when the partially purified nuclease preparations were separated on a sds page, in-gel refolded, and stained for single-strand specific nuclease activity .
this activity co-migrated with a native cel i control , implying that the recombinant enzyme contains a similar quantity of n-linked glycans.
we also detected the recombinant protein by a western blot experiment with an anti-hexahistidine monoclonal antibody .
the ni2+ affinity column-purified nucleases were stable on ice for at least a week and infinitely stable when stored in 50% glycerol at -20°c.
the enzyme activities were reduced by freezing/thawing cycles, decreasing by roughly 50% after each cycle .
test of recombinant nucleases in mutation detection
to test the efficacy of our recombinant nuclease preparations in mutation detection, a highly polymorphic section of exon  <dig> of brca <dig> was used as the substrate  <cit> .
a pcr product derived from one patient contained three single base pair polymorphisms as revealed by a control experiment with cel i purified from celery.
this substrate is challenging because of multiple single-base substitutions in close proximity to each other, a quality that would render many mutation detection techniques ineffective  <cit> .
while the mismatches  <dig> g → a and  <dig> c → t were well detected by all our recombinant nuclease preparations , little cutting of the nucleotide substitution t → c at position  <dig> of brca <dig> was observed, reflected by the low signal from the  <dig> nt long fragment .
we also observed an additional 303– <dig> nt peak which may have originated from processing of the  <dig> nt peak by cel i and sp i exonuclease activities.
control experiments have indicated efficient cutting of t → c at position  <dig> of brca <dig> by native cel i. since this result was reproduced in all our recombinant sp i and cel i preparations, it indicates that the mismatch sequence preference and possibly the balance between the exo- and endonuclease activities of the expressed enzymes is slightly different from their native counterparts purified from plant tissues.
surprisingly, unlike its native counterpart  <cit> , the recombinant sp i was capable of introducing nicks specifically 3' of an extrahelical g nucleotide .
this result was reproduced on several preparations of sp i and confirmed by mass spectrometry analysis of the incised heteroduplex substrates to exclude the possibility of a non-specific action of sp i on its substrate , further indicating that mismatch preferences of cel i orthologs can be modified by recombinant expression.
sp i h135a was an active nuclease, with mismatch recognition properties similar to those of the wild type sp i , indicating that h <dig> is dispensable for the mismatch nuclease activity of sp i.
discussion
expression of a protein in a heterogeneous host followed by purification is an important step in the study of protein function, allowing the unequivocal assignment of a function to a polypeptide.
plants may contain multiple cel i-like nucleases which often co-purify due to aggregation by lectins in plant extracts  <cit> .
celery has at least two active cel i orthologs, termed cel i and cel ii  <cit> , with similar mismatch cutting activities but with striking differences in ph optima and mg2+ requirements  <cit> .
a blast search of the arabidopsis genome yields several cel i-like orfs  <cit> .
recombinant expression of individual homologs in a heterologous system as shown in this report overcomes the cross-contamination and will facilitate their characterization.
in fact, enhanced enzyme purity is a likely explanation for the modification of the properties of recombinant cel i and sp i enzymes.
the apparently altered sequence specificity of recombinant cel i may be the result of removing the cel ii enzyme normally present in cel i preparations from celery as demonstrated in figure 1b.
in line with this interpretation, the mismatch recognition preferences of mixtures of cel i and cel ii nucleases have been reported to differ slightly from those of homogenous preparations of cel i nuclease or cel ii nuclease  <cit> .
single-strand specific nucleases have been reported to vary widely in their efficiency with different substrates depending on the sequence context  <cit> , size of the single-stranded region within a double-stranded substrate  <cit>  and ph  <cit> .
based on the latter two criteria, two groups of s <dig> homologs may be distinguished: s <dig> orthologs and cel i orthologs  <cit> .
s <dig> orthologs are represented by fungal nucleases s <dig> from aspergillus oryzae and p <dig> from penicillium citrinum, plant mung bean nuclease, and recently, recombinant prokaryotic m <dig> nuclease  <cit> .
these enzymes are highly specific for single-stranded nucleic acids, have acidic ph optima, and are essentially inactive at alkali ph.
they cut double-stranded dna at relatively large distorted regions, such as the site of an insertion mismatch of three nucleotides or larger or at at rich regions of double-stranded dna, but not at base-substitution mismatches.
in contrast, cel i orthologs, represented by cel i from celery, are active dnases at both acidic and alkali ph ranges, may be stimulated by or require mg2+, and cut all dna mismatches including single base substitutions at the phosphodiester bonds specifically 3' of the mismatch nucleotides.
no structural explanations for this divergence of nuclease properties have been published and, to this end, it is unclear to what extent the structure of p <dig> may be relevant for making conclusions about cel i orthologs  <cit> .
the present expression system may help address this question by expression and characterization of other single-strand specific nucleases of the s <dig> and cel i family and with the use of a site-directed mutagenesis approach.
one possibility for the surprising ability of our recombinant sp i nuclease to cut at a mismatch containing a g residue is that the cloned cdna product may not represent the dominant cel i-like activity found in spinach extracts.
this possibility can be distinguished by obtaining sufficient quantities of native sp protein for amino acid sequence determination.
alternatively, sp i expressed in insect cells may assume a more relaxed structure, allowing binding of guanine nucleotides.
such relaxation could result from different number and positioning of disulfide bridges, glycosylation patterns and/or folding environments.
neither native nor recombinant cel i has been characterized with respect to their disulfide bonding patterns.
CONCLUSIONS
in the present study, we reported recombinant expression of plant mismatch endonuclease cel i and a newly cloned cel i ortholog, sp i, in a baculovirus system.
active enzymes were expressed as c-terminal hexahistidine fusions, purified from cell culture media using metal affinity chromatography and used for detection of mutations in brca <dig> gene of patient-derived dna.
unlike its native counterpart purified from plant tissues, recombinant sp i nuclease was able to nick the phosphodiester bond 3' of an extrahelical guanine residue.
thus, recombinant expression of cel i orthologs may result in modification of their activities due to enhanced enzyme purity and/or different pattern of post-translational modiciations.
recent years have seen a major increase in the use of cel i for genetic variance detection.
cel i has become an indispensable instrument in applications where high throughput and capability to recognize all mutations are critical.
all of the mentioned applications would benefit from further improvement of cel i mismatch-cutting properties.
for instance, it would be highly desirable to eliminate the 5' to 3' directional endonuclease activity which results in the removal of a 5'-label and prohibits extended incubation of a substrate with the nuclease.
the present cel i expression system may open a new avenue for developing engineered enzymes with enhanced mutation detection properties.

 nucleases of the s family are widely used as tools for probing single-stranded regions of dna and rna as well as for the removal of single-stranded regions from dsdna. one class of plant homologs of s represented by cel i from celery, are particularly capable of efficient cutting at single base substitutions and loops. several cel i-based mutation detection techniques have been developed. they are relatively simple yet highly reliable and capable of detecting a mutation in pools of several dna samples. adaptation of this approach to the tilling method of recovering chemically derived mutations at target regions has allowed cel i to contribute to many plant genetics programs, as well as zebrafish, drosophila, and mouse es cells research. moreover, it is beginning to be successfully applied to programs of disease mutation detection. a cel i ortholog, cel ii nuclease, is the principal component of the surveyor mutation detection kits. the p nuclease of penicillium citrinum is a close ortholog of the s nuclease. although its crystal structure has provided important clues to the mechanism of phosphodiester bond cleavage and single-stranded oligonucleotide binding, the applicability of this model for cel i orthologs has not been tested. several important questions remain. how is the wide range of mismatch substrates recognized by cel i? what determines the ph optima for rnase and dnase activities? how can these enzymes be engineered into even better mutation-detection tools? clearly, a better mechanistic understanding of single-strand specific nucleases is needed to answer these questions. development of an expression system for this class of enzymes will be an important step in this direction. most single-strand specific nucleases are extracellular glycoproteins containing one or more disulfide bridges per monomer to confer high enzyme stability. such proteins are hard to express in an active form in a heterologous system which often fails to provide the correct pattern of posttranslational modifications. indeed, our attempts to express active cel i in various prokaryotic hosts have not succeeded. in the present study we employed a baculovirus system for expression and site-directed mutagenesis studies of enzymes of the cel i family. active cel i nuclease was purified from the cell culture media and used for detection of single-base substitutions in patient-derived dna. we also report the cloning, expression and site-directed mutagenesis of the cdna of a close homolog of cel i nuclease from spinach, which we termed sp i. we show that the properties of recombinant cel i and sp i nucleases may be different from their native counterparts purified from plant tissues.  cloning of the sp i nuclease cdna a nuclease isolated from spinach, called sp, is a particularly intriguing cel i ortholog, showing properties intermediate between cel i and s it has a strong preference for at-rich regions, yet is able to cut single-base mismatches and has a neutral ph optimum. unlike cel i, it is unable to recognize mismatches with guanine bases at the mismatched site. we cloned this cel i ortholog from spinach mrna. we called this putative nuclease sp i to distinguish the properties of this clonally purified form from the native sp nuclease preparations that may contain more than one homolog that are very difficult to separate during native enzyme purification. the sp i mrna sequence was deposited to genbank under accession no.. sp i and cel i amino acid sequences are % identical. amino acid sequence alignment of sp i with other known s1-like nucleases reveals that all residues identified by structural studies as crucial for binding of the three zn2+ atoms and for catalysis are preserved in sp i. interestingly, the nucleotide binding site shows less sequence conservation. a significant divergence was observed in a fragment within a loop located close to the ligand, comprising residues 134– in sp i and 127– in p respectively. the h residue is one of the most prominent sequence features within this fragment of the sp i nuclease, compared with cel i and p to test if h is important for the sp i mismatch-specific nuclease activity, we produced a recombinant virus expressing a h135a mutant of the sp i nuclease. recombinant expression of cel i and sp i infection of a sf cell culture with recombinant viruses containing the cel i or sp i genes under control of a constitutive promoter resulted in accumulation of a single-strand specific nuclease activity both in the culture media and cell extract. this activity adhered to a ni2+ affinity column and was eluted with mm imidazole. a single major nuclease band was observed when the partially purified nuclease preparations were separated on a sds page, in-gel refolded, and stained for single-strand specific nuclease activity. this activity co-migrated with a native cel i control, implying that the recombinant enzyme contains a similar quantity of n-linked glycans. we also detected the recombinant protein by a western blot experiment with an anti-hexahistidine monoclonal antibody. the ni2+ affinity column-purified nucleases were stable on ice for at least a week and infinitely stable when stored in 50% glycerol at -20°c. the enzyme activities were reduced by freezing/thawing cycles, decreasing by roughly 50% after each cycle. test of recombinant nucleases in mutation detection to test the efficacy of our recombinant nuclease preparations in mutation detection, a highly polymorphic section of exon of brca was used as the substrate. a pcr product derived from one patient contained three single base pair polymorphisms as revealed by a control experiment with cel i purified from celery. this substrate is challenging because of multiple single-base substitutions in close proximity to each other, a quality that would render many mutation detection techniques ineffective. while the mismatches g → a and c → t were well detected by all our recombinant nuclease preparations, little cutting of the nucleotide substitution t → c at position of brca was observed, reflected by the low signal from the nt long fragment. we also observed an additional 303– nt peak which may have originated from processing of the nt peak by cel i and sp i exonuclease activities. control experiments have indicated efficient cutting of t → c at position of brca by native cel i. since this result was reproduced in all our recombinant sp i and cel i preparations, it indicates that the mismatch sequence preference and possibly the balance between the exo- and endonuclease activities of the expressed enzymes is slightly different from their native counterparts purified from plant tissues. surprisingly, unlike its native counterpart, the recombinant sp i was capable of introducing nicks specifically 3' of an extrahelical g nucleotide. this result was reproduced on several preparations of sp i and confirmed by mass spectrometry analysis of the incised heteroduplex substrates to exclude the possibility of a non-specific action of sp i on its substrate, further indicating that mismatch preferences of cel i orthologs can be modified by recombinant expression. sp i h135a was an active nuclease, with mismatch recognition properties similar to those of the wild type sp i, indicating that h is dispensable for the mismatch nuclease activity of sp i. discussion expression of a protein in a heterogeneous host followed by purification is an important step in the study of protein function, allowing the unequivocal assignment of a function to a polypeptide. plants may contain multiple cel i-like nucleases which often co-purify due to aggregation by lectins in plant extracts. celery has at least two active cel i orthologs, termed cel i and cel ii, with similar mismatch cutting activities but with striking differences in ph optima and mg2+ requirements. a blast search of the arabidopsis genome yields several cel i-like orfs. recombinant expression of individual homologs in a heterologous system as shown in this report overcomes the cross-contamination and will facilitate their characterization. in fact, enhanced enzyme purity is a likely explanation for the modification of the properties of recombinant cel i and sp i enzymes. the apparently altered sequence specificity of recombinant cel i may be the result of removing the cel ii enzyme normally present in cel i preparations from celery as demonstrated in figure 1b. in line with this interpretation, the mismatch recognition preferences of mixtures of cel i and cel ii nucleases have been reported to differ slightly from those of homogenous preparations of cel i nuclease or cel ii nuclease. single-strand specific nucleases have been reported to vary widely in their efficiency with different substrates depending on the sequence context, size of the single-stranded region within a double-stranded substrate and ph. based on the latter two criteria, two groups of s homologs may be distinguished: s orthologs and cel i orthologs. s orthologs are represented by fungal nucleases s from aspergillus oryzae and p from penicillium citrinum, plant mung bean nuclease, and recently, recombinant prokaryotic m nuclease. these enzymes are highly specific for single-stranded nucleic acids, have acidic ph optima, and are essentially inactive at alkali ph. they cut double-stranded dna at relatively large distorted regions, such as the site of an insertion mismatch of three nucleotides or larger or at at rich regions of double-stranded dna, but not at base-substitution mismatches. in contrast, cel i orthologs, represented by cel i from celery, are active dnases at both acidic and alkali ph ranges, may be stimulated by or require mg2+, and cut all dna mismatches including single base substitutions at the phosphodiester bonds specifically 3' of the mismatch nucleotides. no structural explanations for this divergence of nuclease properties have been published and, to this end, it is unclear to what extent the structure of p may be relevant for making conclusions about cel i orthologs. the present expression system may help address this question by expression and characterization of other single-strand specific nucleases of the s and cel i family and with the use of a site-directed mutagenesis approach. one possibility for the surprising ability of our recombinant sp i nuclease to cut at a mismatch containing a g residue is that the cloned cdna product may not represent the dominant cel i-like activity found in spinach extracts. this possibility can be distinguished by obtaining sufficient quantities of native sp protein for amino acid sequence determination. alternatively, sp i expressed in insect cells may assume a more relaxed structure, allowing binding of guanine nucleotides. such relaxation could result from different number and positioning of disulfide bridges, glycosylation patterns and/or folding environments. neither native nor recombinant cel i has been characterized with respect to their disulfide bonding patterns.  in the present study, we reported recombinant expression of plant mismatch endonuclease cel i and a newly cloned cel i ortholog, sp i, in a baculovirus system. active enzymes were expressed as c-terminal hexahistidine fusions, purified from cell culture media using metal affinity chromatography and used for detection of mutations in brca gene of patient-derived dna. unlike its native counterpart purified from plant tissues, recombinant sp i nuclease was able to nick the phosphodiester bond 3' of an extrahelical guanine residue. thus, recombinant expression of cel i orthologs may result in modification of their activities due to enhanced enzyme purity and/or different pattern of post-translational modiciations. recent years have seen a major increase in the use of cel i for genetic variance detection. cel i has become an indispensable instrument in applications where high throughput and capability to recognize all mutations are critical. all of the mentioned applications would benefit from further improvement of cel i mismatch-cutting properties. for instance, it would be highly desirable to eliminate the 5' to 3' directional endonuclease activity which results in the removal of a 5'-label and prohibits extended incubation of a substrate with the nuclease. the present cel i expression system may open a new avenue for developing engineered enzymes with enhanced mutation detection properties. 
the insect cell-expressed cel i orthologs may not be identical to their native counterparts purified from plant tissues. previously, cel i-like activities were described only in a variety of plants and could not be expressed in an active form in bacteria. both recombinant enzymes were active in mutation detection in brca gene of patient-derived dna. the present expression system should facilitate further development of cel i-based mutation detection technologies. active cel i and sp i nucleases were expressed as c-terminal hexahistidine fusions and affinity purified from the cell culture media. native sp nuclease purified from spinach is unable to incise at single-nucleotide substitutions and loops containing a guanine nucleotide, but the recombinant sp i nuclease can cut at these sites. we also report the cloning of a cel i ortholog from spinacia oleracea which we termed sp i nuclease.
 the detection of unknown mutations is important in research and medicine. for this purpose, a mismatch-specific endonuclease cel i from celery has been established as a useful tool in high throughput projects. previously, cel i-like activities were described only in a variety of plants and could not be expressed in an active form in bacteria.  we describe expression of active recombinant plant mismatch endonucleases and modification of their activities. we also report the cloning of a cel i ortholog from spinacia oleracea which we termed sp i nuclease. active cel i and sp i nucleases were expressed as c-terminal hexahistidine fusions and affinity purified from the cell culture media. both recombinant enzymes were active in mutation detection in brca gene of patient-derived dna. native sp nuclease purified from spinach is unable to incise at single-nucleotide substitutions and loops containing a guanine nucleotide, but the recombinant sp i nuclease can cut at these sites.  the insect cell-expressed cel i orthologs may not be identical to their native counterparts purified from plant tissues. the present expression system should facilitate further development of cel i-based mutation detection technologies. 