BACKGROUND
functional telomeres are essential for cell survival.
telomeres distinguish natural chromosome ends from dna breaks.
when telomere function is lost, cells arrest and undergo senescence or apoptosis  <cit> .
normal dna replication causes telomere shortening, which is counterbalanced by the extension activity of telomerase  <cit> .
the majority of cancers have telomerase activity, and this activity is needed for ongoing division of tumor cells.
in somatic cells, telomerase activity is absent or low.
when cell division occurs faster than the ability of telomerase to elongate telomeres, the short telomeres limit cell replicative capacity  <cit> .
this limited cell division underlies diseases of tissue renewal such as dyskeratosis congenita, aplastic anemia, and pulmonary fibrosis  <cit> .
thus, telomere length regulation plays a pivotal role in cancer and in age-related degenerative disease.
both telomeric dna structure and telomerase play a role in establishing telomere length and end protection.
telomeres have a g-rich 3'-overhang that was first discovered in ciliates  <cit>  and is a conserved feature of telomeres in all eukaryotes  <cit> .
this 3'-overhang is the binding site for essential telomere proteins, such as cdc <dig> in saccharomyces cerevisiae and pot <dig> in mammals and other species.
loss of the overhangs due to disruption of the telomere complex, termed shelterin, leads to telomere-induced dna damage response and chromosome end-to-end fusion  <cit> .
in addition to maintenance of dna ends, the 3'-overhang is essential for telomerase accessibility.
telomerase recognizes and elongates single-stranded dna substrates, but not double-stranded substrates  <cit> .
thus, the processing of blunt ends, generated by replication, is needed for telomerase to elongate telomeres.
in vivo, blocking 3'-overhang formation in s. cerevisiae blocks the ability of telomerase to elongate a telomere seed sequence  <cit> .
3'-overhang formation is an active process.
in s. cerevisiae and mice, generation of the 3'-overhang occurs in the absence of telomerase  <cit> .
in tetrahymena, a conditional telomerase protein component  knockout cell line showed that telomerase depletion had relatively little effects on absolute g overhang length, indicating c-strand degradation, not telomerase elongation of the g-rich strand, must generate the overhang structures  <cit> .
inhibition of cdk <dig> blocks c-rich strand resection, thereby inhibiting 3'-overhang production  <cit> .
these data suggest that there is a nuclease regulated by cdk <dig> that processes the telomeres prior to elongation by telomerase.
additional evidence for an active processing enzyme comes from the fact that both ends of a chromosome have similar overhang structures.
replication of a chromosome to the very end is predicted to generate one chromosome end with a 3'-overhang and one with a blunt end  <cit> .
however, chromosomes in ciliates as well as in yeast and mammals have 3'-overhangs on both ends,  <cit> , implying at least one end must be processed to generate the overhang structure.
finally, the very precise structure of the telomere overhangs implies that it is formed by a regulated process.
the length and sequence of the 3'-overhangs in oxytricha and tetrahymena are surprisingly homogeneous  <cit> .
in oxytricha, there is a precise overhang of two  <dig> nucleotide 5'-ttttgggg-3' repeats, whereas in tetrahymena, the overhangs are either 14- <dig> or 20- <dig> nucleotides in length, and the majority of 5'-strands end in 3'-ccaacc-5', or 3'-caaccc-5'  <cit> .
in mammals, this overhang length is also conserved  <cit> .
this functional and structural evidence argues for the existence of a sequence-specific nuclease that processes telomeres.
to investigate the role of 3'-overhang processing, we set out to identify a telomere-specific nuclease activity.
we chose tetrahymena to look for processing activity because it has uniform telomere repeat sequence, a precise 3'-overhang structure, and a history as an excellent biochemical source for telomere-processing factors.
here we describe a sequence-dependent 5' to 3' exonuclease activity from tetrahymena that processes the c-strand of telomere substrates in vitro.
RESULTS
to identify an overhang-processing nuclease, we assayed cell extracts from tetrahymena  <cit> .
we designed specific duplex oligonucleotide substrates to detect a telomere-specific nuclease activity .
the duplex oligonucleotide substrate contains specific non-telomeric sequences with a biotin moiety that binds to streptavidin to block non-specific degradation from one end .
on the other end, the substrates contained either telomere repeats  or non-telomeric sequence.
we tested substrates that were either blunt-ended, or with a 5'- or a 3'-overhang.
to distinguish size changes of the substrates on each strand, we labeled each strand of the substrate separately in order to monitor processing occurred on the corresponding strand .
identification of a nuclease activity from tetrahymena crude extracts with preference for telomere substrates
we initially incubated the non-telomeric and telomeric blunt-ended substrates with crude tetrahymena cell extracts  and analyzed processing by gel electrophoresis and autoradiography.
we observed a nuclease activity that degraded the telomeric substrate to a greater degree than the non-telomeric substrate .
this activity was robust, strand-specific, and sequence-dependent.
the nuclease cleaved the 5'-strand of a blunt-ended substrate containing telomere repeats.
to further characterize this nuclease activity, we performed biochemical purification.
extracts were precipitated with ammonium sulfate, and then enriched on several fast protein liquid chromatography  columns in the following order: phenyl sepharose, affi-gel blue gel , mono-q, gel filtration-superdex  <dig>  and hydroxyapatite  .
the final active fractions were concentrated by a mono-q or a q-spin column.
the activity of the fractions from each step of the purification was analyzed, and the purification was monitored by bradford protein assay and sds-page .
the final q concentration step showed that the nuclease activity was highly enriched in three fractions .
the active fraction contained low amount of protein, which could only be visualized on a silver-stained gel .
the gel filtration step of the purification indicated the active nuclease was approximately  <dig> kda in size .
after obtaining highly-active fractions, we characterized the specificity of the enzyme; both enzyme titration and time-course assays showed significant preference for telomeric over non-telomeric substrates .
the telomere repeat-containing substrates were efficiently processed by the nuclease  throughout the incubation time while the non-telomeric and 5'-strand labeled substrates were only minimally degraded .
cleavage was specific to telomeric 5'-strands: the substrates that were labeled on the 3'-strand showed no degradation by the nuclease .
thus, processing of the blunt substrates by this nuclease leads to 3'-overhang formation.
the tetrahymena nuclease is a dna exonuclease
we examined whether the tetrahymena nuclease could cleave rna substrates.
we tested both non-telomeric and telomeric dna  and the corresponding rna  in substrate structures as either heteroduplex dna/rna or homoduplex dna/dna or rna/rna.
the nuclease cleaved the telomeric dna substrates in a dna/dna homoduplex , but showed a lower level of cleavage activity on the telomeric dna/rna in a heteroduplex structure where the c strand was the dna strand .
no cleavage activity was observed when the c strand was made of rna in either rna/rna or rna/dna duplexes .
thus, we conclude that the tetrahymena nuclease is a dnase, specific to cleaving dna linkages.
to determine whether the repeat length affected nuclease cleavage activity, we examined the consequence of varying the repeat numbers in the telomeric substrates .
we observed substantial cleavage at similar level on 3-repeat, 4-repeat, and 5-repeat oligonucleotide, indicating that the cleavage by this tetrahymena nuclease is not affected by telomeric repeat length.
the lack of length dependence suggested that the enzyme may be degrading the substrates from the end, like an exonuclease.
exonuclease activity removes a mononucleotide from free dna ends while an endonuclease removes oligonucleotide at each cleavage event  <cit> .
to examine the type of degradation this nuclease exhibits, we used end-labeled substrates rather than the internally-labeled substrates described above .
with these end-labeled phosphorylated substrates, we observed cleavage on both non-telomeric and telomeric substrates , and the cleavage product was mononucleotide.
the decreased specificity for telomere repeats was due to the addition of the phosphate on the 5' strand as described in detail below.
because the first nucleotides on the non-telomeric and telomeric substrates were different, the mononucleotide products exhibited different mobility on a gel.
this data suggested that tetrahymena nuclease is an exonuclease.
to further examine the potential exonuclease activity of the tetrahymena enzyme, we used oligonucleotides with specific bonds containing phosphorothioate linkage modification.
this modification blocks cleavage activity of nucleases.
we first tested the internally-labeled substrates that each contained one to several adjacent phosphorothioate linkages at positions 1- <dig>  5- <dig>  6- <dig>  7- <dig>  and 8- <dig>  .
while the unmodified non-telomeric and telomeric substrates behaved as expected, we saw complete blockage of nuclease activity when there were phosphorothioate linkages at position 1- <dig> .
when phosphorothioate linkages were placed at more internal positions, we saw significant cleavage that did not extend as far into the dna as the unmodified substrates, indicating cleavage only occurred up to the blockage sites .
to further examine the exonuclease activity, we synthesized substrates with individual bonds blocked with phosphorothioate modification and labeled at the 5'-end .
we tested these 5'-labeled substrates with the tetrahymena nuclease.
blockage between nucleotide positions 1- <dig> significantly reduced the cleavage by the nuclease  whereas terminal cleavage still occurred on all other substrates .
we concluded that the tetrahymena nuclease activity we isolated is an exonuclease.
when using end-labeled substrates, we also detected a band that appeared to be larger than a mononucleotide .
while the position of this band first suggested that it might be an endonucleolytic cleavage product, it was blocked by the phosphorothioate at the 1- <dig> position.
we tried to map the size of this product, but its mobility varied depending on the acrylamide concentration, and it did not line up with either 3'-phosphate or 5'-phosphate marker ladders .
we conclude that this may be a modified nucleotide given that its generation is blocked by the terminal phosphorothioate linkage.
the tetrahymena nuclease only cleaves specific permutations of the 5'-telomeric c-rich strand
to further examine the sequence specificity of the exonuclease activity, we examined all the permutations of the tetrahymena 6-nucleotide telomeric repeat sequence.
surprisingly, there was significantly more cleavage of telomeric substrates with sequences ending in 5'-aa or 5'-ca while other permutations were not cleaved .
we also tested telomeric repeat sequences from human  and yeast  and observed cleavage on both of these substrates .
as a negative control, we tested a random non-telomeric 6-nucleotide repeat  and found no significant degradation .
the specificity for certain permutations of the ccccaa repeat suggested that there may be an end-nucleotide sequence preference.
many exonucleases have been biochemically characterized and have been grouped based on their processing polarity, domain similarity, and substrate specificity, such as structure-specific 5' nucleases, 3'-5' exonucleases, metallo-beta-lactamase family, and single-stranded-specific nucleases .
none of these exonucleases that have been previously characterized show sequences specificity.
thus, we sought to characterize the sequence specificity of this tetrahymena exonuclease in detail.
since our nuclease is an exonuclease, to examine the sequence specificity, we compared the activity of the tetrahymena exonuclease to the activity of the well-characterized exo <dig> that has been implicated in telomere processing.
exo <dig> has a strong preference for cleavage of dna with a 5'-phosphate  <cit> .
thus, we first compared the cleavage of 5'-phosphorylated and unphosphorylated substrates with the purified tetrahymena nuclease .
both phosphorylated and unphosphorylated telomeric substrates were cleaved to a similar extent by the tetrahymena nuclease .
there was also an increase in cleavage activity on the 5'-phosphorylated, non-telomeric substrate.
while this non-telomeric cleavage on 5'-phosphorylated substrate was less extensive than cleavage on the telomeric substrate, this data implies a reduced specificity for telomeric substrates on 5'-phosphorylated dna.
we next utilized human exo <dig> in cleavage assays to compare the biochemical properties of our tetrahymena nuclease.
for convenience, whenever exo <dig> activity is mentioned in this study, it refers to human exo <dig>  enzyme titration of exo <dig> with phosphorylated substrates showed that it robustly cleaves dna from 5'-end into the substrate up to the internal labeling site, releasing the internally labeled nucleotide that runs at the bottom of the gel .
however, exo <dig> did not show any preference for telomeric versus non-telomeric substrates .
besides cleavage on single-stranded dna, exo <dig> also exhibits structural preferences on certain substrates.
as an exonuclease, exo <dig> prefers 5'-recessed structures, and as an endonuclease, it cleaves flapped structures  <cit> .
because of these structural preferences for exo <dig>  we tested the specificities of these two nucleases with substrates that had different end structures .
we designed telomeric, and non-telomeric substrates ending in either 5'-cgaa or 5'-aatg that were either blunt, or had a 3'- or 5'-overhang.
using non-phosphorylated substrates, all of the telomeric substrates were actively cleaved by the tetrahymena nuclease .
the exo <dig> digestions showed a preference for substrates that contained a 3' overhang  as described previously  <cit> .
we also examined the activities of both enzymes on phosphorylated substrates and found similar results, although the level of exo <dig> cleavage was higher .
since there was no preference for the different structures tested with the tetrahymena exonuclease, we concluded that this enzyme does not show the structure-dependent activity of exo <dig>
tetrahymena exonuclease shows end-nucleotide specificity
because the tetrahymena exonuclease showed a preference for aa and ca terminal nucleotides on nonphosphorylated telomeric substrates , we next ask whether end-sequence specificity is preserved in the presence of 5'-phosphorylated substrates.
we examined the six different permutations of the telomere repeat using both phosphorylated and nonphosphorylated telomeric substrates.
the tetrahymena nuclease showed sequence preference for aa and ca ending substrates , while exo <dig> did not show significant sequence specificity.
we next tested a series of non-telomeric substrates with all possible permutations of the 5'-terminal two nucleotides .
the substrates were all internally-labeled.
when testing unphosphorylated substrates , there was some cleavage of all substrates, except when g was the terminal nucleotide.
there was a clear preference for substrates ending with an a residue.
the order of cleavage preference for the terminal nucleotide was a>t>c>g .
however, the cleavage pattern on these non-telomeric substrates was limited to terminal nucleotides compared to the more extensive cleavage of the telomeric substrate .
this difference in the extent of cleavage may be due to the specific nucleotide that is exposed next or may be influenced by contacts with internal nucleotides in the telomere repeats.
the possibility of contacts with internal nucleotides could also explain the low cleavage observed on yeast repeat sequences ending in 5'-ac when compared to the tetrahymena repeat permutation ending in 5'-ac .
incubation of the tetrahymena exonuclease with the same set of sequences that were 5'-phosphorylated showed a similar trend of sequence specificity .
exo <dig> was tested along side the tetrahymena exonuclease, and it cleaved all phosphorylated substrates well.
however, exo <dig> did not show any specificity for the terminal nucleotide sequences , note the similar intensity of the mononucleotide band in every 3rd lane on the gel.
unphosphorylated substrates were also cleaved by exo <dig> without any sequence preferences, except that the level of cleavage was lower .
together, this data suggests that unlike exo <dig>  which is a structure-specific nuclease, the tetrahymena nuclease is a sequence-dependent exonuclease.
discussion
we have identified a sequence-dependent exonuclease activity that possesses the biochemical properties to process c-rich telomere strands and generate 3' g-strand overhangs.
this 3'-overhang is a ubiquitous structure of eukaryotic telomeres, and is essential for telomere end protection and for elongation by telomerase.
the tetrahymena nuclease we described is a  <dig> kda enzyme that shows preference for cleaving aa- or ca-ending residues that are present in the c-strand of telomeric repeats.
the existence of a telomere-specific nuclease has been postulated for years.
a number of nucleases including mre <dig>  sae <dig>  dna <dig>  artemis, apollo, werner syndrome protein , and exo <dig> have been examined for their role in different aspects of telomere processing  <cit> .
mre <dig> and wrn are both 3'-5' nucleases that have been found associated with telomeres  <cit> .
while the directionality of mre <dig> and wrn is not consistent with 5'-strand resection at the telomere, recent biochemical studies of wrn show a sequence-specific cleavage on telomeric 3'-overhang  <cit> , suggesting wrn may pay a role in processing the g-rich overhang strand.
sae <dig>  artemis, apollo, and dna <dig> are 5' nucleases, but none of these nucleases show biochemical specificity for either blunt-ended or telomeric substrates.
apollo, interacts with the telomere-binding protein, trf <dig>  and cleaves single-stranded dna but shows no sequence preferences in vitro between telomeric repeats and unique sequences  <cit> .
recent studies have showed that apollo is recruited by trf <dig> and plays a role in processing leading-strand overhangs in mice  <cit> , and human apollo cooperates with trf <dig> to help resolve topological stress during telomere replication  <cit> .
sae <dig> is an endonuclease with substrate specificity on 3' overhangs, hairpins, and branched dna  <cit> , and it processes hairpin structures and meiotic double-strand breaks together with mrx/n complex  <cit> .
dna <dig> is a multi-functional enzyme that possesses helicase and single-stranded dna specific endonuclease activities.
in s. pombe, dna <dig> was implicated in telomere 3'-overhang generation  <cit> , but in vitro, dna <dig> shows no sequence specificity for cleavage of single-stranded dna .
these differences in biological specificity indicate that the tetrahymena exonuclease is not the same enzyme as any of these known 5'-3' nucleases.
because our enzyme has a 5'-3' exonuclease activity, we compared its biochemical properties with exo <dig>  exo <dig> is a well-characterized 5'-3' nuclease that plays a role in multiple dna metabolic pathways  <cit> , including telomere maintenance  <cit> .
in vivo evidence suggests that exo <dig> contributes to telomere maintenance  <cit>  and may participate in the generation of ssdna at dysfunctional telomeres  <cit> .
studies have also shown that exo <dig> is important in processing ends for the recombination pathways that allow survivors to grow in the absence of telomerase in yeast  <cit> .
the biochemical specificity we report for the tetrahymena exonuclease is different from the properties of exo <dig>  the tetrahymena nuclease has specificity for telomere repeats ending in 5'-aa and 5'-ca, but exo  <dig> did not show sequence specificity.
in addition, exo <dig> shows much higher activity on phosphorylated substrates  while the tetrahymena exonuclease has significant activity on both unphosphorylated and phosphorylated substrates.
these differences in specificity indicate that the tetrahymena exonuclease that we purified is distinct from exo <dig>
several lines of evidence indicate that c-strand processing is regulated; the precise structure of 3'-overhangs in ciliates  <cit> , the cell cycle-specific generation of 3'-overhangs in yeast, and the formation of overhangs in the absence of telomerase  <cit> .
this regulation occurs through both cdk <dig>  <cit>  and through telomere-specific binding proteins.
telomere end-binding proteins, such as tebp αβ heterodimer, first discovered in oxytricha  <cit> , bind to single-stranded telomeric dna through an ob fold and protect the telomeres from degradation, recombination, and end-joining  <cit> .
in yeast, cdc <dig> binds to the single-stranded overhang, and together with stn <dig> and ten <dig>  <cit>  protect the ends and play a role in end-processing and telomere elongation.
in mammalian cells, pot <dig>  a component of the shelterin complex, binds the single-stranded overhang through an ob fold domain similar to the tebp α  <cit> .
pot <dig> also plays a role along with other shelterin complex components in regulating access of processing nucleases to telomeres  <cit> .
thus, the specific action of telomere processing nucleases on telomeres will involve interaction with these telomere-specific binding proteins.
CONCLUSIONS
telomere structure and function is conserved throughout eukaryotes.
telomerase is present in most eukaryotes and is essential for telomere elongation.
the negative feedback mechanism in which telomere-binding proteins inhibit elongation of long telomeres and allow the establishment of a telomere length equilibrium  <cit>  is also conserved from yeast to mammals.
while the sequence of telomere-binding proteins in different organisms differ, the function of these proteins in end protection and regulation of telomere elongation is highly conserved  <cit> .
given this conservation, we think it is likely that telomere processing is also conserved.
while there are likely multiple factors involved in telomere processing, the sequence-specific properties of the tetrahymena exonuclease described here suggest it may play a role in telomere processing.

 functional telomeres are essential for cell survival. telomeres distinguish natural chromosome ends from dna breaks. when telomere function is lost, cells arrest and undergo senescence or apoptosis. normal dna replication causes telomere shortening, which is counterbalanced by the extension activity of telomerase. the majority of cancers have telomerase activity, and this activity is needed for ongoing division of tumor cells. in somatic cells, telomerase activity is absent or low. when cell division occurs faster than the ability of telomerase to elongate telomeres, the short telomeres limit cell replicative capacity. this limited cell division underlies diseases of tissue renewal such as dyskeratosis congenita, aplastic anemia, and pulmonary fibrosis. thus, telomere length regulation plays a pivotal role in cancer and in age-related degenerative disease. both telomeric dna structure and telomerase play a role in establishing telomere length and end protection. telomeres have a g-rich 3'-overhang that was first discovered in ciliates and is a conserved feature of telomeres in all eukaryotes. this 3'-overhang is the binding site for essential telomere proteins, such as cdc in saccharomyces cerevisiae and pot in mammals and other species. loss of the overhangs due to disruption of the telomere complex, termed shelterin, leads to telomere-induced dna damage response and chromosome end-to-end fusion. in addition to maintenance of dna ends, the 3'-overhang is essential for telomerase accessibility. telomerase recognizes and elongates single-stranded dna substrates, but not double-stranded substrates. thus, the processing of blunt ends, generated by replication, is needed for telomerase to elongate telomeres. in vivo, blocking 3'-overhang formation in s. cerevisiae blocks the ability of telomerase to elongate a telomere seed sequence. 3'-overhang formation is an active process. in s. cerevisiae and mice, generation of the 3'-overhang occurs in the absence of telomerase. in tetrahymena, a conditional telomerase protein component knockout cell line showed that telomerase depletion had relatively little effects on absolute g overhang length, indicating c-strand degradation, not telomerase elongation of the g-rich strand, must generate the overhang structures. inhibition of cdk blocks c-rich strand resection, thereby inhibiting 3'-overhang production. these data suggest that there is a nuclease regulated by cdk that processes the telomeres prior to elongation by telomerase. additional evidence for an active processing enzyme comes from the fact that both ends of a chromosome have similar overhang structures. replication of a chromosome to the very end is predicted to generate one chromosome end with a 3'-overhang and one with a blunt end. however, chromosomes in ciliates as well as in yeast and mammals have 3'-overhangs on both ends,, implying at least one end must be processed to generate the overhang structure. finally, the very precise structure of the telomere overhangs implies that it is formed by a regulated process. the length and sequence of the 3'-overhangs in oxytricha and tetrahymena are surprisingly homogeneous. in oxytricha, there is a precise overhang of two nucleotide 5'-ttttgggg-3' repeats, whereas in tetrahymena, the overhangs are either 14- or 20- nucleotides in length, and the majority of 5'-strands end in 3'-ccaacc-5', or 3'-caaccc-5'. in mammals, this overhang length is also conserved. this functional and structural evidence argues for the existence of a sequence-specific nuclease that processes telomeres. to investigate the role of 3'-overhang processing, we set out to identify a telomere-specific nuclease activity. we chose tetrahymena to look for processing activity because it has uniform telomere repeat sequence, a precise 3'-overhang structure, and a history as an excellent biochemical source for telomere-processing factors. here we describe a sequence-dependent 5' to 3' exonuclease activity from tetrahymena that processes the c-strand of telomere substrates in vitro.  to identify an overhang-processing nuclease, we assayed cell extracts from tetrahymena. we designed specific duplex oligonucleotide substrates to detect a telomere-specific nuclease activity. the duplex oligonucleotide substrate contains specific non-telomeric sequences with a biotin moiety that binds to streptavidin to block non-specific degradation from one end. on the other end, the substrates contained either telomere repeats or non-telomeric sequence. we tested substrates that were either blunt-ended, or with a 5'- or a 3'-overhang. to distinguish size changes of the substrates on each strand, we labeled each strand of the substrate separately in order to monitor processing occurred on the corresponding strand. identification of a nuclease activity from tetrahymena crude extracts with preference for telomere substrates we initially incubated the non-telomeric and telomeric blunt-ended substrates with crude tetrahymena cell extracts and analyzed processing by gel electrophoresis and autoradiography. we observed a nuclease activity that degraded the telomeric substrate to a greater degree than the non-telomeric substrate. this activity was robust, strand-specific, and sequence-dependent. the nuclease cleaved the 5'-strand of a blunt-ended substrate containing telomere repeats. to further characterize this nuclease activity, we performed biochemical purification. extracts were precipitated with ammonium sulfate, and then enriched on several fast protein liquid chromatography columns in the following order: phenyl sepharose, affi-gel blue gel, mono-q, gel filtration-superdex and hydroxyapatite. the final active fractions were concentrated by a mono-q or a q-spin column. the activity of the fractions from each step of the purification was analyzed, and the purification was monitored by bradford protein assay and sds-page. the final q concentration step showed that the nuclease activity was highly enriched in three fractions. the active fraction contained low amount of protein, which could only be visualized on a silver-stained gel. the gel filtration step of the purification indicated the active nuclease was approximately kda in size. after obtaining highly-active fractions, we characterized the specificity of the enzyme; both enzyme titration and time-course assays showed significant preference for telomeric over non-telomeric substrates. the telomere repeat-containing substrates were efficiently processed by the nuclease throughout the incubation time while the non-telomeric and 5'-strand labeled substrates were only minimally degraded. cleavage was specific to telomeric 5'-strands: the substrates that were labeled on the 3'-strand showed no degradation by the nuclease. thus, processing of the blunt substrates by this nuclease leads to 3'-overhang formation. the tetrahymena nuclease is a dna exonuclease we examined whether the tetrahymena nuclease could cleave rna substrates. we tested both non-telomeric and telomeric dna and the corresponding rna in substrate structures as either heteroduplex dna/rna or homoduplex dna/dna or rna/rna. the nuclease cleaved the telomeric dna substrates in a dna/dna homoduplex, but showed a lower level of cleavage activity on the telomeric dna/rna in a heteroduplex structure where the c strand was the dna strand. no cleavage activity was observed when the c strand was made of rna in either rna/rna or rna/dna duplexes. thus, we conclude that the tetrahymena nuclease is a dnase, specific to cleaving dna linkages. to determine whether the repeat length affected nuclease cleavage activity, we examined the consequence of varying the repeat numbers in the telomeric substrates. we observed substantial cleavage at similar level on 3-repeat, 4-repeat, and 5-repeat oligonucleotide, indicating that the cleavage by this tetrahymena nuclease is not affected by telomeric repeat length. the lack of length dependence suggested that the enzyme may be degrading the substrates from the end, like an exonuclease. exonuclease activity removes a mononucleotide from free dna ends while an endonuclease removes oligonucleotide at each cleavage event. to examine the type of degradation this nuclease exhibits, we used end-labeled substrates rather than the internally-labeled substrates described above. with these end-labeled phosphorylated substrates, we observed cleavage on both non-telomeric and telomeric substrates, and the cleavage product was mononucleotide. the decreased specificity for telomere repeats was due to the addition of the phosphate on the 5' strand as described in detail below. because the first nucleotides on the non-telomeric and telomeric substrates were different, the mononucleotide products exhibited different mobility on a gel. this data suggested that tetrahymena nuclease is an exonuclease. to further examine the potential exonuclease activity of the tetrahymena enzyme, we used oligonucleotides with specific bonds containing phosphorothioate linkage modification. this modification blocks cleavage activity of nucleases. we first tested the internally-labeled substrates that each contained one to several adjacent phosphorothioate linkages at positions 1- 5- 6- 7- and 8-. while the unmodified non-telomeric and telomeric substrates behaved as expected, we saw complete blockage of nuclease activity when there were phosphorothioate linkages at position 1-. when phosphorothioate linkages were placed at more internal positions, we saw significant cleavage that did not extend as far into the dna as the unmodified substrates, indicating cleavage only occurred up to the blockage sites. to further examine the exonuclease activity, we synthesized substrates with individual bonds blocked with phosphorothioate modification and labeled at the 5'-end. we tested these 5'-labeled substrates with the tetrahymena nuclease. blockage between nucleotide positions 1- significantly reduced the cleavage by the nuclease whereas terminal cleavage still occurred on all other substrates. we concluded that the tetrahymena nuclease activity we isolated is an exonuclease. when using end-labeled substrates, we also detected a band that appeared to be larger than a mononucleotide. while the position of this band first suggested that it might be an endonucleolytic cleavage product, it was blocked by the phosphorothioate at the 1- position. we tried to map the size of this product, but its mobility varied depending on the acrylamide concentration, and it did not line up with either 3'-phosphate or 5'-phosphate marker ladders. we conclude that this may be a modified nucleotide given that its generation is blocked by the terminal phosphorothioate linkage. the tetrahymena nuclease only cleaves specific permutations of the 5'-telomeric c-rich strand to further examine the sequence specificity of the exonuclease activity, we examined all the permutations of the tetrahymena 6-nucleotide telomeric repeat sequence. surprisingly, there was significantly more cleavage of telomeric substrates with sequences ending in 5'-aa or 5'-ca while other permutations were not cleaved. we also tested telomeric repeat sequences from human and yeast and observed cleavage on both of these substrates. as a negative control, we tested a random non-telomeric 6-nucleotide repeat and found no significant degradation. the specificity for certain permutations of the ccccaa repeat suggested that there may be an end-nucleotide sequence preference. many exonucleases have been biochemically characterized and have been grouped based on their processing polarity, domain similarity, and substrate specificity, such as structure-specific 5' nucleases, 3'-5' exonucleases, metallo-beta-lactamase family, and single-stranded-specific nucleases. none of these exonucleases that have been previously characterized show sequences specificity. thus, we sought to characterize the sequence specificity of this tetrahymena exonuclease in detail. since our nuclease is an exonuclease, to examine the sequence specificity, we compared the activity of the tetrahymena exonuclease to the activity of the well-characterized exo that has been implicated in telomere processing. exo has a strong preference for cleavage of dna with a 5'-phosphate. thus, we first compared the cleavage of 5'-phosphorylated and unphosphorylated substrates with the purified tetrahymena nuclease. both phosphorylated and unphosphorylated telomeric substrates were cleaved to a similar extent by the tetrahymena nuclease. there was also an increase in cleavage activity on the 5'-phosphorylated, non-telomeric substrate. while this non-telomeric cleavage on 5'-phosphorylated substrate was less extensive than cleavage on the telomeric substrate, this data implies a reduced specificity for telomeric substrates on 5'-phosphorylated dna. we next utilized human exo in cleavage assays to compare the biochemical properties of our tetrahymena nuclease. for convenience, whenever exo activity is mentioned in this study, it refers to human exo enzyme titration of exo with phosphorylated substrates showed that it robustly cleaves dna from 5'-end into the substrate up to the internal labeling site, releasing the internally labeled nucleotide that runs at the bottom of the gel. however, exo did not show any preference for telomeric versus non-telomeric substrates. besides cleavage on single-stranded dna, exo also exhibits structural preferences on certain substrates. as an exonuclease, exo prefers 5'-recessed structures, and as an endonuclease, it cleaves flapped structures. because of these structural preferences for exo we tested the specificities of these two nucleases with substrates that had different end structures. we designed telomeric, and non-telomeric substrates ending in either 5'-cgaa or 5'-aatg that were either blunt, or had a 3'- or 5'-overhang. using non-phosphorylated substrates, all of the telomeric substrates were actively cleaved by the tetrahymena nuclease. the exo digestions showed a preference for substrates that contained a 3' overhang as described previously. we also examined the activities of both enzymes on phosphorylated substrates and found similar results, although the level of exo cleavage was higher. since there was no preference for the different structures tested with the tetrahymena exonuclease, we concluded that this enzyme does not show the structure-dependent activity of exo tetrahymena exonuclease shows end-nucleotide specificity because the tetrahymena exonuclease showed a preference for aa and ca terminal nucleotides on nonphosphorylated telomeric substrates, we next ask whether end-sequence specificity is preserved in the presence of 5'-phosphorylated substrates. we examined the six different permutations of the telomere repeat using both phosphorylated and nonphosphorylated telomeric substrates. the tetrahymena nuclease showed sequence preference for aa and ca ending substrates, while exo did not show significant sequence specificity. we next tested a series of non-telomeric substrates with all possible permutations of the 5'-terminal two nucleotides. the substrates were all internally-labeled. when testing unphosphorylated substrates, there was some cleavage of all substrates, except when g was the terminal nucleotide. there was a clear preference for substrates ending with an a residue. the order of cleavage preference for the terminal nucleotide was a>t>c>g. however, the cleavage pattern on these non-telomeric substrates was limited to terminal nucleotides compared to the more extensive cleavage of the telomeric substrate. this difference in the extent of cleavage may be due to the specific nucleotide that is exposed next or may be influenced by contacts with internal nucleotides in the telomere repeats. the possibility of contacts with internal nucleotides could also explain the low cleavage observed on yeast repeat sequences ending in 5'-ac when compared to the tetrahymena repeat permutation ending in 5'-ac. incubation of the tetrahymena exonuclease with the same set of sequences that were 5'-phosphorylated showed a similar trend of sequence specificity. exo was tested along side the tetrahymena exonuclease, and it cleaved all phosphorylated substrates well. however, exo did not show any specificity for the terminal nucleotide sequences, note the similar intensity of the mononucleotide band in every 3rd lane on the gel. unphosphorylated substrates were also cleaved by exo without any sequence preferences, except that the level of cleavage was lower. together, this data suggests that unlike exo which is a structure-specific nuclease, the tetrahymena nuclease is a sequence-dependent exonuclease. discussion we have identified a sequence-dependent exonuclease activity that possesses the biochemical properties to process c-rich telomere strands and generate 3' g-strand overhangs. this 3'-overhang is a ubiquitous structure of eukaryotic telomeres, and is essential for telomere end protection and for elongation by telomerase. the tetrahymena nuclease we described is a kda enzyme that shows preference for cleaving aa- or ca-ending residues that are present in the c-strand of telomeric repeats. the existence of a telomere-specific nuclease has been postulated for years. a number of nucleases including mre sae dna artemis, apollo, werner syndrome protein, and exo have been examined for their role in different aspects of telomere processing. mre and wrn are both 3'-5' nucleases that have been found associated with telomeres. while the directionality of mre and wrn is not consistent with 5'-strand resection at the telomere, recent biochemical studies of wrn show a sequence-specific cleavage on telomeric 3'-overhang, suggesting wrn may pay a role in processing the g-rich overhang strand. sae artemis, apollo, and dna are 5' nucleases, but none of these nucleases show biochemical specificity for either blunt-ended or telomeric substrates. apollo, interacts with the telomere-binding protein, trf and cleaves single-stranded dna but shows no sequence preferences in vitro between telomeric repeats and unique sequences. recent studies have showed that apollo is recruited by trf and plays a role in processing leading-strand overhangs in mice, and human apollo cooperates with trf to help resolve topological stress during telomere replication. sae is an endonuclease with substrate specificity on 3' overhangs, hairpins, and branched dna, and it processes hairpin structures and meiotic double-strand breaks together with mrx/n complex. dna is a multi-functional enzyme that possesses helicase and single-stranded dna specific endonuclease activities. in s. pombe, dna was implicated in telomere 3'-overhang generation, but in vitro, dna shows no sequence specificity for cleavage of single-stranded dna. these differences in biological specificity indicate that the tetrahymena exonuclease is not the same enzyme as any of these known 5'-3' nucleases. because our enzyme has a 5'-3' exonuclease activity, we compared its biochemical properties with exo exo is a well-characterized 5'-3' nuclease that plays a role in multiple dna metabolic pathways, including telomere maintenance. in vivo evidence suggests that exo contributes to telomere maintenance and may participate in the generation of ssdna at dysfunctional telomeres. studies have also shown that exo is important in processing ends for the recombination pathways that allow survivors to grow in the absence of telomerase in yeast. the biochemical specificity we report for the tetrahymena exonuclease is different from the properties of exo the tetrahymena nuclease has specificity for telomere repeats ending in 5'-aa and 5'-ca, but exo did not show sequence specificity. in addition, exo shows much higher activity on phosphorylated substrates while the tetrahymena exonuclease has significant activity on both unphosphorylated and phosphorylated substrates. these differences in specificity indicate that the tetrahymena exonuclease that we purified is distinct from exo several lines of evidence indicate that c-strand processing is regulated; the precise structure of 3'-overhangs in ciliates, the cell cycle-specific generation of 3'-overhangs in yeast, and the formation of overhangs in the absence of telomerase. this regulation occurs through both cdk and through telomere-specific binding proteins. telomere end-binding proteins, such as tebp αβ heterodimer, first discovered in oxytricha, bind to single-stranded telomeric dna through an ob fold and protect the telomeres from degradation, recombination, and end-joining. in yeast, cdc binds to the single-stranded overhang, and together with stn and ten protect the ends and play a role in end-processing and telomere elongation. in mammalian cells, pot a component of the shelterin complex, binds the single-stranded overhang through an ob fold domain similar to the tebp α. pot also plays a role along with other shelterin complex components in regulating access of processing nucleases to telomeres. thus, the specific action of telomere processing nucleases on telomeres will involve interaction with these telomere-specific binding proteins.  telomere structure and function is conserved throughout eukaryotes. telomerase is present in most eukaryotes and is essential for telomere elongation. the negative feedback mechanism in which telomere-binding proteins inhibit elongation of long telomeres and allow the establishment of a telomere length equilibrium is also conserved from yeast to mammals. while the sequence of telomere-binding proteins in different organisms differ, the function of these proteins in end protection and regulation of telomere elongation is highly conserved. given this conservation, we think it is likely that telomere processing is also conserved. while there are likely multiple factors involved in telomere processing, the sequence-specific properties of the tetrahymena exonuclease described here suggest it may play a role in telomere processing. 
we used biochemical purification to identify a sequence-dependent exonuclease activity in tetrahymena thermophila cell extracts. the nuclease activity showed specificity for 5'-ends containing aa or ac sequences, unlike exo which showed sequence-independent cleavage. the tetrahymena nuclease was active on both phosphorylated and unphosphorylated substrates whereas exo requires a 5'-phosphate for cleavage. telomere function requires a highly conserved g rich 3'- overhang.
 telomere function requires a highly conserved g rich 3'- overhang. this structure is formed by 5'-resection of the c-rich telomere strand. however, while many nucleases have been suggested to play a role in processing, it is not yet clear which nucleases carry out this 5'-resection.  we used biochemical purification to identify a sequence-dependent exonuclease activity in tetrahymena thermophila cell extracts. the nuclease activity showed specificity for 5'-ends containing aa or ac sequences, unlike exo which showed sequence-independent cleavage. the tetrahymena nuclease was active on both phosphorylated and unphosphorylated substrates whereas exo requires a 5'-phosphate for cleavage.  the specificities of the enzyme indicate that this novel tetrahymena exonuclease is distinct from exo and has properties required for 3'-overhang formations at telomeres. 