From the
Telomerase is an unusual ribonucleoprotein that synthesizes new
telomeres onto chromosome ends. The enzyme has been most extensively
characterized in ciliates, where the RNA component has been cloned from
several species, and its elongation properties have been characterized
in detail. To understand the substrate specificity and protein
composition of telomerase, we have used gel shift and UV cross-linking
to characterize the enzyme from the ciliate Tetrahymena
thermophila. In a mobility shift assay, a complex was identified
that contained telomerase RNA, co-purified with telomerase activity,
and was sensitive to nuclease treatment. The mobility shift complexes
specifically formed using several different single-stranded, telomeric
sequences but not non-telomeric primers. These results suggest that the
specificity of telomerase for G-rich primer sequences occurs at least
in part at the level of primer binding. UV cross-linking analysis
identified a 100-kDa cross-linked protein that may be a telomerase
component.
Chromosome ends are maintained by the unusual DNA polymerase,
telomerase. This polymerase, first discovered in Tetrahymena,
adds telomeric sequences de novo to chromosome ends to make up
for sequence loss during replication (Greider and Blackburn, 1985; Yu
et al., 1990). Telomerase represents a unique class of DNA
polymerase in that it contains an essential RNA component, which serves
as an internal template for the addition of telomeric repeat sequences.
In Tetrahymena, telomerase processively adds hundreds of
telomeric d(TTGGGG)
Unlike the binding specificity
of many DNA-binding proteins, telomerase primer specificity is not
precise. Telomerase can elongate G-rich single-stranded DNA
oligonucleotides regardless of the exact sequence of the primer.
However, non-telomeric sequence primers are not efficiently elongated
(Blackburn et al., 1989; Greider and Blackburn, 1987). For a
given primer 3` end, telomerase will correctly add the next base in the
telomeric sequence. The ability to correctly ``fill out'' any
permutation of the telomeric sequence d(TTGGGG) is likely due to primer
alignment with the telomerase RNA sequence CAACCCCAA (Greider and
Blackburn, 1989).
The Tetrahymena telomerase enzyme can be
reconstituted in vitro using synthetic telomerase RNA and
partially purified telomerase proteins (Autexier and Greider, 1994).
The reconstituted enzyme has similar elongation specificities as the
wild type telomerase. Mutations in the 5`-CAACCCCAA-3` sequence showed
that the 5` six nucleotides provide template information while the 3`
most CAA sequence is required for aligning primer 3` ends with the
template region. Thus the template region helps determine the
specificity for both primer 3` end sequence and the nucleotides
incorporated by telomerase.
The ``hybridization-directed''
model of telomerase elongation predicts that alignment of the substrate
3` end with telomerase RNA is important for elongation. However, in
vivo chromosome healing by telomere addition has been observed in
ciliates, yeast, and humans at broken ends that have little or no
G-rich sequence (reviewed in Greider (1991a)). In vitro,
telomerase will elongate ``chimeric'' oligonucleotides
containing two d(TTGGGG) repeats at the 5` end followed by up to 36
bases of non-telomeric DNA at the 3` end (Harrington and Greider,
1991). A similar analysis of human telomerase primer specificity in
vitro showed that the 3` end of a primer does not have to be
strictly complementary to the RNA template to be elongated (Morin,
1991). These results suggest that the sequences necessary for primer
recognition can be spatially separated from the site of telomere
addition, and led to the hypothesis that there may be a site of primer
recognition, or binding, that is distinct from the site of telomere
synthesis (Harrington and Greider, 1991; Morin, 1991). Kinetic analysis
using primers with a fixed 3` but different 5` end sequences also
suggests that there must be a contribution of a site distinct from the
template region in primer binding (Lee and Blackburn, 1993). Finally,
the non-processive elongation of short primers implies that there must
be a second site, or ``anchor site'' for primer binding
(Collins and Greider, 1993; Lee and Blackburn, 1993). By analogy to RNA
polymerases, a primer 10 nucleotides or longer can always be bound at
one site or the other during processive elongation, while shorter
primers may only be bound at the template site (Collins and Greider,
1993).
To date all experiments on primer specificity have assayed
elongation as the end point. The relative contribution of primer
binding and elongation have not been separately determined. We have
used a mobility shift assay to directly examine whether the telomerase
primer recognition step, like primer elongation, is specific for G-rich
sequences. In addition, we have identified a 100-kDa protein which
cross-links to telomerase products. The UV cross-linking and gel shift
analysis may be useful for the identification of telomerase proteins in
other organisms.
Tetrahymena cells
were grown and harvested after 12-18 h of starvation, without
subsequent mating (Avilion et al., 1992). Typically, 36 liters
of Tetrahymena cells were grown to a density of 3.0-3.5
Some purifications were carried out using a similar procedure as
above, except the order of columns was changed to DEAE-agarose,
phenyl-Sepharose, and heparin-agarose, followed by spermine-agarose,
concentration on a DEAE-agarose column, and sucrose gradient
sedimentation (Harrington, 1993). Briefly, S100 extract prepared from
approximately 10
We next assayed directly for telomerase RNA
in the mobility shift complex. Telomerase fractions were incubated
either with or without radiolabeled telomeric primer and
electrophoresed on adjacent lanes on a nondenaturing gel (Fig.
4 A). The two lanes were subsequently excised, cut into 10
slices and RNA eluted from each gel slice. Reverse transcription and
PCR using telomerase RNA specific primers showed that telomerase RNA
was present in gel slice number 3 that contained the mobility shift
complex in the presence of primer (Fig. 4 B). Telomerase
RNA was not detected at this position in the gel when telomerase
extracts were not incubated with the
The
competition for primer binding in the gel shift and cross-linking
assays using oligonucleotides complementary to telomerase RNA also
suggests two possible modes for primer binding. The oligonucleotides
oligo 3 and oligo 8 are both elongated by telomerase. When oligo 3,
which binds adjacent to and covers the template region, is preincubated
with telomerase it inhibits subsequent elongation of d(TTGGGG), while
preincubation with oligo 8 which hybridizes adjacent to the template
does not (Greider and Blackburn, 1989). Consistent with this, in the
gel shift and cross-linking studies oligo 3 competed for
d(TTGGGG)
In vivo, telomerase will add (TTGGGG)
Polymerase interactions with primer
and template have been studied extensively using gel mobility shift
assays. Kinetic parameters of primer and template binding (Hsieh et
al., 1993), analysis of DNA binding specificities (Ng et
al., 1993), identification of auxiliary factors (Flores et
al., 1992), and mutational analysis of amino acids involved in
primer and template binding (Date et al., 1991; Joung and
Engler, 1992) have been defined using this assay. Having extensively
characterized the telomerase primer complex, we can now use this assay
to address the mechanism by which telomerase recognizes primer
substrates. In addition, the initial binding affinity and the initial
rate of nucleotide addition can be separately compared for a wide
variety of telomeric and non-telomeric primers.
If the
100-kDa protein is a telomerase component, the sedimentation of
telomerase between 200 and 400 kDa in sucrose gradients suggests that
other polypeptides in these fractions may also be telomerase
components, but are not cross-linked during elongation (Harrington,
1993). Further purification is underway to unambiguously define the
polypeptides that make up the telomerase RNP and to clone the genes for
these subunits.
We thank Winship Herr and Bruce Stillman for helpful
advice and the following people for critical reading of the manuscript:
Winship Herr, Bruce Stillman, Erich Grotewold, Brenda Andrews, Mike
Tyers, Kathleen Collins, Stephanie Smith, and Chantal Autexier. We also
thank Jim Duffy, Phillip Renna, and Mike Ockler for photography and
artwork.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
repeats, one nucleotide at a
time, using an internal RNA sequence CAACCCCAA as a template (Greider
and Blackburn, 1989). The role of the telomerase RNA was demonstrated
in two ways. First, inactivation of Tetrahymena telomerase
using RNA-complementary oligonucleotides and RNase H cleavage abolished
telomerase activity in vitro (Greider and Blackburn, 1989).
Second, when a telomerase RNA gene mutation in the CAACCCCAA template
region was introduced into Tetrahymena, telomeres
corresponding to the mutant sequence were synthesized (Yu et
al., 1990). These results demonstrated that not only is the
CAACCCCAA sequence used as a template but also that telomerase is the
enzyme which synthesizes telomere sequences in vivo.
Telomerase activities with similar properties to the Tetrahymena enzyme have been identified in several different organisms,
including hypotrichous ciliates, human, mouse, and Xenopus laevis cells (Lingner et al., 1994; Mantell and Greider, 1994;
Morin, 1989; Prowse et al., 1993; Shippen-Lentz and Blackburn,
1989; Zahler and Prescott, 1988). The RNA component of telomerase has
so far been cloned only in ciliates (Greider and Blackburn, 1989;
Lingner et al., 1994; Romero and Blackburn, 1991;
Shippen-Lentz and Blackburn, 1990).
Purification of Tetrahymena Telomerase
At all
steps in the purification, telomerase elongation activity was assayed
as described previously (Harrington and Greider, 1991). Briefly, 20
µl of extract was incubated with 20 µl of a 2 reaction
buffer, such that the final concentration of the added components was
100 µ
M TTP (Pharmacia), 1 µ
M [
P]dGTP, 0.1 µg of d(GGGGTT)
in 1
telomerase buffer (1 m
M spermidine, 60
m
M K-acetate, 5 m
M
-mercaptoethanol, and 50
m
M Tris acetate, pH 8.5). Incubation at 30 °C for 1 h was
followed by phenol extraction and ethanol precipitation of the
radiolabeled products. The dried pellets were resuspended in 2.5 µl
of sequencing gel loading buffer (37%, v/v, formamide, and
approximately 0.25%, w/v, bromphenol blue, 0.25%, w/v, xylene cyanol),
and resolved on a 10%, w/v, denaturing acrylamide gel as described
previously (Greider and Blackburn, 1987).
10
cells/ml in PPYS (2% w/v proteose peptone
(Difco), 0.2%, w/v, yeast extract (Difco), 10 m
M FeCl
) at 30 °C, pelleted, and starved by
resuspension in 8-12 liters of Dryls medium (1.3 m
M Na
HPO
, 1.2 m
M NaH
PO
, 1.7 m
M Na-citrate, 2
m
M CaCl
) at 30 °C for 12-18 h. Cells
were harvested and the cell pellet was lysed at 4 °C in 3 volumes
of TMG (10 m
M Tris, pH 8.0, 1 m
M MgCl
,
10%, v/v, glycerol, 5 m
M
-mercaptoethanol, 100 µ
M phenylmethylsulfonyl fluoride, and 0.25 µg/ml each of
leupeptin, pepstatin, chymostatin, and antipain) and 0.2%, v/v, Nonidet
P-40, and centrifuged at 100,000
g in a SW41 Beckman
rotor at 4 °C for 1 h. All subsequent purification was carried out
at 4 °C, and telomerase fractions were quick-frozen in liquid
nitrogen and stored at -70 °C. Heparin-agarose-purified
telomerase was obtained as described previously (Greider and Blackburn,
1987) or as follows. The S100 was loaded onto a heparin-agarose
(Bio-Rad) column equilibrated with TMG, washed with TMG, and eluted
with TMG containing 0.2
M potassium glutamate. The eluate was
loaded onto DEAE-agarose (Bio-Rad) column equilibrated with TMG, washed
with TMG, 0.15
M potassium glutamate, and eluted with a linear
salt gradient of 0.15-0.6
M potassium glutamate in TMG.
-10
cells was loaded onto
DEAE-agarose (Bio-Rad) equilibrated in TMG, 0.15
M potassium
glutamate (KGlu), and eluted with a linear salt gradient of 0.15
M KGlu to 0.6
M KGlu in TMG. Active fractions were pooled
and loaded onto phenyl-Sepharose equilibrated in TMG, 0.4
M KGlu, and eluted with TM, 50%, v/v, ethylene glycol. The pooled
active fraction was adjusted to approximately 0.05
M KGlu,
loaded onto heparin-agarose (Pharmacia) equilibrated in TMG, and eluted
with TMG, 0.2
M KGlu. Active fractions were pooled and loaded
onto spermine-agarose (Sigma) equilibrated in TMG, 0.2
M KGlu,
and eluted with TMG, 0.6
M KGlu. To concentrate the enzyme,
active fractions were diluted to 0.15
M KGlu with TMG, loaded
onto a small DEAE-agarose column, and eluted with TMG, 0.4
M KGlu. A portion of the most active eluate (100-150 µl)
was loaded onto a 3.6-3.8-ml 7-30%, w/v, sucrose gradient
in TMG, 0.4
M KGlu, and centrifuged in a Beckman SW50.1 rotor
for 17 h, 150,000
g at 4 °C. Fractions were
collected from the bottom to the top of the gradient using a glass
capillary tube and a fraction collector, and the samples were
immediately assayed for telomerase activity before storage in liquid
nitrogen. In this purification scheme, telomerase was purified
approximately 100-1000-fold based on measuring the enrichment for the
telomerase RNA component (Harrington, 1993). The fold purification
relative to activity was not possible to determine accurately because
activity increased over the first few columns suggesting that
inhibitors were being removed.
Affinity Purification
Affinity purification of
telomerase extracts was carried out by a modification of the method of
Franza et al. (1987). Approximately 1 ml of partially purified
extract (sizing column, heparin-agarose, DEAE-agarose) was incubated
with 75 µl of streptavidin-agarose beads (Bethesda Research
Laboratories), and rocked at 4 °C for 30 min. The agarose beads
were pelleted in a microcentrifuge, and the supernatant was removed to
a new tube. To this supernatant was added 30-100 µg of the
nonspecific competitor, pBR oligonucleotide (Harrington and Greider,
1991), and the sample was rocked at 4 °C for another 30 min. Fifty
to 100 µg of biotinylated (TTGGGG)was added, and the
sample was rocked for 1 h at 4 °C (Photoprobe Biotin obtained from
Vector Laboratories and DNA prepared according to manufacturer's
instructions). The supernatant was then divided into three, and
serially precipitated with approximately 200 µl of
streptavidin-agarose for 20 min at 4 °C. After each incubation, the
beads were pelleted, the supernatant was removed, and another one-third
supernatant was added. After all supernatant had been exposed to the
agarose beads, the beads were extensively washed in TMG. A small amount
of TMG remained over the washed pellet. The beads were assayed directly
for telomerase activity and UV cross-linking.
Oligonucleotides and Mobility Shift Probes
All
oligonucleotides were obtained from Operon Technologies, Inc. and were
gel-purified as described previously (Harrington and Greider, 1991).
For generation of probes for the mobility shift assay, 0.1 µg of
gel-purified oligonucleotide was added to 1 kinase buffer
(Sambrook et al., 1989), 100 µCi of
[
-
P]ATP (DuPont NEN), and 5 units of T4
polynucleotide kinase (BRL) in a final volume of 20 µl, and
incubated at 37 °C for 15-30 min. The reaction was stopped by
addition of 80 µl of 1
TE (10 m
M Tris-Cl, pH 8.0,
1 m
M EDTA) and stored at -20 °C. The probe was
diluted to a final concentration of 1 ng/µl and 1 µl was used
for each reaction in the mobility shift assay.
Telomerase Mobility Shift Assay
For the standard
mobility shift, 10 µl of extract was incubated with 10 µl of a
mixture containing 1 µl of P-d(TTGGGG)
(or
P-d(GGGGTT)
in some experiments), 3.0 µl
of mobility shift buffer (33 m
M Tris acetate, pH 8.0, 3.3
m
M MgCl
, 33%, v/v, glycerol), 1.5 µl of 100
ng/ml d(T)
, and 4.5 µl of sterile water. The sample
was incubated on ice for 10-30 min, and was loaded directly onto
a pre-run 6%, w/v, nondenaturing acrylamide gel (20:1
acrylamide:bis-acrylamide) in 1
TBE (Sambrook et al.,
1989), electrophoresed for 4 h at 240 V, dried, and exposed to XAR-5
film. For the competition assays, the standard mobility shift assay was
performed as described above, except that 1 µl of the appropriate
concentration of unlabeled competitor oligonucleotide was added to the
probe mixture. For the nuclease experiments, 10 µl of telomerase
extract was incubated with 1-2 units of micrococcal nuclease and
1 m
M CaCl
(final concentration) at 30 °C for
10 min. The sample was returned to ice, and brought to 10 m
M EGTA and 10 m
M MgCl
, followed by addition of
10 µl of probe mixture as described above. Control samples were
treated identically, except micrococcal nuclease was omitted. For
treatment with ribonuclease ONE (Promega) or ribonuclease CL3
(Pharmacia Biotech Inc.), the appropriate units as indicated in
Fig. 3
were added to 10 µl of partially purified telomerase
extract, and incubated on ice for 10 min prior to the addition of 10
µl of probe mixture.
Figure 3:
RNase sensitivity of the mobility shift
complex. Lane 1, P-labeled 1-kbp marker, with
base pairs as shown at the left. Lane 2, untreated telomerase
extracts assayed for
P-d(TTGGGG)
complex
formation; lanes 3-6, telomerase extracts preincubated
on ice with increasing units of RNase ONE prior to probe addition.
Lanes 7-9, preincubation with increasing units of RNase
CL3. The position of the telomerase mobility shift is indicated with an
arrow. The labeled input probe was run off the
gel.
Reverse Transcriptase-PCR
Telomerase RNA was isolated from telomerase
preparations with one phenol extraction, and ethanol precipitated with
5 µg of yeast tRNA (Sigma) as a carrier. In vitro transcribed telomerase RNA was used as a positive control for the
PCR, and was prepared using T7 polymerase as described previously
(Avilion et al., 1992). For isolation of RNA from excised gel
slices as indicated in Fig. 4, the gel slices were crushed into
0.3
M sodium acetate, 0.2%, w/v, SDS and eluted overnight at
30 °C, and the RNA was precipitated from the supernatant with 600
µl of absolute ethanol. The sequence of the primers used in the PCR
reaction are: ``oligo 10`,''
5`-AAAAATAAGACATCCATTGATAAATAGTGTATCAAATG-3`, and ``oligo
9`,'' 5`-ATACCCGCTTAATTCATTCAGA-3`. Telomerase RNA samples
(ranging from 50 pg to 1 ng) were brought to 4.5 µl with diethyl
pyrocarbonate-treated sterile water, and added to a 20 µl of
mixture containing 1 Analysis of
Telomerase RNA
reaction buffer (50 m
M Tris-Cl,
pH 8.3, 6 m
M MgCl
, 40 m
M KCl), 100 pmol
of primer (oligo 10), 1.0 m
M each of dATP, dCTP, dGTP, and
TTP, 1.0 m
M dithiothreitol, 20 units of RNasin (Promega), and
2-5 units of avian myeloblastosis virus reverse transcriptase
(Life Sciences, Inc.). The sample was incubated at room temperature for
10 min, and subsequently at 52 °C for 60 min, and 95 °C for 5
min. To this sample was added a 80-µl mixture containing 1
PCR buffer (20 m
M Tris-Cl, pH 8.4, 50 m
M KCl, 1.2
m
M MgCl
), 200 µ
M each of dATP, dCTP,
dGTP, and TTP, 1.0 µ
M of each primer (oligo 10, oligo 9),
and 2.5 to 5 units of AmpliTaq polymerase (Perkin-Elmer). The sample
was overlaid with 100 µl of light white mineral oil (Sigma), and
amplified in a Perkin-Elmer PCR machine for 30 cycles of 1 min at 94
°C, 1 min at 46 °C, and 1 min at 72 °C. To visualize the
products, 10-30 µl of each reaction were resolved on a 1%,
w/v, agarose gel and the gel was stained in 1 µg/ml ethidium
bromide.
Figure 4:
Telomerase RNA is present in the shifted
complex. A, a mobility shift gel was run and each lane was cut
into 10 sections to determine the position of telomerase RNA in the
gel. Lane 1, P-labeled 1-bp marker, with base
pairs indicated at the left. Lane 2, partially
purified telomerase (200-fold) was incubated under standard conditions
with
P-d(TTGGGG)
, and electrophoresed on a 6%,
w/v, nondenaturing acrylamide gel. Lane 3, telomerase extract
was incubated with all components of the mobility shift mixture except
the probe. The wet gel was wrapped and briefly exposed on a
PhosphorImager imaging plate to visualize the telomerase complex, and
both lanes were divided into several gel fragments, 1-10 for lane 2 (gel slice number 3 contained the telomerase
complex and is indicated at right with an arrow), and
11-20 for lane 3. B, the products of reverse
transcriptase-PCR using telomerase RNA-specific primers for each gel
slice are shown after electrophoresis on a 1%, w/v, agarose gel and
stained with ethidium bromide. Lane 1, 1-bp marker, with base
pairs as indicated at the left. Lanes 2-11, gel slices
1-10, corresponding to lane 2 in A. Lanes
12-21, gel slices 11-20, corresponding to lane 3 in A. Lane 22, 100 pg of in vitro transcribed telomerase RNA used as a positive control in the PCR
reaction. The position of the expected product is indicated to the
right with an arrow. Lane 23, as a negative control,
no RNA added in the reverse transcriptase-PCR
reaction.
UV Cross-linking of Telomerase
For the
NRdUTP elongation assay, all reactions were carried out in
subdued light. Ten µl of telomerase extract was incubated with a
4-µl reaction mixture, consisting of 1 µl of 10
telomerase buffer, 1 µl of [
P]dGTP (400
Ci/mmol; NEN), 1.0 µl of 1 m
M N
RdUTP
(synthesized according to Bartholomew et al. (1990, 1991)),
and 0.5 µg of gel-purified d(G
T
)
primer. Elongation reactions proceeded for 6-10 min at 30
°C. Samples to be cross-linked were placed in a 96-well microtiter
plate pre-blocked with 1 mg/ml bovine serum albumin or approximately 50
µl of Sigmacote (Sigma), covered in plastic wrap, and placed at
room temperature directly underneath a hand-held UV monitor at 254 nm
for 5-10 min. For unirradiated controls, the samples remained at
room temperature in the dark during the course of cross-linking. The
sample was removed, and approximately 5 units each of RNase ONE, DNase,
and MNase were added to the sample, and incubated at 30 °C for 10
min. The sample was boiled in protein gel loading buffer and resolved
on a 7-16%, w/v, acrylamide linear gradient protein gel, with a
3%, w/v, acrylamide stack.
Telomerase Primer Recognition
The specificity of
Tetrahymena telomerase primer elongation has been extensively
characterized (Blackburn et al., 1989; Collins and Greider,
1993; Greider, 1991b; Greider and Blackburn, 1987; Harrington and
Greider, 1991; Lee and Blackburn, 1993). However, since it is not
possible to distinguish between primer binding and nucleotide addition
in the previously used elongation reactions, we used a mobility shift
assay to directly study the primer binding specificity of highly
purified telomerase. Radiolabeled telomeric d(TTGGGG)oligonucleotide was incubated with fractions containing
telomerase and resolved on a nondenaturing gel. Using partially
purified extracts, we identified a protein-DNA complex that was
specific for telomeric DNA and that migrated with the 344-bp DNA marker
(Fig. 1). This complex was efficiently competed by a 20-fold excess of
unlabeled competitor telomeric oligonucleotide, d(TTGGGG)
,
but was not competed by up to a 2500-fold excess of non-telomeric
oligonucleotide, pBR (Fig. 1). Other telomeric or telomere-like
oligonucleotides that are substrates for telomerase elongation, such as
d(TTGGGG), d(GGGGTT)
, d(TTAGGG)
,
d(TG)
, d(TGTGTGGG)
TG, and
d(TTGGGG)
pBR (Blackburn et al., 1989; Greider and
Blackburn, 1987; Harrington and Greider, 1991), also competed for
formation of this complex (data not shown). An oligonucleotide
complementary to the telomerase RNA template region, oligo 3, also
competed for
P-d(TTGGGG)
-binding
(Fig. 1). This oligonucleotide contains 21 nucleotides
complementary to the RNA, including the nine nucleotides, TTGGGGTTG,
covering the RNA template (Greider and Blackburn, 1989). A second RNA
complementary oligonucleotide, oligo 8, which hybridizes adjacent to
but does not cover the CAACCCCAA RNA template (Greider and Blackburn,
1989) did not efficiently compete for
P-d(TTGGGG)
binding (data not shown). The telomere-specific mobility shift
did not require Mg
, TTP, or dGTP for complex
formation (data not shown). This complex was not formed when telomerase
extracts were incubated with the
P-labeled non-telomeric
oligonucleotide, pBR, or when telomerase extracts were treated with
Proteinase K (data not shown).
Figure 1:
Gel mobility
shift of d(TTGGGG) by telomerase. Partially purified telomerase was
incubated with 1 ng of P-d(TTGGGG)
and
resolved on a nondenaturing gel. Lane 1,
P-labeled 1-kbp marker, with base pairs indicated at the
left. Lanes 2-16, the indicated amount of unlabled
competitor oligonucleotide was added to the mobility shift mixture; for
d(TTGGGG)
, from 2500 ng ( lane 2) to no competitor
( lane 6), and similarly, competition with oligo 3 and pBR,
lanes 7-11 and 12-16, respectively. The
position of the d(TTGGGG)
-specific mobility shift is
indicated with an arrow at right, and the arrow at bottom right marks the approximate position
of unbound
P-d(TTGGGG)
probe. The sequences of
oligonucleotides used in this experiment are: pBR,
5`-AGCCACTATCGACTACGCGATCAT-3`; oligo 3,
5`-GCA-CTAGATTTTTGGGGTTG-3`.
To determine whether the
P-d(TTGGGG)
-binding activity corresponds to
telomerase or to some other telomere-binding protein, we followed
telomerase elongation activity and the mobility shift complex
throughout telomerase purification (Fig. 2). Tetrahymena S100
extracts were purified over gel filtration, heparin-agarose, and
DEAE-agarose (Fig. 2 A). The
P-d(TTGGGG)
-binding activity further
co-purified with telomerase over the series: DEAE-agarose,
phenyl-Sepharose, heparin-agarose, spermine-agarose, a DEAE
concentrating column, and finally sucrose gradient sedimentation
(Fig. 2 B). Co-purification of the band shift with
telomerase on the final gradient is shown in Fig. 2 C. In
this experiment the band shift appeared as a doublet. This doublet,
seen in several highly purified preparations, has identical specificity
as the single shift and may be due to limited telomerase degradation in
these fractions (data not shown).
Figure 2:
Mobility shift co-purifies with telomerase
activity. A: lanes 1-5, telomerase elongation
activity through a partial purification. Lanes 1, 3, and
5, pooled fractions containing telomerase activity after gel
filtration, heparin-agarose, and DEAE-agarose, respectively. Lanes
2 and 4, flow-through fractions from the heparin-agarose
and DEAE-agarose columns, respectively. Lanes 6-12,
P-d(TTGGGG)
mobility shift assay on respective
fractions throughout the purification at left: lane 6, the
same pooled, active fractions after gel filtration as in lane
1; lane 7, a fraction from the gel filtration column
which did not contain telomerase activity; lane 8, a gel
filtration fraction containing telomerase activity. Lanes
9-12, mobility shift assay using the same fractions as in
lanes 2-5. Markers are indicated at right in
base pairs. B, telomerase activity fractionated over
DEAE-agarose, phenyl-Sepharose, heparin-agarose, spermine-agarose, and
concentrated on a DEAE-agarose column was sedimented on sucrose
gradient. Each fraction was assayed for telomerase elongation activity,
and the DNA products were resolved on a 10%, w/v, denaturing acrylamide
gel. Lanes 1-19, sucrose gradient fractions from the
bottom to the top of the gradient, indicated at bottom. The
sedimentation of native protein markers in the gradient are indicated
with arrows above lanes 3, 10, and 14 in kDa. The
position of the first nucleotide added to the d(GGGGTT)
primer is shown with an arrow at bottom right. C, fractions 1-14 of the sucrase gradient shown in part B were tested for the ability to bind the
P-d(TTGGGG)
probe. Lane 1,
P-labeled 1-kbp marker, with the molecular weight of each
band as indicated to the left. Lane 2, the purified
fraction used to load the sucrose gradient. Lanes 2-16,
sucrose fractions 1-14 from the bottom to the top of the
gradient.
Since telomerase contains an
essential RNA component, we tested whether the complex was sensitive to
RNase treatment. Partially purified telomerase extracts were
preincubated with different ribonucleases, and then assayed for complex
formation. Treatment with either RNase ONE, which cleaves after any
nucleotide, or RNase CL3, which cleaves primarily at C residues,
eliminated the complexes migrating with the 344-bp DNA marker (Fig. 3).
Incubation with each RNase resulted in the formation of other complexes
at lower mobilities than the telomerase complex (Fig. 3). The
reason for this effect is not known. Pre-treatment with active MNase
also eliminated the 344-bp complex, whereas the complex was unaffected
by incubation of telomerase extracts with MNase that had been
inactivated with EGTA (data not shown). These results indicate that the
telomeric primer binding complex contains an RNA component necessary
for complex stability.
P-d(TTGGGG)
probe. Telomerase RNA was also detected near the wells of the
mobility shift gel, possibly as a result of enzyme that did not enter
the acrylamide gel (Fig. 4 B). Thus, the presence of
telomerase RNA in the gel section containing the (TTGGGG) binding
complex suggests that this complex represents telomerase.
UV Cross-linking of Telomerase Products to Potential
Protein Components
To identify the protein components present in
the telomerase complex, we generated a photoreactive telomeric
oligonucleotide probe containing the TTP analog, NRdUTP
(Bartholomew et al., 1990, 1991). We first attempted to
directly bind telomerase to the
P-labeled
N
RdUTP-containing DNA, and then cross-link the complex.
However, using the mobility shift assay it was apparent that telomerase
would not bind a probe that contained N
RdUTP (data not
shown). We therefore took advantage of the ability of telomerase to
incorporate the photoreactive TTP analog, N
RdUTP, during
the elongation of telomeric primers. In this assay, the generation of a
radioactive, cross-linkable primer depends upon the action of
telomerase to incorporate the N
RdUTP and
[
P]dGTP onto unlabeled d(TTGGGG)
primer. Upon irradiation of the telomerase reaction with UV
light, three bands at approximately 100, 50, and 25 kDa were apparent
(Fig. 5). Only the 100-kDa cross-linked protein was specific to the
presence of telomerase elongation products. Telomerase extracts that
were inactive, either through ribonuclease treatment or omission of
telomeric primer, did not generate a 100-kDa band. The cross-linked
protein at 100 kDa was seen only upon exposure to UV, whereas the two
smaller proteins were labeled even in the absence of UV irradiation
(Fig. 5). Proteinase K treatment either before primer elongation
or just prior to UV irradiation abolished the signal at 100 kDa
indicating that this band is a protein (data not shown).
Figure 5:
Cross-linking of a 100-kDa protein to
telomerase products. Telomerase elongation activity was assayed in the
presence of [P]dGTP and N
RdUTP.
Lane 1, standard elongation assay; lane 2, telomeric
primer was omitted from the reaction; lane 3, the telomerase
extract was pretreated with 10 units of ribonuclease ONE for 10 min on
ice prior to the elongation assay; lanes 4 and 5,
standard assay conditions as in lane 1. The position of the
input primer d(G
T
)
is indicated at
the bottom right by an arrow. Aliquots of the same
samples in lanes 1-5 were exposed to UV light and
resolved by SDS-PAGE. Lanes 7, 8, and 10, 10-min UV
treatment. Lane 9, 5-min UV treatment. The 100-kDa
cross-linked protein is indicated at the right by an
arrow; protein standards are at the left in
kDa.
Using
telomerase purified over 4 steps, DEAE, phenyl-Sepharose,
heparin-agarose, spermine-agarose, and concentrated on a DEAE column,
we compared the elongation of different telomeric and non-telomeric
primers in the UV cross-linking assay. Oligonucleotides that showed
significant incorporation of NRdUTP and
[
P]dGTP generated a labeled 100-kDa protein, for
example, d(GGGGTT)
and d(TTGGGG)
(Fig. 6).
Oligonucleotides which competed for elongation activity, such as oligo
3 and to a lesser extent oligo 8, also competed for labeling of the
100-kDa cross-linked protein. In contrast, oligonucleotides which were
not efficiently elongated in the presence of N
RdUTP, such
as pBR and pBRG
, showed little or no detectable
cross-linked protein at 100 kDa (Fig. 6 B). Preincubation of
extracts with the non-telomeric oligonucleotides, pBR and an 18-base
oligo(dT), d(T)
, prior to
d(G
T
)
addition did not compete for
cross-linking to the 100-kDa protein (Fig. 6 B). In
extracts preincubated with the pBR oligonucleotide, both the elongation
products and the 100-kDa cross-link were enhanced, suggesting that pBR
is competing for nonspecific single-stranded DNA binding activity in
the extract (Fig. 6 B).
Figure 6:
Specificity of the 100-kDa cross-link is
similar to telomerase primer specificity. A: lanes 1-4, telomerase was reacted with 0.5 µg of the primer indicated
above each lane. Lanes 1 and 2, telomerase elongation
products using the primers d(GT
)
and d(T
G
)
, respectively.
Lanes 3 and 4, elongation of pBRG
and pBR
oligonucleotides, respectively. Lanes 5-8, samples were
preincubated with 1.0 µg of the oligonucleotides pBR, oligo
d(T)
, oligo 3, and oligo 8, respectively, prior to the
addition of telomeric primer d(G
T
)
(0.5 µg) and reaction mixture. Lane 9, telomerase
was preincubated with 2 units of RNase ONE (Promega) prior to the
elongation assay. Lane 10, telomerase was incubated with 2
units of RNase ONE immediately following the elongation assay, prior to
UV cross-linking. Lane 11, elongation of
d(G
T
)
in the presence of 5 m
M EDTA and 2 m
M MgCl
. B, aliquots of
the same samples as in A were exposed to UV light for 5 min
and analyzed by SDS-PAGE. The position of the 100-kDa cross-linked
protein is indicated with an arrow at the right.
Protein markers are indicated in kDa at the
left.
In this assay the generation
of radiolabeled product for cross-linking depends on the presence of
telomerase activity, thus the 100-kDa protein was not labeled when
fractions were preincubated with RNase (Fig. 6). To determine
whether the cross-linking of the 100-kDa protein to the
NRdUTP probe is RNA-dependent, we carried out a telomerase
reaction and then subsequently treated with RNase before UV
cross-linking. Incubation with ribonuclease after the telomerase
elongation reaction had no effect on the synthesized products yet
labeling of the 100-kDa cross-linked protein was reduced (Fig. 6,
A and B). Thus primer binding may require the
integrity of the telomerase complex.
Co-purification of the 100-kDa Species with
Telomerase
To examine the co-localization of the 100-kDa
cross-linkable protein with telomerase, enzyme purified over
DEAE-agarose, phenyl-Sepharose, spermine-agarose, DEAE concentration,
and sucrose gradient sedimentation was assayed by UV cross-linking
(Fig. 7). Upon sucrose gradient sedimentation, long telomerase
elongation products with NRdUTP incorporated peaked in
fractions 4 and 5 and the 100-kDa cross-linked species was also present
in fractions 4 and 5 (Fig. 7, A and B). SDS gel
analysis of these sucrose gradient fractions showed that a band of
approximately 100 kDa was present in the fraction that contained the
100-kDa cross-linked protein. Because it is not possible to quantitate
the amount of protein that is cross-linking with the reagents currently
available, we cannot determine if the 100-kDa protein seen by silver
staining is the same protein that cross-links to the telomerase
products. A number of other polypeptides were also present in the
active gradient fractions. Quantitative estimates of the fold
purification indicate that telomerase is not purified to homogeneity in
the fractions (data not shown). Thus, from the available data, we
cannot yet conclude that the 100-kDa band seen by silver staining is a
telomerase component. Further purification of telomerase is currently
underway.
(
)
Figure 7:
Sedimentation of 100-kDa cross-linked
protein with telomerase. A, as the last step in the large
scale telomerase purification (see ``Experimental
Procedures''), telomerase extract was sedimented on a 7-30%,
w/v, sucrose gradient, and fractions were collected from the bottom to
the top of the gradient. This gradient is the same as shown in Fig.
2 B. For this experiment, each fraction was assayed in the
presence of NRdUTP, and the DNA products were resolved on a
10%, w/v, denaturing acrylamide gel. B, the same sucrose
gradient samples as shown in A were cross-linked, and resolved
by SDS-PAGE. The position of the 100-kDa cross-linked protein is
indicated at the right with an arrow, and molecular
weight standards are shown at the left in kDa. C,
approximately one-twentieth of each fraction from the sucrose gradient
was loaded onto a SDS-PAGE gel. The fraction numbers are indicated at
the bottom. M indicated the protein size
standards.
To examine whether the 100-kDa
cross-link co-purified with telomerase using a different purification,
we developed an affinity purification for telomerase. Biotinylated
telomeric oligonucleotides were tested for their ability to precipitate
telomerase activity onto streptavidin-agarose. Telomerase fractions
purified over a sizing column, heparin-agarose, and DEAE-agarose were
incubated with a biotinylated 48-base telomeric oligonucleotide,
d(TTGGGG), followed by precipitation of the extract onto
streptavidin-agarose. Using this procedure up to 25% of telomerase
activity was recovered on the agarose beads (Harrington, 1993). The
agarose beads containing the affinity-purified telomerase activity were
incubated with telomeric primer, N
RdUTP,
[
P]dGTP, and subjected to UV cross-linking.
SDS-PAGE and autoradiography of the affinity-purified, cross-linked
extracts also showed radiolabeling of a 100-kDa protein (Fig. 8).
Similar to the extracts purified using standard chromatography,
cross-linking of the 100-kDa protein was dependent on UV treatment and
the generation of telomerase reaction products (Fig. 8). The
100-kDa protein was also labeled in the absence of added
d(GGGGTT)
primer (Fig. 8, lane 2), since
telomerase could elongate the primer bound to streptavidin-agarose,
d(TTGGGG)
(data not shown).
Figure 8:
Affinity purification and cross-linking of
telomerase. Telomerase extracts were purified to approximately
1000-fold by affinity chromatography, and samples were assayed for
elongation and cross-linking. Lane 1, no UV treatment.
Lane 2, no added d(GT
)
primer. Lane 3, affinity-purified extracts were
incubated with approximately 10 µg of ribonuclease A prior to the
telomerase elongation assay. Lanes 2 and 3 were
exposed to UV light for 10 min. Lanes 4 and 5,
telomerase elongation products were exposed to UV light for 5 and 10
min, respectively.
Telomerase Primer Binding Specificity
Telomerase
is an unusual DNA polymerase in that it carries the template sequence
for telomere repeat synthesis as an essential RNA component. Although
the enzyme copies RNA into DNA, like a reverse transcriptase, the
telomerase mechanism is unique since only six nucleotides of the
template are copied and telomeric primers are specifically elongated.
Telomerase elongates telomeric sequences, whereas non-telomeric primers
are very poor substrates (Blackburn et al., 1989; Greider and
Blackburn, 1987; Harrington and Greider, 1991). The primer elongation
specificity may lie in the initial binding of telomerase to primer
substrates or in the ability to elongate primers once they are bound,
or both. To distinguish between these two steps, binding and
elongation, we sought an assay which would directly measure binding of
primer oligonucleotides. We identified a telomeric primer
d(TTGGGG)-specific complex using a mobility shift assay
that was competed by several G-rich oligonucleotides. The specificity
of primer binding reflects that of primer elongation, suggesting that
elongation specificity is in part at the level of binding. The ability
of telomerase to specifically bind telomeric primers was distinct from
elongation, since binding occurred in the absence of nucleotides and
Mg
. This property is similar to those of other RNPs,
such as RNase P, which require Mg
, and nucleotides
only during catalysis, and not for substrate recognition (Smith and
Pace, 1993). It is not yet clear whether the complex we have identified
is a true ``preinitiation'' complex that is competent for
elongation upon addition of nucleotides.
Specificity at Both Anchor and Template
Sites
Current models for telomerase primer binding suggest that
there are two distinct sites on telomerase for binding primer
oligonucleotides (Collins and Greider, 1993; Harrington and Greider,
1991; Lee and Blackburn, 1993; Morin, 1991). The template region
CAACCCCAA plays a role in aligning the 3` end of primer
oligonucleotides (Autexier and Greider, 1994). A second site for primer
binding, the anchor site, possibly on a protein component, is required
for processive elongation of bound primers (Collins and Greider, 1993).
How each of these sites determines primer specificity has not yet been
determined. Telomerase is not restricted to elongating
d(TTGGGG)primers. Oligonucleotides containing the
telomere sequence of other eukaryotes such as d(TTAGGG), d(TTTTGGGG),
d(TGGGTGTG), and even the sequence (TG)
, not found at
telomeres in nature, are elongated (Blackburn et al., 1989;
Greider and Blackburn, 1987). These data suggest that the role of the
RNA CAACCCCAA sequence in primer binding is not strictly through base
pairing. The primer d(TG)
will only form two base pairs
with the template region. In addition, the anchor site, which may play
an important role in primer recognition, must not have strict
specificity for d(TTGGGG) in the way that many DNA-binding proteins do.
The results presented here suggest that telomerase binding primer
specificity matches primer elongation specificity. Thus binding may
play a key step in determining what substrates are elongated.
binding, but oligo 8 did not compete efficiently.
The ability of oligo 3 to compete for telomeric primer binding could
occur at either the anchor site or RNA template since oligo 3 contains
the sequence TTGGGGTTG at its 3` end. Oligo 8, however, is probably
elongated solely by hybridizing adjacent to the CAACCCCAA template.
repeats onto AT-rich DNA with no pre-existing telomeric sequence
(Yu and Blackburn, 1991). This specificity is clearly different than
that seen in vitro. Binding to nontelomeric sequences was not
detectable in the gel shift assay. There may be other factors in the
cell which associate with telomerase to facilitate the recognition of
new or broken chromosome ends, which do not co-purify with telomerase
activity in vitro, or which are expressed only at specific
times in the Tetrahymena life cycle.
Telomerase Has Properties Similar to Other
Polymerases
The proposal for two distinct binding sites for the
elongating chain in telomerase is based on the two-site model for RNA
binding during elongation by RNA polymerase ((Collins and Greider,
1993) reviewed in Chamberlin (1993)). The polymerase moves in saltatory
steps along the template and at each translocation the growing chain is
fed from the template site (site 1) to the anchor site (site 2). The
crystal structure has been solved for T7 RNA polymerase, Klenow
fragment, human immunodeficiency virus reverse transcriptase, and DNA
polymerase (Kohlstaedt et al., 1992; Pelletier et
al., 1994). Each of these polymerases has a separate site for
polymerization and binding of the elongating chain. The overall
arrangement of these sites is remarkably conserved (reviewed in Joyce
and Steitz (1994) and Pelletier et al. (1994)). The
identification of a primer specific cleavage activity further supports
this analogy between telomerase and RNA polymerase (Collins and
Greider, 1993). The band shift complex identified in this study is
analogous to the ternary complexes of RNA polymerase, RNA and DNA
template isolated on native gels (Hagler and Shuman, 1993). Using this
assay it may be possible to physically isolate intermediates in the
telomerase elongation reaction.
Identification of a Potential Telomerase Subunit by UV
Cross-linking
Several observations indicate that a protein of
approximately 100 kDa may be a subunit of the telomerase enzyme. First,
cross-linking of this protein in our assays depended on UV treatment,
telomerase activity, d(GGGGTT)primer, N
RdUTP,
and [
P]dGTP. Second, preincubation with the
nonspecific oligonucleotide pBR prior to telomeric primer addition
enhanced the 100-kDa cross-link, whereas a nonspecific protein might be
competed by excess oligonucleotide. Third, labeling of the 100-kDa
protein was sensitive to ribonuclease treatment of the telomerase
complex after generation of the
P-labeled
N
RdUTP-containing DNA, suggesting this protein is part of
an RNP complex. Fourth, the 100-kDa cross-linked protein was present in
sucrose gradient fractions and affinity purified fractions that
contained extensively purified telomerase activity. One limitation of
the cross-linking approach taken here is that telomerase activity
itself is required to generate the products that cross-link to the
100-kDa protein. The cross-link is present in fractions with telomerase
activity simply because only those fractions that contain telomerase
activity can synthesize the cross-linkable products. To avoid this
problem we attempted to first generate
P-labeled
N
RdUTP-containing DNA and then to bind it to telomerase for
cross-linking. However, gel shift experiments using d(TTGGGG) probes
substituted with N
RdUTP indicated that telomerase would not
bind these probes. Although the polymerase activity of telomerase will
incorporate the analog, the initial binding of the primer is inhibited.
The anchor site on telomerase may be unable to accommodate the large
bulky arylazide group present in the N
RdUTP.
Once the telomerase protein component(s)
are cloned we can use the band shift assay to define the contribution
of the protein to the primer binding specificity.
RdUTP,
5-[ N-( p-azidobenzoyl)-allyl]-deoxyuridine
monophosphate.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.