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J. Biol. Chem., Vol. 275, Issue 31, 24199-24207, August 4, 2000
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From the Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215
Received for publication, April 16, 2000
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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The protein catalytic subunit of telomerase
(TERT) is a reverse transcriptase (RT) that utilizes an internal RNA
molecule as a template for the extension of chromosomal DNA ends. In
all retroviral RTs there is a conserved tyrosine two amino acids
preceding the catalytic aspartic acids in motif C, a motif that is
critical for catalysis. In TERTs, however, this position is a leucine, valine, or phenylalanine. We developed and characterized a robust in vitro reconstitution system for Tetrahymena
telomerase and tested the effects of amino acid substitutions on
activity. Substitution of the retroviral-like tyrosine in motif C did
not change overall enzymatic activity but increased processivity. This
increase in processivity correlated with an increased affinity for
telomeric DNA primer. Substitution of an alanine did not increase
processivity, while substitution of a phenylalanine had an intermediate
effect. The data suggest that this amino acid is involved in
interactions with the primer in telomerase as in other RTs, and show
that mutating an amino acid to that conserved in retroviral RTs makes
telomerase more closely resemble these other RTs.
Telomeres form a protective cap on chromosome ends and are usually
composed of short G-rich DNA repeats complexed with proteins (1). The
telomeres of unicellular eukaryotes and of the germ cells of
multicellular organisms are maintained by the enzyme telomerase, which
was first identified in the ciliated protozoan Tetrahymena
thermophila (2). Telomerase activity has also been detected in
many human cancer cells and appears to be necessary for their continued
growth (3-5).
Telomerase is a reverse transcriptase that utilizes an internal RNA
molecule as a template for the extension of DNA ends (6). The RNA
component of telomerase has been cloned from many different organisms
(reviewed in Ref. 7). The protein catalytic subunit of telomerase,
known as TERT for telomerase reverse
transcriptase, was first identified in the ciliate
Euplotes aediculatus and the yeast Saccharomyces
cerevisiae (8, 9) and has since been identified in humans, mice,
fission yeast, plants, and other ciliated protozoa (10-18). The
evolutionary conservation of
TERT1 suggested that it and
the telomerase RNA form a core catalytic unit. This was supported by
experiments in which in vitro translated TERT and the
telomerase RNA were sufficient to reconstitute both human and
Tetrahymena telomerase activity (15, 19, 20), although
reconstitution was dependent on the presence of chaperone proteins in
the rabbit reticulocyte lysate used for in vitro translation (21, 22).
The derived amino acid sequences of the TERT proteins contain motifs
common to all reverse transcriptases, consistent with the role of
telomerase in RNA-templated DNA polymerization (23). Within these
reverse transcriptase motifs is an invariant trio of aspartic acids
that are directly involved in catalysis (24, 25). Mutation of any of
these three aspartic acids in yeast or human TERT abolished telomerase
activity in vitro and in vivo, confirming the
importance of the RT motifs for telomerase catalysis (9, 10, 19, 26).
While the basic RT catalytic mechanism is undoubtedly conserved in
telomerase, there are many features of telomerase that distinguish it
from other RTs. The current model for the in vitro action of
Tetrahymena telomerase (27, 28) is shown in Fig.
1. The 3' end of the DNA primer first
base pairs with the telomerase RNA, and the primer is then extended using the RNA as a template. When the end of the 9-nucleotide (nt)
template region is reached, the primer either dissociates from the
complex, or translocates on the RNA template and realigns for another
round of elongation. In contrast, retroviral RTs do not form stable
complexes with the RNA template, and they catalyze processive DNA
synthesis that does not normally involve translocation of the template
relative to the primer (for an informative exception, see Ref. 29). The
amino acids that are unique to telomerase presumably contribute to
these differences. Even within the RT motifs, Tetrahymena
TERT and the RT from human immunodeficiency virus (HIV RT) share only
16% amino acid sequence identity. Outside these motifs the sequences
are even less conserved, and the TERTs have long amino- and
carboxyl-terminal regions that are absent in HIV RT. It is therefore of
interest to study the details of telomerase action to determine
similarities and differences from what is already known of the
mechanisms of other RTs.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (21K):
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Fig. 1.
A model for the mechanism of
Tetrahymena telomerase action. A, TERT
and its associated RNA subunit. The sequence of the template portion of
T. thermophila telomerase RNA is shown. B,
telomerase binds to a telomere end (in vivo) or a
telomere-like DNA primer (in vitro). Binding involves the
nucleotides at the 3' end of the primer base pairing with the RNA
template. Telomerase then catalyzes addition of nucleotides onto the
primer until the end of the template is reached. C, after
extension, the newly elongated product may either dissociate from
telomerase, or translocate to the beginning of the template
(D) allowing another round of elongation. Solid
arrows indicate processive synthesis; primer dissociation steps
indicated by dashed arrows decrease processivity.
One amino acid that differs between telomerase and other RTs is located
two residues prior to the two conserved catalytic aspartic acids in RT
motif C (Fig. 2). This residue is a
leucine, valine, or phenylalanine in all TERTs identified to date,
while it is a tyrosine in 29 out of 29 retroviral RTs (30).
Furthermore, this tyrosine is critical for catalytic activity of the
retroviral RTs. Mutation of this residue to alanine or serine in either
HIV RT or murine leukemia virus RT reduced reverse transcriptase
activity to 1-7% of wild type (wt) levels (31-34). Mutation of the
tyrosine to phenylalanine led to a more modest reduction of HIV RT
activity (22-30% of wt levels; Refs. 32 and 33), and had no effect on
murine leukemia virus RT activity (34). Examination of the crystal
structure of HIV RT provides a plausible explanation for these effects.
The shape of the protein resembles a right hand, with a large cleft
between the "fingers" and "thumb" domains in which the template
and primer nucleic acids bind (35, 36). The tyrosine of motif C
protrudes into this cleft and forms several contacts with the template
and primer: two carbon atoms in its side chain phenyl ring show
hydrophobic interactions with the deoxyribose of the terminal primer
nucleotide (37), and the hydroxyl group on its side chain hydrogen
bonds with atoms in the penultimate primer nucleotide (37, 38). It was
hence postulated that the tyrosine is involved in precisely positioning
the template-primer relative to the active site (37). Thus,
substitution of the tyrosine with a phenylalanine (which lacks a
hydroxyl group) would eliminate the hydrogen bond to the primer, and
substitution of an amino acid lacking a phenyl ring such as alanine or
serine would further reduce interaction with the primer.
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It is therefore curious that the TERT proteins do not need a tyrosine
at this site for activity. In order to further investigate the role of
this amino acid (Leu-813) in Tetrahymena telomerase action,
we mutated it to tyrosine, phenylalanine, and alanine. The tyrosine
mutant (L813Y), while not showing any change in telomerase activity
level, did show an increase in enzyme processivity. This is an unusual
case where a single amino acid substitution provides an
"improvement" in enzyme action. Because this substitution does not
occur in any of the nine TERTs that have been identified, we propose
that it may be advantageous to telomerases to synthesize rather small
numbers of repeats in a single enzymatic cycle.
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EXPERIMENTAL PROCEDURES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Construction of a Synthetic Tt_TERT Gene-- The cloning of the T. thermophila TERT gene has been described (14). A synthetic Tt_TERT gene was constructed in order to correct the 66 Tetrahymena Glu codons (TAA and TAG) to the universal genetic code and to change the codon usage to more closely resemble that of both Escherichia coli and rabbit (Oryctolagus cuniculus). 469 of the 3351 base pairs of the gene were changed without changing any encoded amino acids. The sequence of the synthetic gene is available from the authors upon request. A set of 42 100-mer oligonucleotides was synthesized on an ABI DNA synthesizer. These oligonucleotides spanned the Tt_TERT gene, with each alternate oligonucleotide representing the sense and antisense strands of the gene, respectively, and a 20-nt overlap between them. A combination of two approaches was used to assemble the gene. In the first approach, adjacent pairs of 100-mer oligonucleotides were amplified by polymerase chain reaction (PCR) utilizing short (24 nt) oligonucleotides complementary to the ends of the 100-mers, and the enzyme Pwo polymerase (Roche Molecular Biochemicals). The resulting PCR products were combined in pools of adjacent pairs and subjected to another round of PCR using the short primers. This process was repeated for 5 rounds of PCR, resulting in a single product of 2 kilobases representing the 5'-half of the gene, which was cloned into the BamHI and HindIII sites of the vector pCR-ScriptTM (Stratagene). In the second approach, all 42 100-mers were combined in one tube, extended with Klenow polymerase (New England Biolabs), ligated, and run on a 1% agarose gel. The smear at around 600-800 base pairs was cut out of the gel, purified with a gel extraction kit (Qiagen), and amplified with short PCR primers, spaced about 600 base pairs apart in the 3'-half of the gene sequence. The resulting fragments were digested with restriction enzymes, ligated, and cloned into the HindIII and HincII sites of pCR-ScriptTM. The inserts from the two plasmid constructs were combined to form the complete gene. Sequencing of the entire insert revealed 19 PCR-induced errors, which were repaired by site-directed mutagenesis (39-41). The insert was then subcloned into the BamHI and XhoI sites of the pET-28a expression vector (Novagen).
Mutant versions of synthetic Tt_TERT in pET-28a were constructed by site-directed mutagenesis (39-41). The presence of the mutation and the lack of any second site mutations were confirmed by sequencing.
In Vitro Translation of Tt_TERT--
Tt_TERT was expressed in
rabbit reticulocyte lysates using the TNT Coupled Reticulocyte Lysate
kit (Promega). In a typical 400-µl reaction, 8 µg of pET-28a-TERT
DNA was incubated with 432 ng (8 pmol) in vitro transcribed
telomerase RNA (see below), 32 µl of [35S]methionine
(1175 Ci/mmol, 10 µCi/µl; NEN Life Science Products Inc.), and
other kit components as recommended by the manufacturer, at 30 °C
for 60 min. Translation reactions were flash-frozen in liquid nitrogen
and stored at 
80 °C. The telomerase complex was either used in a
telomerase assay directly from the translation reaction, or first
purified on agarose beads coupled to antibodies to the T7-tag encoded
by the pET-28a expression vector. Immunopurification was carried out as
follows. T7-tag antibody-agarose beads (Novagen; 100 µl) were washed
4 times in 1.5 ml of Wash Buffer-100 (20 mM Tris acetate,
pH 7.5, 10% glycerol, 1 mM EDTA, 5 mM
MgCl2, 0.1% Nonidet P-40, 1 mM DTT, 100 mM potassium glutamate), centrifuging at 1,500 × g for 2 min between washes. The beads were incubated twice
with 1 ml of Blocking Buffer (20 mM Tris acetate, pH 7.5, 10% glycerol, 1 mM EDTA, 5 mM
MgCl2, 0.1% Nonidet P-40, 1 mM DTT, 100 mM potassium glutamate, 0.5 mg/ml lysozyme, 0.5 mg/ml
bovine serum albumin, 0.05 mg/ml glycogen, 0.1 mg/ml yeast RNA) for 15 min at 4 °C with agitation. Reticulocyte lysate translation reaction (350 µl) was added to 350 µl of Blocking Buffer and centrifuged at
16,000 × g for 10 min at 4 °C to remove any
particulates. The supernatant from that spin was added to the 100 µl
of blocked beads and agitated at 4 °C for 2 h. The beads were
washed 4 times in 1.3 ml of Wash Buffer-300 (20 mM Tris
acetate, pH 7.5, 10% glycerol, 1 mM EDTA, 5 mM
MgCl2, 0.1% Nonidet P-40, 1 mM DTT, 300 mM potassium glutamate), 2 times in 1.3 ml of TMG (10 mM Tris acetate, pH 8.0, 1 mM
MgCl2, 10% glycerol), and resuspended in 100 µl of TMG
to make a 1:1 slurry. To check protein recovery, 1.5 µl of the slurry
or 2.5 µl of the original translation reaction was added to
Laemmli's sample buffer (125 mM Tris-Cl, pH 6.8, 4% SDS,
0.005% bromphenol blue, 20% glycerol, 0.72 M
-mercaptoethanol), heated to 100 °C for 3 min, and
electrophoresed in an 8% polyacrylamide/SDS gel (42). The gel was
fixed in 25% isopropyl alcohol, 10% acetic acid for 30 min, dried at
80 °C, and exposed to a PhosphorImager screen overnight. The amount
of full-length protein was calculated by comparison to
[35S]methionine standards spotted onto the dried gel
(spots were placed on the front and back of the gel and their intensity
averaged). Immunopurification typically recovered 15-25% of the
full-length TERT protein present in the original translation reaction,
resulting in a final full-length TERT concentration of 2-10
nM in the 1:1 bead slurry.
In Vitro Transcription of Telomerase RNA-- A plasmid containing the Tetrahymena telomerase RNA gene, a promoter for T7 RNA polymerase and a hammerhead ribozyme self-cleavage domain to process the 5' end of the RNA was constructed as described (43). This plasmid was digested with the enzyme Ear1, which cuts at the 3' end of the telomerase RNA gene. Linearized plasmid (25 µg) was transcribed in a 500-µl reaction containing 40 mM Tris-Cl, pH 7.5, 12 mM MgCl2, 10 mM DTT, 2 mM spermidine, 1 mM each rNTP, and 25 µl of T7 RNA polymerase (1 mg/ml), at 37 °C for 60 min. Hammerhead cleavage was stimulated by the addition of an extra 10 mM MgCl2 and incubation at 50 °C for 30 min. The reaction was centrifuged briefly at 16,000 × g to remove the precipitate, ethanol precipitated, and resuspended in 100 µl of loading buffer (0.05% bromphenol blue, 0.05% xylene cyanol in formamide). The entire reaction was heated to 70 °C for 10 min, loaded on a 1.5-mm thick 4% polyacrylamide, 8 M urea gel and electrophoresed at 25 W for 1 h. UV shadowing allowed visualization of the band representing processed RNA, which was then excised and the RNA was eluted in 1 ml of TEN (10 mM Tris-Cl, pH 7.5, 1 mM EDTA, 0.25 M NaCl) at 4 °C overnight before ethanol precipitation.
Tetrahymena Extract Preparation--
T. thermophila
strain B7 (from the American Type Culture Collection) was grown
vegetatively and harvested as described (14). S100 extracts were made
from the cells as described (6). The extracts were partially purified
on a 2-ml DEAE-agarose (Bio-Rad) column. The column was equilibrated
with TMG before loading 5 ml of S100 extract, washed with 6 ml of TMG
plus 0.2 M potassium glutamate, and eluted with 12 ml of
TMG plus 0.35 M potassium glutamate in 1-ml fractions.
Fractions containing active telomerase were pooled, dialyzed against
TMG at 4 °C overnight, flash-frozen, and stored at 80 °C.
Telomerase Assay--
A standard telomerase assay included 10 µl of partially purified Tetrahymena extract, reticulocyte
lysate translation reaction, or 1:1 slurry of immunopurification beads
(see above). Where different translation reactions or
immunopurifications were being assayed in the same experiment, the
amounts used were adjusted in order to equalize their TERT protein
contents. Typical reactions contained full-length TERT to a
concentration of 1-5 nM. A standard 20-µl telomerase
assay included 1 × Telomerase Buffer (50 mM Tris-Cl, pH 8.3, 1.25 mM MgCl2, 5 mM DTT),
250 nM to 1 µM primer
[(G4T2)3], 100 µM
dTTP, and 10 µM [
-32P]dGTP at 80 Ci/mmol
(0.4 µl of nonradioactive dGTP at 450 µM and 1.6 µl
of [-32P]dGTP at 10 µCi/µl, 800 Ci/mmol). The
reaction was incubated at 30 °C for 60 min and terminated by the
addition of 80 µl of TES (50 mM Tris-Cl, pH 8.0, 20 mM EDTA, 0.2% SDS). The reaction was phenol/chloroform
extracted, ethanol precipitated, and resuspended in 5 µl of loading
buffer (0.05% bromphenol blue, 0.05% xylene cyanol in formamide). A
2.5-µl portion was loaded on a 12% polyacrylamide, 8 M
urea sequencing gel and electrophoresed in 1 × TBE at 50 watts for 2 h. The gel was dried at 80 °C and exposed overnight to a PhosphorImager screen. As a control for recovery and loading, a 100-mer
oligonucleotide (or an 18-mer for assays of Tetrahymena extracts) was end-labeled with [
-32P]ATP and
polynucleotide kinase (New England Biolabs), and 5,000 cpm was added to
the reaction prior to phenol/chloroform extraction. As a size marker,
(G4T2)3 primer was labeled with
terminal deoxynucleotidyltransferase (Amersham Pharmacia Biotech) and
either [-32P]dGTP or [-33P]ddGTP, and
5,000 or 10,000 cpm, respectively, were loaded on the gel.
For the pulse-chase telomerase assays (Figs. 4 A and
E, and 7, A and D) a 5' biotinylated
primer ((G4T2)3) (Integrated DNA Technologies) was used as a substrate. The biotinylated primer (250 nM) was incubated with immunopurified telomerase (the
volume adjusted to result in equal protein concentrations) under
standard assay conditions (1 × Telomerase Buffer (see above), 100 µM dTTP, and 10 µM
[-32P]dGTP, 80 Ci/mmol) for 2 min at 30 °C. A
"chase" of 40 µM non-biotinylated (G4T2)3 or
(G4T2)5G primer was then added, and
incubation at 30 °C continued for a total of 10, 20, or 30 min. A
control with no chase and a control in which non-biotinylated chase
primer was added prior to incubation were both incubated for 30 min at 30 °C. All reactions were terminated with 80 µl of TES, extracted with 100 µl of phenol/chloroform, and the biotinylated primer was
recovered on magnetic Streptavidin Dynabeads (Dynal) as follows. Dynabead slurry (50 µl; 10 mg/ml) was prewashed twice in 50 µl of
0.1 M NaOH, 0.05 M NaCl, once in 50 µl of 0.1 M NaCl, once in 50 µl of 2 × Binding Buffer (10 mM Tris-Cl, pH 7.5, 1 mM EDTA, 2 M
NaCl) and then resuspended in 100 µl 2 × Binding Buffer. The 100 µl of phenol-extracted telomerase reaction was added to the Dynabeads and incubated at room temperature for 10 min with rotation. The beads were then washed 3 times with 100 µl of 2 × Binding Buffer, resuspended in 5 µl of formamide loading buffer, and heated at 95 °C for 5 min to release the biotinylated products; 2.5 µl was electrophoresed as above. As a recovery and loading control, a 3'
biotinylated 12-mer primer was end-labeled with polynucleotide kinase
and 5,000 cpm was added to the reaction prior to phenol/chloroform extraction.
The 5' biotinylated primer
((G4T2)3) was also used as a
substrate for the bind-and-chase telomerase assay (Figs. 4C
and 7C). It was prebound at 250 nM with
immunopurified telomerase in 1 × Telomerase Buffer for 15 min at
30 °C. Non-biotinylated
((G4T2)3) chase primer (40 µM) and nucleotides dTTP and [-32P]dGTP
(concentrations as in standard assay) were then added (or nucleotides
alone for the no-chase controls). The reaction was incubated for a
further 10 or 30 min, terminated, and the biotinylated products were
isolated as above.
Primer Dissociation Assay--
A modified telomerase assay was
used to measure the rate of dissociation of primer from Tt_TERT (Fig.
8). An 18-mer oligonucleotide (GT2(G4T2)2G3)
was preincubated at a concentration of 100 nM with 10 µl
of immunopurified telomerase (1:1 bead slurry) in 1 × Telomerase Buffer at 30 °C for 15 min. A 21-mer competitor primer
(GT2(G4T2)3) was added
(final concentration 20 µM) and incubated with the
enzyme/primer mixture at 30 °C for varying lengths of time (see Fig.
8). Telomerase activity was initiated by the addition of nucleotides
(ddTTP at 100 µM and [-32P]dGTP at 10 µM) and allowed to extend the primers at 30 °C for 10 min. As a control to ensure that the excess of 21-mer did not allow the
enzyme to rebind 18-mer, both primers were preincubated with enzyme
simultaneously prior to telomerase extension. The reactions were
terminated and electrophoresed as above. To determine koff, the intensity of the major (larger)
extension band (indicated by * in Fig. 8A) was quantitated,
normalized to the combined intensities of all three major extension
products (18- and 21-mer) and plotted against time. The data were fit
to the equation y = ae
kt + b, where k = koff and
b represents a background in the experiment of about
10%.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Reconstitution and Characterization of Telomerase Activity in
Vitro--
It has previously been demonstrated that
Tetrahymena TERT protein, when coexpressed with
Tetrahymena telomerase RNA in rabbit reticulocyte lysates,
forms a complex capable of telomerase activity (15) and that formation
of this complex is dependent on factor(s) present in the reticulocyte
lysate (22). We were also able to reconstitute telomerase activity
in vitro (Fig. 3A).
In ciliates the codons TAA and TAG encode glutamine, whereas these are
stop codons in the "universal" genetic code. A synthetic
Tt_TERT gene was constructed both to correct these Glu
codons and to adjust the codon usage to more closely resemble that of
rabbit. The synthetic gene was cloned into the pET-28a expression
vector. When expressed in rabbit reticulocyte lysates in the presence
of [35S]methionine, a major protein band of ~130 kDa
was observed, corresponding to full-length protein. In addition,
several smaller proteins were visible, representing either products of
proteolytic degradation or proteins resulting from internal translation
start sites (Fig. 3B). The protein was expressed in the
presence of in vitro transcribed telomerase RNA, and
telomerase activity was assayed in the presence of a
(G4T2)3 telomeric primer, dTTP and
[-32P]dGTP. A ladder of primer extension products with
the 6-nt pausing pattern characteristic of Tetrahymena
telomerase was observed upon denaturing polyacrylamide gel
electrophoresis (Fig. 3A, lane 1). Activity was abolished by
the use of either a telomerase RNA lacking the template domain
(lane 2) or a TERT protein with the catalytic aspartate in
motif A substituted by alanine (lane 3), demonstrating that
the activity observed is indeed telomerase activity.
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The pET-28a-TERT plasmid encodes an 11-amino acid tag (T7-tag) at the NH2 terminus of the TERT protein. Agarose beads coupled to antibodies to this tag were used to immunoaffinity purify the telomerase complex from the reticulocyte lysate after expression, which resulted in an enhancement of the proportion of full-length TERT (Fig. 3B, Beads). This immunopurification step resulted in an increase in apparent processivity of telomerase (Fig. 3A, lanes 4 and 5), as observed by the increase in intensity of larger products relative to the +1 product. This increase in apparent processivity was eliminated by the addition of rabbit reticulocyte lysate to the immunopurified telomerase, indicating that factor(s) in the reticulocyte lysate inhibit telomerase processivity (Fig. 3A, lane 6).
In the standard telomerase assay, the multiple repeat products are
typically thought to result from processive extension (i.e. the product remains associated with the same enzyme molecule), and this
has been demonstrated to be the case for Tetrahymena telomerase from extracts (27). However, such a pattern could also arise
from a distributive mode of action (i.e. the products dissociate from the enzyme and rebind for further addition). To distinguish between these possibilities for reconstituted telomerase, we used a pulse-chase variation of the telomerase assay similar to that
described by Maine et al. (44). A biotinylated
(G4T2)3 substrate primer was
incubated with immunopurified telomerase for 2 min under standard
reaction conditions. A 160-fold excess of non-biotinylated
(G4T2)3 competitor primer was then
added, and the reaction continued for a total of 10, 20, or 30 min. The biotinylated products were then isolated using magnetic streptavidin beads and electrophoresed (Fig.
4A). Only the biotinylated
products were recovered; a control using only non-biotinylated
substrate primer showed no recovered products (data not shown). When
competitor primer was added at the beginning of the reaction it almost
completely prevented extension of biotinylated primer molecules (Fig.
4A, lane 6), demonstrating that the concentration of
competitor primer was sufficient to prevent rebinding of biotinylated
primer during the chase period. Thus, the products visible on the gel
represent only processive synthesis. The size of products increased
between the 2-min pulse reaction and the chased reactions, indicating that the enzyme is truly processive, albeit to a modest extent (Fig.,
4A, lanes 2-4). A control in which no competitor primer was
added during a 30-min reaction showed a greater intensity of products,
indicating that the enzyme can carry out multiple turnovers of the
processive elongation reaction (lane 5). A graphical representation of the processivity in this experiment is shown in Fig.
4B, where the intensity of each successive repeat is
expressed relative to the intensity of the first repeat. (It should be
noted that longer products are less abundant than they appear from
their intensities, since they incorporate increasing amounts of
radiolabeled nucleotides and hence have a higher specific activity.
Thus, the data in Fig. 4B and all subsequent such graphs
have been adjusted for the specific activity of each repeat band,
i.e. the intensity of the second repeat was divided by 5, that of the third repeat divided by 9, and so on.) The data are linear
on a semi-log plot, with the slope of the line being inversely related
to the processivity. The processivity increased between the 2-min pulse
and the 10-min chased reaction, but showed no further increase and was
identical to that of the unchased reaction (Fig. 4B).
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A variation on the pulse-chase assay was carried out to confirm that
reconstituted telomerase is processive. In this bind-and-chase experiment, immunopurified enzyme was allowed to bind to biotinylated primer at 30 °C for 15 min. After binding, a 160-fold excess of competitor non-biotinylated primer and nucleotides were added, and the
reaction was allowed to proceed for a further 10 or 30 min (Fig.
4C, + chase lanes). The pattern of extension was
compared with controls in which competitor primer was omitted (Fig.
4C, chase lanes). Since the chase was almost
completely effective (Fig. 4C, control lane), any products
larger than +1 appearing in the chased reactions represent processive
synthesis. Once again, reconstituted telomerase displayed true processivity.
The results in Fig. 4, A and C, could also be
obtained if telomerase utilized an "efficiency enhanced"
distributive mechanism (45), i.e. if partially elongated
primers had a greater affinity for telomerase and thus became
preferentially elongated even in the presence of excess chase primer.
To investigate this possibility, we incubated telomerase with the
original 18-mer primer together with various ratios of a 31-mer primer
((G4T2)5G) that is identical to the
largest product visible from the 2-min pulse reaction in Fig.
4A. Primers with four or more G-rich repeats have the
potential to form G-quartet secondary structures which can inhibit
telomerase activity (46). To minimize formation of these structures,
primers were heated to 95 °C prior to use, and the reaction buffer
contained no monovalent cations, which induce G-quartet formation (47). When the two primers were incubated at a ratio of 1:1 and extended by
telomerase with [-32P]dGTP and ddTTP, both primers
were elongated (Fig. 4D). The presence of ddTTP terminates
the reaction after the addition of 4 and 5 nucleotides to the 31- and
18-mer, respectively, allowing the extension products of each to be
distinguished. The fact that the 18-mer can be extended in the presence
of an equal concentration of the 31-mer indicates that the latter does
not have a greater affinity for telomerase. When the 18-mer is present
at a 160-fold excess over the 31-mer, a situation mimicking the chase
in Figs. 4, A and C, the 31-mer is not visibly
extended, indicating that the extension products seen in the chased
reactions are not due to a greater affinity for longer substrates. The
31-mer in this experiment probably partially exists in a G-quartet
structure, since it was extended less efficiently than the 18-mer in a
1:1 ratio (Fig. 4D). However, the conditions in this
experiment and the pulse-chase experiments are identical and hence any
long products would be expected to form the same structure as the
31-mer primer.
To confirm the above conclusion, we also carried out a pulse-chase experiment using the 31-mer as the chase primer (Fig. 4E). A biotinylated 18-mer was extended for 2 min, followed by incubation with a 160-fold excess of nonbiotinylated 31-mer for a total of 30 min and recovery of the biotinylated products. The chase was effective (lane 4), and once again the chased reaction showed an increase in size of products over the 2-min pulse (lanes 1 and 2). Thus we conclude that the processivity observed on telomerase assay gels, while limited in extent, does represent true processivity.
An increase in dGTP concentration in the telomerase assay has been
shown to increase processivity in Tetrahymena,
Euplotes, and mammalian systems (15, 44, 48, 49), an effect
that may be due to stimulation of enzyme translocation by dGTP (50). Since we observed that immunopurification of telomerase from
reticulocyte lysates increased its processivity, we therefore
determined whether immunopurified telomerase was still responsive to
changes in dGTP concentration. Our immunopurified reconstituted
Tetrahymena telomerase did demonstrate an increase in
processivity with dGTP concentration, as did native activity from a
Tetrahymena cell extract (Fig.
5A). A graphical
representation of the processivity of reconstituted telomerase is shown
in Fig. 5B, where the intensity of each successive repeat is
expressed relative to the intensity of the first repeat after adjusting
for specific activity. A dGTP concentration of 10 µM,
which gives maximum processivity with the reconstituted enzyme, was
chosen as the standard concentration for all other experiments in this
study. The processivity at 10 µM dGTP varies slightly in
different experiments, but on average each band is about 30% as
intense as the previous one, indicating that the efficiency of
continuing extension from one repeat to the next is about 30%. The
average product length synthesized before one-half of the enzyme
molecules have dissociated or stalled is approximately 0.3 repeats, or
about 2 nt.
|
Telomerase activity from extracts was much more processive than the reconstituted enzyme at all dGTP concentrations (Fig. 5A). Previous studies have estimated that telomerase from Tetrahymena extracts synthesizes about 521 nt before one-half of the enzyme dissociates (27), although this calculation did not take into account the greater specific activity of longer products and hence is an overestimate. Addition of reticulocyte lysate did reduce the processivity of telomerase from extracts, but the processivity remained much higher than that of reconstituted telomerase (Fig. 3A).
In addition to affecting the processivity of telomerase, reticulocyte lysate also inhibits total telomerase activity (data not shown). Therefore, due to the impurity of the reticulocyte lysate system, most of the following experiments have been carried out using immunopurified telomerase.
Leucine-Tyrosine Mutant at Position 813 Has Increased
Processivity--
The L813Y mutant was constructed by site-directed
mutagenesis and expressed in reticulocyte lysates. The amount of
protein produced and its stability at 30 °C were approximately equal
to wt (Fig. 6E). In the
in vitro telomerase assay, the L813Y mutant showed levels of
activity equal to wt (Fig. 6A). Quantitation of the
intensity of all radiolabeled products on the gel and comparison to
standards of known specific activity demonstrate that both wt and L813Y
enzymes synthesize approximately 0.2-0.4 fmol of extended primer per
min per fmol of enzyme. However, the apparent processivity of L813Y
telomerase was increased (Fig. 6A).
|
Since immunopurification of wt TERT increased its processivity, we next examined whether the L813Y mutant remained more processive than wt after immunopurification (Fig. 6B, compare "beads" lanes). The results are represented graphically in Fig. 6C, which shows the intensity of each repeat band relative to the first repeat after adjusting for specific activity. Mutant L813Y was more processive than wt both before and after immunopurification. After immunopurification, the average product length synthesized before one-half of the enzyme molecules had dissociated or stalled was approximately 0.6 repeats, or about 4 nt. Thus, this mutant is about twice as processive as wt telomerase.
Position Leu-813 was also mutated to alanine and phenylalanine. Fig. 6, B and D, show that L813A is less processive than wt, while L813F has a processivity intermediate between wt and L813Y. The expression levels and stability of all mutant proteins were approximately equal to wt (Fig. 6E).
Mutant L813Y Also Demonstrates True Processivity--
It seemed
unlikely that the increased apparent processivity of mutant L813Y could
result from a switch to a distributive mode of action. Nevertheless,
pulse-chase and bind-and-chase assays identical to those already
described for wt telomerase were performed to test for processivity. A
2-min pulse with a biotinylated
(G4T2)3 primer (Fig.
7A, lane 1) was followed by a
chase with a 160-fold excess of nonbiotinylated
(G4T2)3 primer (lanes
2-4) and recovery of the biotinylated products on streptavidin
magnetic beads. Since the chase was effective (lane 6), the
increase in size of products is due to a processive mode of action.
Graphical representation of the processivity of the reactions indicates
that, as for wt enzyme, the chased reactions have a processivity
identical to the unchased control (Fig. 7B). A
bind-and-chase assay in which the L813Y enzyme was prebound to
biotinylated (G4T2)3 primer
followed by extension in the presence of an excess of non-biotinylated (G4T2)3 primer (Fig. 7C)
confirmed the processivity of the reaction. Finally, to confirm that
these results were not due to an increased affinity for longer
substrates, a pulse-chase experiment was performed with an excess of
nonbiotinylated 31-mer ((G4T2)5G)
as the chase primer (Fig. 7D). Once again, an increase in
size of products was seen after 30 min in the presence of the long
chase primer (lane 2).
|
Primer Dissociates More Slowly from Mutant L813Y Than from wt
Telomerase--
We postulated that the greater processivity of mutant
L813Y might result from an increased affinity for primer, resulting in
a greater probability of the enzyme translocating for another round of
synthesis rather than dissociating (Fig. 1). As a measure of primer
affinity for enzyme, we used a modified telomerase assay to determine
the rate constant for primer dissociation
(koff). An 18-mer primer
(GT2(G4T2)2G3)
was preincubated with immunopurified wt or L813Y telomerase at 30 °C
for 15 min. A 200-fold excess of 21-mer competitor primer
(GT2(G4T2)3) was added
and incubated with the enzyme/primer mixture at 30 °C for varying
lengths of time. Telomerase activity was then initiated by the addition
of nucleotides (ddTTP and dGTP) and allowed to proceed at 30 °C for 10 min. The inclusion of ddTTP resulted in termination of extension after incorporation of the first T, i.e. after 2 nucleotides
for the 18-mer, and after 5 nucleotides for the 21-mer (Fig.
8A). As a control to ensure
that the excess of 21-mer did not allow the enzyme to rebind 18-mer,
both primers were preincubated with enzyme simultaneously prior to
telomerase extension; there was almost no detectable extension of the
18-mer (Fig. 8A, chase control lanes). Thus, the amount of
addition to the 18-mer after varying lengths of chase time is a
reflection of the amount of the original primer still bound to enzyme
at that time. Fig. 8A shows that the 18-mer dissociates
rapidly from the wt enzyme (it is almost entirely dissociated after 10 min), but more slowly from the L813Y enzyme. The intensity of the major
(larger) extension band (indicated by * in Fig. 8A) was
quantitated, normalized to the combined intensities of all three major
extension products (18- and 21-mer) to control for differences in
enzyme activity between lanes, and plotted against time (Fig.
8B). The data fit well to a single exponential, giving a
koff of 0.68 ± 0.08 min1 for
wt and 0.21 ± 0.02 min1 for L813Y. These correspond
to half-lives of the primer-enzyme complexes of 1.0 ± 0.2 min for
wt and 3.3 ± 0.3 min for L813Y. Thus primer dissociates
approximately 3-fold more slowly from the L813Y mutant than from wt
enzyme.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Telomerase reverse transcriptases are related to retrotransposon and retroviral RTs (9, 23), yet telomerase has a distinct mode of action including repetitive copying of its template RNA and relatively low processivity. The protein structural features that account for these differences are completely unknown. We now identify an amino acid in the conserved motif C of TERT that affects the intrinsic processivity of the enzyme.
Mutating a leucine to a tyrosine at a position that is likely to be close to the active site caused Tetrahymena telomerase to increase in processivity. We demonstrated that both wt telomerase and the mutant show true processivity rather than distributive action. The 2-fold increase in processivity correlated with a 3-fold slower dissociation rate of primer from the enzyme. Although these are modest increases, it is rare for a site-directed mutation to "improve" an enzyme that has undergone natural selection over evolutionary time. Indeed, none of 28 other site-directed amino acid substitutions we have tested in Tetrahymena telomerase increased either activity or processivity.2
Our results, together with the crystal structure of the homologous region from HIV RT, suggest a possible mechanism for the increase in processivity. The side chain of tyrosine 813 could be hydrogen bonding with bases at the end of the primer as well as engaging in hydrophobic interactions with the primer, as the equivalent tyrosine 183 does in HIV RT (37). These interactions may increase the affinity of the enzyme for primer and make it more likely that the primer will undergo translocation for another round of extension rather than dissociating (see Fig. 1). This conclusion is supported by the observation that changing the same amino acid to phenylalanine resulted in intermediate processivity and changing it to alanine resulted in reduced processivity, since this emulates the pattern of activity displayed by these amino acids at the equivalent position in HIV RT.
All variants of telomerase tested showed approximately equal amounts of activity and were affected only in their processivity. In contrast, mutants at this position in other RTs were greatly affected in their activity levels (31-34). This difference may be due to additional stabilization of the enzyme-primer complex provided by other regions of telomerase such as the anchor region (51, 52). Thus, loss of the primer interaction provided by the tyrosine in HIV RT is catastrophic for enzyme activity, while in telomerase this loss may be compensated by other primer-enzyme interactions.
It is difficult to directly compare the levels of processivity reached by the tyrosine mutant of telomerase and HIV RT since in the former case processive synthesis involves repeated translocations on a short region of template, whereas HIV RT normally acts continuously along a template molecule. The processivity of HIV RT also varies greatly depending on the template sequence (53, 54) but can reach up to 300 nt on an RNA template in vitro (53). In the in vitro reconstitution assay described here, the tyrosine mutant of TERT more closely resembles classical RTs in this respect than does wt TERT.
Neither the wt nor the tyrosine mutant of reconstituted telomerase was as processive as telomerase from Tetrahymena extracts. The reason for the difference in processivity of reconstituted and native Tetrahymena telomerase is unknown, but may be due to factors that copurify with telomerase from extracts that are missing in reticulocyte lysates. Indeed, differences in purification schemes have resulted in differences in processivity of telomerase from S. cerevisiae and Euplotes crassus (55, 56). There is also precedence for processivity increasing factors associated with other polymerases, e.g. the processivity of T7 DNA polymerase is increased by the associated protein thioredoxin (57). Tetrahymena telomerase is not very processive in vivo, however, synthesizing only a few repeats before dissociating (58), suggesting that the extreme processivity of Tetrahymena telomerase from extracts may itself be an in vitro artifact.
The in vivo effects of this tyrosine mutation of telomerase in Tetrahymena are unknown. We predict that it would be more processive in vivo, i.e. each enzyme molecule would synthesize more telomeric repeats before dissociating from the telomere. It is unclear whether this would have an effect on steady-state telomere length, however, since there are likely to be many factors regulating telomere length besides telomerase, and there may be selective pressure in Tetrahymena cells against long telomeres (59, 60).
To investigate the effects of amino acid substitutions in TERT we have
used an in vitro reconstitution system based on translation in rabbit reticulocyte lysates, as has been described by others (15,
19, 20). Chaperone proteins or other factors in the reticulocyte
lysates contribute to the assembly of the telomerase ribonucleoprotein
complex (21, 22). We have shown that the reticulocyte lysates also have
a negative effect on telomerase activity, impacting both processivity
and the total levels of in vitro activity.
Immunopurification of the telomerase complex from the reticulocyte
lysates abrogated these effects and thus will provide a useful system
for investigation of other site-directed mutants of telomerase.
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ACKNOWLEDGEMENTS |
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We thank Dr. Art Zaug for the gift of the telomerase RNA plasmids, Anne Gooding for preparation of T7 RNA polymerase, Elaine Podell for oligonucleotide synthesis, YuMing Han for automated sequencing, and many members of the Cech laboratory for discussions and reading of the manuscript.
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FOOTNOTES |
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* This work was supported by a Human Frontier Science Program Long-term Fellowship (to T.M.B) and the Howard Hughes Medical Institute.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Howard Hughes Medical
Institute, Dept. of Chemistry and Biochemistry, University of Colorado,
Boulder CO 80309-0215. Tel.: 303-492-8606; Fax: 303-492-6194; E-mail:
thomas.cech@colorado.edu.
Published, JBC Papers in Press, May 11, 2000, DOI 10.1074/jbc.M003246200
2 T. Bryan, K. Goodrich, and T. Cech, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: TERT, telomerase reverse transcriptase; HIV, human immunodeficiency virus; PCR, polymerase chain reaction; RT, reverse transcriptase; wt, wild type; nt, nucleotide; DTT, dithiothreitol.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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, 2 min unchased; 
, 10 min chased; 
, 20 min chased; 
, 30 min chased; 
, 30 min unchased. C, bind and
chase assay. Immunopurified wt telomerase was prebound to biotinylated
(G4T2)3 primer for 15 min. An
excess of non-biotinylated competitor primer was added (+ chase
lanes) along with dTTP and [
, 0.33 µM;
, 10 µM; 
