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INTRODUCTION |
The ends of chromosomes in most eukaryotes are capped with tandem
simple sequence repeats. These telomeric repeats and their associated
proteins are necessary and sufficient to distinguish a stable linear
chromosome end from a highly unstable DNA break (reviewed in Ref. 1).
However, telomeres are incompletely replicated by
DNA-dependent DNA polymerases. The resulting loss of
telomeric repeats with cell proliferation induces senescence or
apoptosis of cultured human primary cells (reviewed in Ref. 2).
Telomeric repeats eroded by proliferation can be restored by the enzyme telomerase, a specialized reverse transcriptase
(RT)1 that uses a defined
region within its integral RNA component to template telomeric repeat
synthesis (reviewed in Refs. 3, 4). Although telomerases in most
organisms recognize only established telomeres as substrates, ciliate
telomerases also recognize nontelomeric sites of developmentally
programmed chromosome fragmentation. This ciliate telomerase chromosome
healing activity is required to generate a transcriptionally competent
macronucleus containing thousands of amplified, telomere-capped
minichromosomes (reviewed in Ref. 5).
Most biochemical characterization of telomerase has been done in
ciliate systems because of the relative abundance of enzyme (reviewed
in Ref. 6). Ciliate telomerases have been shown to catalyze at least
three activities. In a standard reaction, the template is copied by
successive dNTP additions to synthesize a telomeric repeat. Second, if
dTTP is reduced or omitted, telomerase reiteratively copies a template
cytidine residue to synthesize product DNA composed of poly(dG). This
template slippage-dependent polynucleotide synthesis
resembles that catalyzed by human immunodeficiency virus RT in the
presence of MnCl2 and the absence of a complete set of
dNTPs (7). Finally, telomerase can catalyze nucleolytic cleavage of
substrates or products. All of these nucleotide addition and removal
activities appear to occur at the same active site.
Endogenous Tetrahymena thermophila telomerase assayed in
cell extracts can add hundreds of repeats to a single primer before product dissociation (8). This high degree of repeat addition processivity requires primer and product interaction with a
template-independent substrate anchor site (9, 10). Under standard
reaction conditions, a primer bound only by interaction at the
template, such as T2G4, will be elongated to
the template 5'-end but then will dissociate from the enzyme when
dissociated from the template. In contrast, longer primers such as
G4T2G4 or
(G4T2)3 can remain bound at the anchor site even when dissociated from the template. With anchor site
interaction, a product 3'-end released from the template 5'-end can
reposition at the template 3'-end to allow processive repeat addition.
Anchor site interaction also substantially decreases the
Km for primer in vitro (9, 10).
Recombinant Tetrahymena telomerase, composed of only the
telomerase RNA and telomerase reverse transcriptase (TERT) subunits that are essential for activity in vitro, demonstrates a
more limited repeat addition processivity than the endogenous enzyme (11). In addition, unlike the endogenous enzyme, recombinant telomerase
repeat addition processivity requires high micromolar dGTP
concentrations, much higher than the submicromolar dGTP concentration required for processive nucleotide addition within a repeat. In this
dGTP-dependence of repeat addition processivity, recombinant Tetrahymena telomerase resembles the endogenous
Euplotes aediculatus and Chinese hamster ovary cell
telomerases (12, 13). The extent of repeat addition processivity varies
among telomerase enzymes of different species and also between
telomerase complexes of the same species (14, 15). It seems most likely
that dGTP-stimulated repeat addition processivity is an inherent
property of all telomerase RNPs, with the species-specific addition of
other substrate anchor sites in a telomerase holoenzyme conferring the
differences in repeat addition processivity observed.
For all telomerases known to demonstrate repeat addition processivity,
dGTP is the first nucleotide added to a product repositioned from the
template 5'- to 3'-end. One possible model to explain the dGTP
dependence of repeat addition processivity would be that the
stimulatory dGTP binds at the active site and enhances the probability
of its addition as the first nucleotide in the second repeat.
Alternately, dGTP could interact with a site entirely separable from
the active site to stimulate repeat addition processivity as previously
suggested (12). Experimentally testable predictions can be made that
discriminate these models. For example, if dGTP must be the first
nucleotide to add to a repositioned product to observe
dGTP-dependent repeat addition processivity, then altering the template residue dictating this nucleotide addition specificity should abrogate the dGTP-dependence of repeat addition processivity. On
the other hand, if the dGTP interaction required for repeat addition
processivity is independent of the template, any template sequence
change allowing wild-type levels of product synthesis to the template
5'-end, rebinding of the product 3'-end at the template 3'-end and
product-anchor site interaction should allow processive repeat addition
stimulated by dGTP. Our data indicate that dGTP-stimulated repeat
addition processivity is accomplished in a manner different from
envisioned by either model above.
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MATERIALS AND METHODS |
Recombinant Telomerase Production--
T. thermophila
telomerase RNA expression constructs were derived from pT7159 (16) by
site-specific mutagenesis. Pairs of complementary mutagenic
oligonucleotides were used in a linear amplification reaction with
Pfu polymerase and double-stranded DNA templates (17). All
telomerase RNA expression constructs were sequenced to confirm the
presence of only the intended change. To express telomerase RNAs with
the wild-type RNA 3'-end, plasmids were digested with FokI
and reprecipitated after organic extraction. T. thermophila
TERT was expressed from the plasmid p133CITE (11). Equal masses of
telomerase RNA-encoding plasmid and telomerase protein-encoding plasmid
were combined for coupled transcription/translation in rabbit
reticulocyte lysate (Promega TNT). This produces roughly equimolar
amounts of telomerase RNAs and TERT (about 10 nM). For the
determination of relative activity in Table I, expression reactions were analyzed by Northern blot to verify comparable levels of
telomerase RNAs and by immunoblot to verify comparable levels of TERT.
For the experiment shown in Fig. 7, N-terminal hemagglutinin
epitope-tagged TERT was coexpressed with telomerase RNA, purified by
binding to immobilized HA antibody and eluted with peptide.
Telomerase Activity Assays--
Activity assays were performed
under conditions similar to those previously described for recombinant
Tetrahymena telomerase (11). Typically, 2 µl or less of
reticulocyte lysate expression reaction was diluted to 10 µl in T2MG
(20 mM Tris-HCl, pH 8.0, 1 mM
MgCl2, 10% glycerol, ± 2.5 mM dithiothreitol)
and used in a 20-µl final assay volume in 1× assay buffer (50 mM Tris acetate, pH 8.0, 2 mM
MgCl2, 5 mM
-mercaptoethanol, 10 mM spermidine). Nucleotides were included as indicated in
the figure legends. DNA oligonucleotide primers were used at 1 µM final concentration unless otherwise specified. Assays
were incubated at 30 °C for ~1 h unless otherwise specified.
Product DNA was extracted with phenol/cholorofom/isoamyl alcohol,
precipitated with ammonium acetate, and resolved by denaturing gel
electrophoresis. In some experiments, a radiolabeled 80-nt
oligonucleotide was added before extraction and precipitation as an
internal control for sample recovery (see Fig. 7).
Quantitation of Repeat Addition Processivity--
Quantitation
of product intensity was done by phosphorimager (Fuji). Relative
product intensities were converted to relative molar amounts of product
by normalizing for the number of incorporated dGTPs. The molar amount
of second repeat addition product divided by the sum of the first and
second repeat addition products is a measure of repeat addition
processivity. For example, for primer (TG)8TTG, repeat
addition processivity is determined as molar amount (primer +12)/(sum
of primer +6 and primer +12). This calculation does not include the
small amount of product lost by elongation with additional repeats,
which is difficult to quantitate reliably and would not substantially
affect the values obtained. Similar values for repeat addition
processivity were obtained comparing first and second repeat addition
products, second and third repeat addition products, or third and
fourth repeat addition products. Also, repeat addition processivity
values were similar when calculated from a 15 or 60 min reaction,
indicating that use of these reaction times did not limit repeat
addition processivity.
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RESULTS |
The Influence of Template Position 48 on Repeat Addition
Processivity--
Tetrahymena telomerase repeat addition
processivity was assayed by elongation of an excess of the DNA primer
(TG)8TTG in the presence of [32P]dGTP and
dTTP. This primer binds with its 3'-end at the template 3'-end (Fig.
1A). Products with lengths of
primer +1 to primer +6 can be synthesized in the addition of the first
repeat (Fig. 1A). Because primer is in greater than
1,000-fold excess over telomerase enzyme and telomerase products (data
not shown), products longer than primer +6 derive from processive
repeat addition. In 0.6 µM dGTP, which is sufficient for
high nucleotide addition processivity within a repeat, recombinant
Tetrahymena telomerase added predominantly one repeat to
each bound (TG)8TTG primer (Fig. 1B,
WT lane 0.6 µM dGTP). Because of
differences in absolute dGTP concentration in different shipments of
[32P]dGTP, some variation in the absolute repeat addition
processivity attained with 0.6 µM dGTP assays containing
only radiolabeled dGTP stock was observed (compare Figs. 1-4 and 6).
With increasing dGTP concentration, increasing repeat addition
processivity allowed the elongation of some primers by many repeats
before dissociation (Fig. 1B, WT lanes
2.6-40 µM dGTP). Because unlabeled dGTP was added to
dilute the radiolabeled dGTP stock for dGTP concentrations greater than
0.6 µM, dGTP specific activity was reduced as dGTP concentration increased, and product intensity decreased accordingly. However, if product intensity was adjusted for specific activity, the
total amount of product was similar at all dGTP concentrations. In this
study, we quantitated repeat addition processivity by determining the
amount of first repeat addition product that was extended by addition
of a second repeat on a molar basis (see "Materials and Methods").
For the wild-type enzyme reactions shown in Fig. 1, repeat addition
processivity increased from 3.1% at 0.6 µM dGTP to 22%
at 40 µM dGTP.

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Fig. 1.
The repeat addition processivity of all C48N
enzymes is similarly dependent on the concentration of dGTP.
A, template region of the T. thermophila
telomerase RNA spans positions C-43 to A-51. The primer
(TG)8TTG binds at the template 3'-end and is elongated by
up to 6 nt in first repeat addition. Repositioning of a product 3'-end
from the template 5'- to 3'-end can allow second repeat addition.
B, each C48N enzyme was assayed with the primer
(TG)8TTG. Reactions contained 0.6 µM
[32P]dGTP and extra unlabeled dGTP to obtain 0.6-40
µM total dGTP. All reactions contained 200 µM dTTP, reactions with C48G also contained 200 µM dCTP, and reactions with C48U also contained 200 µM dATP. The migration of primer extended by addition of
various numbers of nucleotides is indicated. Note that the first
radiolabeled product of the C48N enzymes other than wild-type has a
length of primer +2 because of the initial addition of an unlabeled
nucleotide. Repeat addition processivity as a function of dGTP
concentration is indicated (values at 0.6 µM dGTP for
C48U and C48A are not possible to calculate accurately because of the
poly(dG) product background). A shorter exposure of C48U
lanes and a longer exposure of C48A lanes are
also shown below.
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The first nucleotide added to a product repositioned for processive
repeat synthesis is templated by the RNA residue C-48. To assess the
influence of this template position on repeat addition processivity, we
created expression constructs encoding the altered telomerase RNAs
C48G, C48U, and C48A. Coexpression of these RNAs with TERT produced
recombinant telomerase enzymes, which still bound the
(TG)8TTG primer at the template 3'-end (Fig.
1B). The C48N telomerases were assayed with
[32P]dGTP, dTTP, and dCTP (C48G) or dATP (C48U). Initial
elongation of the primer (TG)8TTG occurred by addition of
dCTP (C48G), dATP (C48U), dTTP (C48A), or dGTP (WT). In 0.6 µM dGTP, repeat addition processivity was low for all
enzymes (Fig. 1B). If dGTP concentration was increased to
2.6, 10, or 40 µM, the repeat addition processivity of
all C48N enzymes was stimulated (Fig. 1B). The maximum
attainable repeat addition processivity varied with template sequence
(Table I). For example, the C48U and C48A
enzymes had lower maximal repeat addition processivity than the C48G
and WT enzymes (3.4 and 5.8% versus 14 and 22%). This is
likely to originate from the reduced nucleotide addition processivity
of the C48U and C48A enzymes, evident in the enhanced accumulation of
mid-template dissociation products. This reduced nucleotide addition
processivity is expected, based on the reduced stability of an A-U or
T-A product-template hybrid compared with the wild-type G-C. Although
maximal repeat addition processivity varied with template sequence, the
concentration of dGTP required to stimulate maximal repeat addition
processivity was similar for all C48N enzymes. We conclude that the
dGTP concentration dependence of repeat addition processivity does not
depend on the sequence of template position 48.
Next, dGTP concentration was fixed at 0.6 µM whereas the
concentration of the nucleotide cognate to template position 48 was varied from 0 to 125 µM (Fig.
2). In no case did increasing the concentration of a dNTP other than dGTP stimulate repeat addition processivity, even if nucleotide concentration was raised to the threshold at which telomerase activity was inhibited nonspecifically (data not shown). In the absence of the dNTP cognate to template position 48, none of the altered templates allowed substantial incorporation of [32P]dGTP (Fig. 2, lanes 0 µM dNTP). This establishes that dGTP was not
misincorporated by the C48G, C48U, and C48A enzymes at template position 48, which could have allowed a dGTP-stimulated repeat addition
processivity still requiring the first nucleotide addition of dGTP.
Trace product synthesis with C48G enzyme in the absence of dCTP may
derive from incorporation of dTTP at the template position 48G, whereas
the primer-sized product of C48A synthesized in the absence of dTTP
derives from cleavage and readdition of the primer 3'G, templated by
either 43C or 49C. From the assays in Fig. 2, we conclude that the
first nucleotide added to a template-repositioned product does not
determine the nucleotide dependence of repeat addition
processivity.

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Fig. 2.
The repeat addition processivity of C48N
enzymes is not stimulated by dCTP, dATP, or dTTP. Each C48N enzyme
was assayed with the primer (TG)8TTG. Reactions contained
0.6 µM [32P]dGTP and concentrations of the
indicated nucleotides from 0-125 µM. Reactions with C48G
and C48U also contained 200 µM dTTP. The migration of
primer extended by addition of various numbers of nucleotides is
indicated. A longer exposure of C48A lanes is also shown
below.
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In the experiments described above, a surprising lack of correlation
was observed between the relative activity level and the relative
repeat addition processivity directed by different template sequences.
Based on product intensity, the C48U enzyme outperformed the wild-type
enzyme, which in turn outperformed the C48G and C48A enzymes (Table I).
This rank order differs from that of repeat and nucleotide addition
processivities, in which the WT and C48G enzymes outperformed the C48A
and C48U enzymes. Unlike the differences in processivity, the
differences in overall activity cannot be explained by predicted
alterations in the stability of the product-template hybrid.
Nucleotide Structural Requirements for Stimulation of Repeat
Addition Processivity--
To investigate which features of the dGTP
nucleotide were important for its ability to stimulate repeat addition
processivity, we tested whether other purine nucleotides could enhance
repeat addition processivity in reactions with 0.6 µM
dGTP. Addition of the ribonucleotide triphosphate GTP or deoxyinosine
triphosphate (dITP) at concentrations up to 125 µM failed
to enhance repeat addition processivity (Fig.
3A). GTP differs from dGTP by
the presence of a ribose C-2'-hydroxyl group, whereas dITP differs from
dGTP by the lack of the amino group on base C-2. Thus, addition of a
C-2' hydroxyl or loss of the C-2 amino group both prevent dGTP from
stimulating repeat addition processivity. GTP is incorporated poorly by
Tetrahymena telomerase (18), and neither GTP nor dITP inhibited incorporation of [32P]dGTP under the conditions
assayed (Fig. 3A).

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Fig. 3.
Structural variants of dGTP have different
abilities to stimulate repeat addition processivity. Wild-type
enzyme was assayed with the primer (TG)8TTG. The migration
of primer extended by addition of various numbers of nucleotides is
indicated. A, reactions contained 0.6 µM
[32P]dGTP, 200 µM dTTP, and concentrations
of the indicated nucleotides from 0-125 µM. Some
reactions also contained 10 µM extra unlabeled dGTP as
indicated. A longer exposure of the right 6 lanes is also
shown below. B, reactions contained 0.6 µM [32P]dGTP, no dTTP, and concentrations
of the indicated nucleotides from 1-100 µM.
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We also examined 7-deaza-dGTP, which differs from dGTP by carbon
substitution of the base N-7. Addition of 7-deaza-dGTP to reactions
with 0.6 µM dGTP had three consequences: stimulation of
repeat addition processivity, inhibition of [32P]dGTP
incorporation, and alteration of the product profile to promote
accumulation of products with lengths of primer +3, +9, etc. (Fig.
3A). Each of these effects of 7-deaza-dGTP was competed by
addition of extra dGTP, with the ratio of products altered as a
reflection of the ratio of dGTP to 7-deaza-dGTP in the reaction (Fig.
3A, right). The altered product profile was also
observed in assays with [32P]dTTP and 7-deaza-dGTP alone,
without any dGTP (data not shown). In reactions with 0.6 µM [32P]dGTP without dTTP, 7-deaza-dGTP and
dGTP both stimulated synthesis of a polynucleotide ladder (Fig.
3B). These results suggest that 7-deaza-dGTP is efficiently
incorporated by telomerase into both telomeric repeats and a
polynucleotide ladder, but that 7-deaza-dGTP incorporation into
telomeric repeats affects the product profile. The change in product
pattern is likely to reflect a change in the stability of the
product-template hybrid. Independent of its possible impact on hybrid
stability, however, the repeat addition processivity stimulated by
7-deaza-dGTP indicates that the dGTP nitrogen at base position 7 is not
an essential property of a processivity-stimulatory nucleotide.
We next examined the role of the dGTP triphosphate group. Surprisingly,
addition of 2.6, 10, or 40 µM of dGMP, dGDP, or dGTP to
assays with 0.6 µM [32P]dGTP stimulated
repeat addition processivity (Fig.
4A). The maximal repeat
addition processivity obtained with titration of the concentration of
each of these three nucleotides was strikingly similar, as was the
concentration of nucleotide required to obtain it. In contrast,
addition of up to 250 µM deoxyguanosine did not stimulate
repeat addition processivity (Fig. 4B). Although dGMP and
dGDP cannot be incorporated, they inhibited product synthesis (Fig. 4,
A and B). This inhibition was eliminated by
addition of extra dGTP (data not shown). In contrast, deoxyguanosine
did not inhibit product synthesis (Fig. 4B). We conclude
that the nucleotide-stimulating repeat addition processivity requires
only a monophosphate group. In addition, we note that all nucleotides that stimulate repeat addition processivity reduce dGTP incorporation, whether or not they can be incorporated themselves. This suggests that
the processivity-stimulatory dGTP (or dGTP analog) is binding in the
active site with a specificity that parallels that of dGTP binding for
nucleotide incorporation (see "Discussion").

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Fig. 4.
Only one phosphate group of dGTP is required
to stimulate repeat addition processivity. Wild-type enzyme was
assayed with the primer (TG)8TTG. The migration of primer
extended by addition of various numbers of nucleotides is indicated.
A, reactions contained 0.6 µM
[32P]dGTP, 400 µM dTTP, and concentrations
of the indicated nucleotides from 0-40 µM. Repeat
addition processivity as a function of dGTP concentration is indicated.
B, reactions contained 1.2 µM
[32P]dGTP, 400 µM dTTP, and concentrations
of dGMP or deoxyguanosine (dG) from 0-250 µM.
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Template Requirements for Repeat Addition Processivity--
In
addition to template position 48 and a substrate dNTP, the active site
elongating a repositioned product would be strongly influenced by the
terminal product-template base pair. To investigate the role of this
base pair, composed of a product G and template C in the wild-type
enzyme, we created expression constructs encoding C4349U and C4349G
telomerase RNAs. It was necessary to coordinately change both positions
C-43 and C-49 to obtain processive repeat addition, because a product
3'-end synthesized at the template 5'-end must be able to rebind at the
template 3'-end (Fig. 5A). Telomerases with wild-type, C4349U, or C4349G templates were assayed with the primers (TG)8TTG, (TG)8TTA, or
(TG)8TTC, respectively. The C4349U enzyme assayed with
(TG)8TTA synthesized predominantly short products from
incomplete first repeat addition and is not described further. The
C4349G enzyme assayed with (TG)8TTC had robust activity
(Table I) and enough nucleotide addition processivity for substantial
complete first repeat synthesis (Fig. 5B). In contrast with
the C48N enzymes, however, the addition of up to 40 µM
dGTP stimulated only a trace amount of second repeat addition (Fig.
5B). Various concentrations of dCTP (data not shown) or 100 µM dGMP (Fig. 5C) did not significantly
stimulate the repeat addition processivity of the C4349G enzyme.

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Fig. 5.
Repeat addition processivity requires more
than wild-type levels of overall activity, product-template hybrid
stability, and product-anchor site interaction. A,
C4349G enzyme binds the primer (TG)8TTC at the 3'-end of
the template. The first repeat addition product can reposition from the
template 5'- to 3'-end. B, the C4349G enzyme was assayed
with the primer (TG)8TTC. Reactions contained 0.6 µM [32P]dGTP, extra unlabeled dGTP to
obtain 0.6-40 µM total dGTP, 200 µM dCTP,
and 200 µM dTTP. The migration of primer extended by
addition of various numbers of nucleotides is indicated. A longer
exposure is also shown at right. C, the wild-type
and C4349G enzymes were assayed with the primers (TG)8TTG
and (TG)8TTC, respectively. Reactions contained 3.0 µM dGTP, 200 µM dTTP, and for C4349G also
200 µM dCTP. Aliquots of a single reaction were stopped
after 12, 24, or 48 min. Separate assays contained an additional 100 µM dGMP. The migration of primer extended by addition of
various numbers of nucleotides is indicated. D, the
wild-type enzyme was assayed with the indicated primers at 1 µM concentration for either a 15- or 60-min reaction
time. Reactions contained 2.6 µM dGTP and 400 µM dTTP. The migration of primer extended by addition of
various numbers of nucleotides is indicated.
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There were several possible explanations for the inhibited repeat
addition processivity of the C4349G enzyme. As a first possibility, product elongated to the template 5'-end could have failed to dissociate, preventing additional product elongation. In fact, low
product turnover limits budding yeast telomerase to a single round of
repeat addition per primer binding event in vitro (19). One
simple method to investigate failed dissociation was to assay a time
course of product synthesis, because only if product turnover occurs
could product continue to accumulate with time. Product accumulation
occurred continuously over the entire time course of a standard assay
for both the wild-type and C4349G enzymes (Fig. 5C),
suggesting that the inhibition of C4349G enzyme repeat addition
processivity does not derive from a failure of product turnover. As a
second possibility, the repositioned product 3'-end could have formed a
weaker hybrid with the template 3' end. However, the primers
(TG)8TTG and (TG)8TTC were elongated at similar
concentrations by the wild-type and C4349G enzymes, respectively (data
not shown). This suggests that the product-template hybrid formed at
the C4349G template 3'-end was sufficiently stable to allow repeat
addition processivity.
As a third possibility, the part of the repositioned product
sequence that does not bind the template could have formed a weaker
interaction at the substrate anchor site, resulting in product
dissociation. To address this possibility, primers with anchor site
sequences corresponding to the products of first repeat addition with
wild-type and C4349G templates ((TG)8TTGGGGTTG
and (TG)8TTCGGGTTG) were assayed with wild-type
enzyme. The (TG)8TTCGGGTTG primer promoted synthesis of
slightly more product than (TG)8TTGGGGTTG, but the two
primers were elongated with a similar Km and similar
repeat addition processivities (Fig. 5D and data not shown).
The lower maximal activity with (TG)8TTGGGGTTG likely derives from inhibitory binding of some primer across the entire template, rather than at the template 3'-end. This mode of binding would also account for the enhanced nucleolytic cleavage of this primer, evident in the accumulation of radiolabeled product with a
length of primer +0 (Fig. 5D). Cleavage occurs
preferentially for substrates aligned at the template 5'-end (6), and
the (TG)8TTGGGGTTG primer, unlike the
(TG)8TTCGGGTTG primer, can align with the template 5'-end
without template-primer mismatch. Considering all the data for the
C4349G enzyme, we conclude that its low repeat addition processivity
does not reflect the compromise of any molecular event previously known
to be required for repeat addition processivity: product-template
dissociation, rebinding of the product 3'-end at the template 5'-end or
product interaction at the substrate anchor site. This suggests that
these activities are not sufficient for repeat addition processivity.
We also investigated the role of a particular length of template and/or
product repeat in promoting repeat addition processivity. We created
telomerase RNA expression constructs that truncated or extended the
template, encoding either a 5-nt T2G3 repeat
(3C) or a 7-nt T2G5 repeat (5C) instead of the
wild-type 6-nt T2G4 repeat (Fig.
6, A and B).
Similar template substitutions have been assayed by reconstitution of
recombinant RNA with endogenous, micrococcal nuclease-treated
Tetrahymena proteins in vitro (20) or by
expression of a recombinant Tetrahymena telomerase RNA gene in vivo (21). Our 3C enzyme, composed of recombinant TERT
and telomerase RNA, had strong telomerase activity (Table I) and the
expected product ladder of 5-nt rather than 6-nt periodicity (Fig.
6A). In addition, the 3C enzyme catalyzed nucleolytic
cleavage of the primer (TG)8TTG to generate substantial
radiolabeled product with length of primer +0. Maximal repeat addition
processivity was within 2-fold of that of the wild-type enzyme (15 versus 24% in parallel reactions at 40 µM
dGTP; Fig. 6A) and required a similar dGTP concentration.
In vitro reconstitution of a 3C template with endogenous
proteins also produced a telomerase enzyme with some repeat addition
processivity (20).

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Fig. 6.
Maximal repeat addition processivity and
overall activity level vary independently with template
alterations. A, B, D, the wild-type, 3C, 5C, and 5' +2U
enzymes were assayed with the primer (TG)8TTG. Reactions
contained 0.6 µM [32P]dGTP, extra unlabeled
dGTP to obtain 0.6-40 µM final dGTP, 200 µM dTTP and for 5' +2U(+dATP) also 200 µM
dATP. The migration of primer extended by addition of various numbers
of nucleotides is indicated. Repeat addition processivity as a function
of dGTP concentration is indicated for WT, 3C, and 5' +2U(-dATP).
C, the wild-type enzyme was assayed with the indicated
primers at 50 nM concentration. Reactions contained 5 µM dGTP and 200 µM dTTP. All
lanes are from the same exposure of a single gel.
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In contrast, the 5C recombinant enzyme had both reduced activity and
dramatically reduced repeat addition processivity (Table I, Fig.
6B). A 5C template reconstituted with endogenous proteins in vitro had similarly little if any repeat addition
processivity (20), whereas the repeat addition processivity of 5C
enzyme reconstituted in vivo was inhibited but still
substantial (21). The very low repeat addition processivity that could
be detected for our recombinant 5C enzyme still required a dGTP
concentration over micromolar. Overall activity and maximal repeat
addition processivity were not improved by using primers capable of
more initial hybridization with the template (data not shown). Also, if
the first repeat addition product of the 5C enzyme was assayed as a
primer for the wild-type enzyme, it was as efficiently and processively
elongated as a primer with wild-type or 3C template product sequence
(Fig. 6C and data not shown). These results suggest that the
inhibition of 5C enzyme activity and repeat addition processivity does
not derive from formation of a weaker product-template hybrid or a
weaker product-anchor site interaction. Furthermore, product
accumulation continued over the time course of a standard 5C enzyme
reaction, demonstrating that the inhibition of repeat addition
processivity does not derive from a failure of product turnover (data
not shown). We conclude that as for the C4349G enzyme, no molecular
event known to be required for repeat addition processivity compromises
the repeat addition processivity of the 5C enzyme.
The 5C template change altered both template length and product repeat
length. To determine whether altered template length alone inhibited
overall activity or maximal repeat addition processivity, we created a
telomerase RNA expression construct with two uridines inserted
immediately adjacent to the last template residue of the wild-type RNA
(5'+2U; Fig. 6D). In assays containing dATP, the primer
(TG)8TTG was elongated by 8 nt in first repeat addition. In
assays lacking dATP, however, the wild-type 6-nt repeat was synthesized. Assayed with or without dATP, the overall activity of the
5'+2U enzyme was reduced compared with the wild-type enzyme (Table I).
Repeat addition processivity was negligible in reactions with dATP
(Fig. 6D), responsible at least in part to the inability of
the first repeat addition product to rebind at the template 3'-end. In
reactions without dATP, however, repeat addition processivity was
within 2-fold of that of the wild-type enzyme (Table I, Fig. 6D). We conclude that increased template length inhibits
overall activity independent of any change in product length or repeat addition processivity. However, increased template length alone, without a change in product length, does not affect
dGTP-dependent repeat addition processivity.
Anchor Site Independence of Repeat Addition Processivity--
The
inhibited repeat addition processivity of C4349G and 5C enzymes, above
shown to be independent of any previously known processivity
requirement, prompted us to examine whether the
dGTP-dependent repeat addition processivity of recombinant
Tetrahymena telomerase was accomplished by a fundamentally
different mechanism than the dGTP-independent repeat addition
processivity of the endogenous enzyme. To test this, we compared the
concentration dependence and repeat addition processivity of primer
elongation for a set of primers with the same 3'-end but different 5'
lengths or sequences. Because each primer forms the same 3' interaction
with the template, differences in a primer Km for
elongation or in elongation repeat addition processivity derive from
differences in template-independent enzyme-product interactions. For
endogenous Tetrahymena telomerase, increasing the length of
a primer from 6 to 10 nt substantially increases primer binding
affinity because of anchor site interactions (9, 10). For the
recombinant enzyme, increasing the length of a primer from 6 to 10 nt
also slightly increased apparent primer binding affinity (Fig.
7 and data not shown).

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Fig. 7.
Repeat addition processivity does not require
the previously defined type of anchor site interaction. Primers at
the indicated concentrations were assayed with telomerase affinity
purified from expression lysate. Reactions contained 5 µM
dGTP and 200 µM dTTP. Products corresponding to
completion of first and second repeat synthesis are indicated (+3 and
+9 or +6 and +12). The radiolabeled oligonucleotide added after the
activity assay as an internal control for sample recovery is also
indicated (IC). Repeat addition processivity was calculated
for the 1 µM primer concentration of each titration
series. At low primer concentration in particular, an enhanced
nucleolytic cleavage of product is evident for telomerase purified from
expression lysate (most lanes) compared with unpurified
telomerase or purified telomerase supplemented with fresh lysate
(first lane).
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For endogenous Tetrahymena telomerase, increasing the length
of a primer from 6 to 10 nt increases the repeat addition processivity of elongation dramatically (9, 10). In contrast, for the recombinant
enzyme, even a 6-nt primer had maximal repeat addition processivity
(Fig. 7). If anything, increasing primer length slightly decreased
repeat addition processivity for primers composed of perfect telomeric
repeats (16% with T2G4, 12% with
G4T2G4, 11% with
(T2G4)3). Processive elongation of
all primers, independent of length or sequence, required greater than
micromolar concentrations of dGTP (data not shown). We conclude that
the repeat addition processivity of recombinant Tetrahymena
telomerase does not depend on an anchor site interaction similar to
that described for the endogenous enzyme. Instead, there is a distinct,
dGTP-dependent mechanism for repeat addition processivity
that involves enzyme interaction with nascent product DNA (Fig.
8). It is the grip of this nascent
product DNA binding site on product released from the template, rather
than the grip of an anchor site on more 5' product sequence, that
allows dGTP-dependent repeat addition processivity. The
modest extent of repeat addition processivity gained by the dGTP-dependent mechanism may not be evident in assays of
endogenous Tetrahymena telomerase with substrates that are
capable of anchor site interaction. However, using short primer DNAs
with the endogenous enzyme, a stimulation of repeat addition
processivity at greater than micromolar dGTP concentrations was indeed
observed (data not shown). The quantitative impact of dGTP stimulation
is difficult to assess for the endogenous enzyme, because any product
that gains a second repeat will efficiently engage the dGTP-independent repeat addition processivity mechanism.

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Fig. 8.
Working model for the molecular basis of
dGTP-dependent repeat addition processivity. A primer
(such as TTGGGG) anneals with the telomerase RNA template region
(3'-AACCCCAAC-5'), positioning the primer 3'-end in the active site
(hatched box). As template-product hybrid incorporates
successively more 5' regions of the template during repeat synthesis
(step 1), the template-product hybrid moves relative to the
active site and product DNA can become engaged by a nascent product
binding site (NPS). Alternately, the NPS could engage
nascent product constitutively during repeat synthesis. Upon
template-product hybrid dissociation, the template returns to a more
favorable position with respect to the active site and the product
remains bound at the NPS (step 2). Realignment of the
product 3'-end at the template 3'-end allows a second round of repeat
synthesis. New product synthesis or some other event could displace a
previous repeat from the NPS.
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DISCUSSION |
Template- and Nucleotide-dependence of Repeat Addition
Processivity--
We have defined molecular requirements for the
repeat addition processivity of a recombinant Tetrahymena
telomerase RNP composed of telomerase RNA and TERT. Changes in the
template sequence that reduce nucleotide addition processivity,
including the substitutions C48U and C48A, also reduce maximal repeat
addition processivity. Apart from its influence on nucleotide addition
processivity, however, the identity of the base at template position 48 is not important for the basic mechanism of repeat addition
processivity or for establishing the dependence of repeat addition
processivity on dGTP. Insertion of extra telomerase RNA nucleotides
immediately 5' of the template also does not inhibit repeat addition
processivity, indicating that there is not a strict spacing required
between the template and the TERT binding site 5' of the template (22). In contrast, despite maintaining high nucleotide addition processivity, the template sequence alterations C4349G and 5C inhibit repeat addition
processivity almost completely. The RNA residues affected by these
substitutions may be critical for repeat addition processivity directly
or it may be that the products of these templates are the basis of the
inhibition observed. Because the C4349G and 5C substitutions allow
complete first repeat addition but inhibit repeat addition
processivity, these substitutions may affect the same molecular event
as the processivity-stimulatory binding of dGTP.
In addition to template requirements, we also investigated nucleotide
structural requirements for repeat addition processivity. We found that
deoxyguanosine monophosphate promotes as much repeat addition
processivity as deoxyguanosine triphosphate and has a similar
Km for stimulation of repeat addition processivity as well. This suggests that triphosphate hydrolysis is not required to
stimulate repeat addition processivity. In addition, we found that the
C-2 amino group of dGTP is essential but N-7 is not. Purine
ribonucleotides including GTP (shown above) and XTP (data not shown) do
not stimulate repeat addition processivity, indicating a specificity
for the deoxyribose sugar of dGTP. Notably, if the steric gate
excluding rNTPs from the active site is removed with the TERT
substitution Y623A, rGTP can be incorporated, and rGTP now stimulates
repeat addition processivity as well (23). This observation, combined
with those described above, convincingly argues that a
processivity-stimulatory dGTP molecule binds at the active site.
Tetrahymena telomerase has a Km for dGTP
incorporation that is about 10-fold lower than the
Km for incorporation of dTTP (18). Although the
Km for dATP and dCTP incorporation has not been
measured explicitly, the use of altered templates containing U or G
appears to require a concentration of dATP or dCTP more similar to that
required for dTTP than dGTP (reviewed in Ref. 6). The lower
Km for incorporation of dGTP than for other dNTPs
suggests that the telomerase active site harbors a specialization,
which specifically increases dGTP binding affinity. Binding of dGTP in
the active site in the absence of optimally positioned template and/or
product could be facilitated by this active site specialization, would
occur with a higher Km than measured for dGTP
incorporation and could stimulate repeat addition processivity.
Two Different Mechanisms for Repeat Addition
Processivity--
Among telomerase enzymes, the highest degree of
repeat addition processivity appears to be catalyzed by endogenous
Tetrahymena telomerase. However, recombinant
Tetrahymena telomerase does not share the very efficient,
dGTP concentration-independent repeat addition processivity of the
endogenous enzyme (11). The simplest explanation for this discrepancy
would be that the recombinant telomerase RNP, composed of only TERT and
telomerase RNA, fails to reconstitute at least some of the substrate
anchor sites present in the endogenous enzyme. Precisely because of
this difference, recombinant Tetrahymena telomerase may be
more representative of telomerase enzymes in general. Like the
recombinant Tetrahymena enzyme, endogenous telomerases from
another ciliate (12) and from mammalian cells (13, 24) demonstrate a
more limited repeat addition processivity than the endogenous
Tetrahymena enzyme that is stimulated by dGTP.
In this work, we investigated the mechanism by which recombinant
Tetrahymena telomerase can elongate products as short as T2G4T2G, derived from elongation of
the primer T2G4, with repeat addition
processivity. Our results suggest that product hybridized or formerly
hybridized at the template can associate with a nascent product binding
site after dissociation of the template-product hybrid (Fig. 8). The
specificity of the nascent product interaction would be reflected in
the template and/or product sequence requirements described above and
possibly by the inhibition of primer use upon dimethyl sulfate
modification of terminal guanosines at the N-7 position, which is not
predicted to affect base pairing with the template (25). If nascent
product interaction can persist in the absence of a template hybrid,
the proposed telomerase nascent product binding site would more closely
resemble that of an RNA polymerase than a viral RT, which interacts
with product in the context of the minor groove of a template-product
hybrid (26).
Our data argue strongly for binding of the processivity-stimulatory
dGTP at the active site but leave open the nature of the processivity-determining molecular event. It is possible that dGTP
either promotes the interaction of nascent product with enzyme (Fig. 8,
step 1) or promotes a subsequent conformational change required to reposition the product 3'-end (Fig. 8, step 2).
We have shown that the dGTP-dependence of repeat addition processivity does not derive from its role as the first nucleotide added to a
repositioned product. Our results also indicate that an influence of
dGTP on the dissociation of product-template hybrid is unlikely, because similar amounts of product are synthesized at all dGTP concentrations, and substantial product turnover occurs even when repeat addition is not processive. Thus, nascent product binding or
product 3'-end repositioning are more likely targets of stimulation by
dGTP. Additional insight will require a more direct investigation of
the product interaction sites in recombinant telomerase.