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Molecular and Cellular Biology, November 2001, p. 7775-7786, Vol. 21, No. 22
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.22.7775-7786.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
N-Terminal Domains of the Human Telomerase Catalytic Subunit
Required for Enzyme Activity in Vivo
Blaine N.
Armbruster,
Soma
S. R.
Banik,
Chuanhai
Guo,
Allyson C.
Smith, and
Christopher
M.
Counter*
Department of Pharmacology and Cancer
Biology, Department of Radiation Oncology, Duke University
Medical Center, Durham, North Carolina 27710
Received 1 May 2001/Returned for modification 4 June 2001/Accepted 7 August 2001
 |
ABSTRACT |
Most tumor cells depend upon activation of the ribonucleoprotein
enzyme telomerase for telomere maintenance and continual proliferation. The catalytic activity of this enzyme can be
reconstituted in vitro with the RNA (hTR) and catalytic (hTERT)
subunits. However, catalytic activity alone is insufficient for the
full in vivo function of the enzyme. In addition, the enzyme must
localize to the nucleus, recognize chromosome ends, and orchestrate
telomere elongation in a highly regulated fashion. To identify domains of hTERT involved in these biological functions, we introduced a panel
of 90 N-terminal hTERT substitution mutants into
telomerase-negative cells and assayed the resulting cells for
catalytic activity and, as a marker of in vivo function, for cellular
proliferation. We found four domains to be essential for in vitro and
in vivo enzyme activity, two of which were required for hTR binding.
These domains map to regions defined by sequence alignments and
mutational analysis in yeast, indicating that the N terminus has also
been functionally conserved throughout evolution. Additionally, we
discovered a novel domain, DAT, that "dissociates activities of
telomerase," where mutations left the enzyme catalytically
active, but was unable to function in vivo. Since mutations in this
domain had no measurable effect on hTERT homomultimerization, hTR
binding, or nuclear targeting, we propose that this domain is involved in other aspects of in vivo telomere elongation. The discovery of these
domains provides the first step in dissecting the biological functions
of human telomerase, with the ultimate goal of targeting this
enzyme for the treatment of human cancers.
 |
INTRODUCTION |
A fundamental difference between
normal somatic cells and malignant cells is the ability of the latter
to proliferate beyond the normally defined set of cell divisions,
through a process known as cellular immortalization. The ability of
cancer cells to become immortal is linked to the replication of
chromosome termini or telomeres. Telomeres are DNA-protein structures
that protect chromosome ends from degradation and
inappropriate recombination (8). The DNA portion of
this structure in most eukaryotes is comprised of tandem
repeats of a short G-rich sequence that extends past the
complementary C strand, forming a 3'G-rich overhang that can adopt
higher-ordered structures (8, 23). During DNA replication in normal human somatic cells, there is a loss of telomeric DNA, which
eventually elicits a growth arrest signal in cultured cells termed
senescence (26, 28, 55). If such a signal is disrupted, as
it is in transformed cells, further telomere shortening eventually denudes chromosome ends of its protective DNA, leading to a period of
crisis characterized by massive genomic instability and cell death (12, 55). Telomere loss may therefore serve as a
protective mechanism to prevent sustained proliferation of abnormal
cells that have a neoplastic predisposition.
Most cancer cells overcome the proliferative blockade of telomere
shortening through activation of the normally dormant
telomerase enzyme (3, 58). Human
telomerase is a reverse transcriptase containing a ~127-kDa
catalytic protein (hTERT) (27, 32, 41, 47) that
reverse transcribes the template region of the associated RNA
subunit (hTR) (18) onto the 3' end of telomeric DNA,
thereby elongating telomeres. Normally, somatic cells express only the hTR subunit (2, 18), but during tumorigenesis the
hTERT gene is illegitimately activated, restoring
telomerase activity, preventing further telomere shortening and
thereby immortalizing cells (14, 33, 35, 41, 47, 48).
hTERT is both required for the tumorigenic transformation of normal
cells (16, 24, 54) and the continual proliferation of
cancer cells (20, 25, 64). Since telomerase is
activated in as many as ~85% of tumors but is absent in most normal
tissues (3, 58), inhibition of hTERT could represent a
specific means of targeting a broad range of cancers. Understanding how
hTERT functions in human cells could be important for developing
antitelomerase therapies.
Enzyme catalysis can be reconstituted in vitro with hTERT and hTR,
suggesting that these subunits form the core of a more complex
holoenzyme (4-7, 40, 43, 60, 61); however, the exact
stochiometry of this core complex is uncertain. Biochemical purification of telomerase activity from the ciliate
Euplotes suggests that the enzyme is composed of a single
RNA, catalytic protein subunit, and associated protein
(38). However, accumulating evidence suggests that
telomerase may be a multimeric complex. For example, certain
template mutations of the RNA were found to be copied in yeast and
human cells only when a wild-type telomerase complex was
present (51, 52, 60), and telomerase activity was
immunoprecipitated with catalytically inactive hTERT fragments produced in telomerase-positive cells (7).
TERT proteins from a variety of organisms are defined by a large
central catalytic domain, encompassing approximately one third to one
half of the protein, which contains reverse transcriptase motifs
essential for catalysis (46). C-terminal to this domain is
a short highly divergent region, where the comparison of yeast and
human proteins reveals little to no obvious sequence conservation or
functional similarity (5, 7, 19; S. S. R. Banik
et al., unpublished data). On the other hand, the N terminus of yeast telomerase contains four domains termed I, II, III, and the
T-motif that are essential for yeast viability, with the latter two
domains being necessary for RNA binding (9, 19, 63). In
Tetrahymena spp., the N terminus is also essential for
telomerase activity and a 321-amino-acid region encompassing
the T-motif and domains II and III (as defined in yeast) can bind the
telomerase RNA (36). Similarly, in vitro, deletion
of the first 350 amino acids of human TERT abolishes telomerase
activity, and a large 287-amino-acid N-terminal fragment of hTERT
that maps to RNA-binding regions in the yeast and
Tetrahymena protein has been shown to bind hTR (5,
7). More recently, an alignment of ~500 amino acids of the N
terminus from an array of phylogenic TERT proteins identified five amino acids that are identical, clustering in three regions termed GQ, CP, and QFP, which overlap with yeast domains I, II, and
III, respectively (63). Thus, the N terminus may
contain evolutionarily conserved regions essential for RNA binding and telomerase activity.
Recent studies suggest that telomere elongation by hTERT involves
more than the association with hTR or catalytic activity. Addition of a
double hemagglutinin (HA) epitope tag to the C terminus of
hTERT (hTERT-HA) results in a catalytically active enzyme that cannot maintain telomere length or immortalize cells in vivo (13, 49, 66). More recently, three different alanine substitution mutations in the N terminus of yeast catalytic subunit of
telomerase, Est2p, have been found to dissociate catalytic from
biological activity (19, 63). The biological function
disrupted by these mutations is uncertain, since telomerase
activity appears to be regulated at multiple levels in vivo. For
example, the enzyme must localize to the nucleus to be functional, a
process recently shown to be regulated during T-cell activation
(39), possibly by phosphorylation or association of the
protein 14-3-3 (39, 56). Telomerase is also targeted
specifically to telomeres, and in yeast this process is mediated
through a number of proteins (17, 22, 31, 50, 53, 65).
Lastly, telomere elongation is known to be cell cycle regulated and
tightly coupled to the synthesis of the complementary C strand
(1, 15, 53). Biochemical analysis of in vitro
reconstituted enzyme activity would not be expected to identify domains
of TERT responsible for most of these other cellular functions. To
elucidate the domains of hTERT required for such functions in human
cells, we studied the consequence of mutations to hTERT in vivo. In
addition to identifying domains essential for catalytic activity,
we discovered a domain essential for another cellular function of
telomerase. This DAT domain is dispensable for catalytic
activity, but is required for in vivo telomerase function. This
represents the first domain of hTERT linked to the biological
regulation of telomerase.
 |
MATERIALS AND METHODS |
Plasmids.
By using pairs of complementary oligonucleotides
bearing the sequence AATGCTGCTATACGATCG (encoding for the sequence
NAAIRS for the sense oligonucleotides) in place of the sequence
encoding the six amino acids to be mutated (flanked on either side by
15 nucleotides complementary to native hTERT sequence), each NAAIRS substitution was introduced into either the
EcoRI-MluI or the MluI-NcoI
fragment of a N-terminal FLAG-tagged hTERT (FLAG-hTERT) by
QuikChange site-directed mutagenesis (Stratagene). Accordingly, 90 separate oligonucleotide pairs were used to systematically substitute
every six amino acids from the +2 position up to +547, with the
exception of position +260. Mutated regions were sequenced to confirm
correct substitution. To create retroviral constructs, all 90 of the
mutated fragments were removed and cloned back into EcoRI-MluI or MluI-NcoI
sites of full-length FLAG-hTERT cloned in the
EcoRI-SalI sites of the plasmid pBluescript
SK(
) (Stratagene), after which the mutated open reading frame was
excised and cloned into the EcoRI-SalI sites in
the retroviral vector pBabehygro (45). To create in vitro
expression constructs, selected mutants were similarly extracted and
cloned into the EcoRI-MluI or
MluI-NcoI sites of an N- and a C-terminal
FLAG-tagged hTERT cDNA (FLAG-hTERT-FLAG) that was inserted in
the plasmid pCIneo (Promega). GST-pCIneo was made by digesting pGEX-4T1
(Amersham Pharmacia Biotech) with SspI and SalI
and then cloning this fragment into pCIneo digested with
EcoRI (blunted with Mung bean nuclease) and SalI.
GST-hTERT-pCIneo (wild type; positions +50, +92, and +152) was
produced by introducing FLAG-hTERT into GST-pCIneo with
EcoRI and SalI and then removing the FLAG
sequence by PCR cloning with primers
5'-CGAATTCCAAACCGCCCCCTCCTTCCGCCAG and
5'-GTCCACGCGTCCTGCCCG. GST-hTERT-pCIneo (+386 and
+512) were made by inserting MluI and SalI
fragments, digested from corresponding FLAG-hTERT-pBabehygro
plasmids, into wild-type GST-hTERT-pCIneo cut with MluI
and SalI.
The hTR-expressing plasmid pBluescriptSK-hTR was created by inserting
the EcoRI-digested hTR PCR product, generated by amplifying plasmid phTRA (10) with primers
5'-CGGAATTCGGGTTGCGGAGGG and 5'-CGGAATTCGCATGTGTGAGCCGAGTCCTGG into the same site
downstream of the T7 promoter in pBluescript SK(
) (Stratagene).
Cell culture and apoptosis assays.
The simian
immunodeficiency virus (SV40) T/t-Ag transformed human embryonic kidney
cell line HA5 (59) was infected at population doubling
(pd) ~51 to 56 with the amphotropic retroviruses derived from
the above-described pBabehygro constructs encoding each of the 90 NAAIRS mutant FLAG-hTERT cDNAs or, as controls, wild-type FLAG-hTERT or no insert, after which stable polyclonal populations were selected in media supplemented with 100 µg of hygromycin B
(Sigma)/ml as previously described (13). A population
doubling of 0 was arbitrarily assigned to the first confluent
plate under selection. Cells were continually passaged at 1:4 or 1:8
under selection until either crisis or until the culture divided more than 2.5 times longer than vector control cell lines. Crisis was defined as the period when cultures failed to become confluent within
25 days and exhibited massive cell death.
For apoptosis studies, infected HA5 cell lines were split 1:4 or 1:8,
and 3 days later the adherent cells were trypsinized
and pooled with
nonadherent cells from the media. These cells
were washed twice in cold
1× phosphate-buffered saline (PBS) and
stained with annexin V
and propidium iodide according to manufacturer's
instructions using the Annexin V-FITC Apoptosis Detection Kit
II
(Pharmingen). Flow analysis was performed at the Duke Comprehensive
Cancer Center Flow Cytometry Shared Resource facility by using
a
FACSCaliber (Becton
Dickinson).
hTERT mRNA detection, telomerase activity, and
telomere length assays.
For quantitative reverse transcription-PCR
(RT-PCR), total RNA from each of the described infected HA5 cells was
isolated with the RNAzol reagent according the manufacturer's
instructions (Teltest), and 250 ng of RNA was RT-PCR amplified to
detect either total hTERT or PBGD mRNA by using the LightCycler
TeloTAGGG hTERT Quantification Kit and LightCycler (Roche).
hTERT signals were normalized to PBGD mRNA levels, and the
number of hTERT transcript was determined by using a standard curve
generated from RT-PCR of known concentrations of in vitro-transcribed
hTERT mRNA, in accord with the manufacturer's instructions
(Roche). Conversion to transcript per cell was determined based on the
number of cell equivalents of RNA assayed.
To specifically detect endogenous or ectopic hTERT mRNA or the
GAPDH (glyceraldehyde-3-phosphate dehydrogenase) mRNA, the
RNA
described above was amplified by using semiquantitative RT-PCR
as
previously described (
24) with primers specific for the
following:
endogenous hTERT, 5'-ACTCGACACCGTGTCACCTA and
5'-GTGACAGGGCTGCTGGTGTC;
ectopic hTERT,
5'-GACACACATTCCACAGGTCG and
5'-GACTCGACACCGTGTCACCTAC;
or GAPDH,
5'-GAGAGACCCTCACTGCTG and
5'-GATGGTACATGACAAGGTGC. Reaction
products were resolved on
10% polyacrylamide gels, dried, and
exposed to a phosphorimager
screen.
To detect telomerase activity, lysates were isolated from
infected HA5 cells at two different passages, protein concentration
was
measured by Bradford assay (Bio-Rad), lysates were diluted
in the lysis
buffer to a concentration of 0.1 µg/µl, and 0.2 µg
was assayed
for telomerase activity by using the telomeric repeat
amplification protocol as previously described (
34). As a
negative
control, duplicate reactions were heat treated at 85°C for 2 min
to inactivate telomerase. Reaction products were resolved
on 10%
polyacrylamide gels, dried, and exposed to a phosphorimager
screen
to quantitate enzyme activity as previously described
(
34).
Telomeres were visualized by Southern hybridizing 10 µg of
HinfI and
RsaI restriction
enzyme-digested genomic DNA with the
32P-labeled telomeric
(C
3TA
2)
3
oligonucleotide exactly as previously
described (
12), with
the exception that washes were performed
with 10× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium
citrate).
Western blot and indirect immunofluorescence.
To analyze
mutant hTERT protein expression, 293T cells were transiently
transfected with pCIneo or pCIneo-FLAG-hTERT-FLAG constructs by
calcium phosphate transfection method (21). Cells were
collected at ~48 h posttransfection and lysed in 1× PBS, 5 mM EDTA,
0.2% NP-40, 10% glycerol, 1 mM benzamidine, 1 µg of pepstatin A/ml,
1 µg of leupeptin/ml, 1.5 µg of aprotinin/ml, 0.1 mM
phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and 1 mM
Na3VO4. The protein
concentration was measured by Lowry assay (Bio-Rad), and 30 µg of
soluble lysate was separated by sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE), transferred to polyvinylidene
difluoride membrane (Millipore), and blocked with TBST (1× TBS [50 mM
Tris-HCl, pH 7.4; 150 mM NaCl]-0.02% Tween 20)-5% milk. Blots were
incubated with either anti-FLAG M2 mouse monoclonal antibody (Sigma) or
anti-actin (C-2) mouse monoclonal antibody (Santa Cruz Biotechnology
Inc.) and the goat anti-mouse immunoglobulin G-horseradish peroxidase
(Santa Cruz Biotechnology, Inc.) diluted in TBST-5% milk. Blots were washed three times for 6 min each time in 1× TBS or TBST, and protein
was detected with ECL Reagent according to the manufacturer's protocol
(Amersham Pharmacia Biotech).
Localization of hTERT proteins was visualized in the human
osteosarcoma cell line, U2OS, by indirect immunofluorescence. A
total
of 2 µg of pCIneo, pCIneo-FLAG-hTERT-FLAG wild-type, or
NAAIRS
mutant +92 and +122 constructs were transiently transfected
into U2OS
cells by calcium phosphate and examined ~36 h posttransfection.
Cells
were fixed with 3% paraformaldehyde-2% sucrose, permeablized
with
1× PBS-0.2% Triton X-100, and blocked with PBTN (1× PBS,
0.1%
Triton X-100, 5% goat serum). Ectopic hTERT was detected
by
anti-FLAG M2 mouse monoclonal antibody recognized by a goat
anti-mouse
antibody conjugated with fluorescein isothiocyanate
(Jackson
ImmunoResearch) diluted in PBTN. Nuclei were stained
with 2 µg of
Hoechst 33258 (Sigma)/ml. Cells were examined at
×400 magnification on
a Nikon Eclipse TE300 light
microscope.
hTR-hTERT and hTERT-hTERT
coimmunoprecipitations.
hTR was expressed and
32P labeled with the T7-coupled Maxiscript Kit
(Ambion) by using 1 µg of linearized pBluescriptSK-hTR. Unincorporated nucleotides were removed by using a G-25 Minispin Column
(Amersham Pharmacia Biotech). 35S-labeled
proteins were produced by using the T7 quick coupled TNT System
(Promega) from plasmids pCIneo-FLAG-hTERT-FLAG;
pCIneo-FLAG-hTERT-FLAG-NAAIRS +50, +152, +386, or
+512; and pCMV-HDAC1-FLAG in the presence of 3 µl of hTR RNA.
For coimmunoprecipitations, 4.4 µg of the M2 anti-FLAG monoclonal
antibody was prebound to 25 µl of GammaBind G-Sepharose
(Amersham
Pharmacia Biotech) in S-100 buffer (9 mM Tris, pH 7.5;
0.9 mM
MgCl
2; 0.9 mM EGTA, pH 8; 1.5 mM dithiothreitol;
0.5% CHAPS
{3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate};
10% glycerol; 1 mM benzamidine; 0.1 mM phenylmethylsulfonyl fluoride)
in the presence of blocking agents (100 ng of bovine serum albumin
/ml,
100 ng of casein/ml, 100 ng of tRNA/ml, 250 ng of yeast total
RNA/ml,
100 ng of glycogen/ml) as previously described with minor
modifications
(
44). Coated beads were added to completed TNT
reactions,
diluted with S-100 buffer in a final volume of 750
µl, and incubated
for 1 h at room temperature in the presence
of 200 U of RNasin
(Promega) and nonspecific blocking agents described
above. The beads
were washed three times with prechilled S-100
buffer, heated in SDS
buffer, and resolved by SDS-PAGE.
For the immunoprecipitation of hTERT-hTERT complexes, wild-type
and NAAIRS-substituted (+50, +152, +386, or +512) FLAG-hTERT-FLAG
and N-terminal glutathione
S-transferase (GST)-tagged
hTERT were
separately transcribed and translated as described above
in the
presence of 0.5 µl of [
35S]methionine
and 1 µl of cold methionine, or 4 µl of
[
35S]methionine, respectively, supplemented
with 20 pmol of Ts oligonucleotide
(
34) and 1 µl of
trace-labeled hTR RNA expressed in vitro with
the Maxiscript Kit
(Ambion) by using 0.17 µl of [
32P]UTP and 6 µM cold UTP. Reactions were incubated 30°C for 40
min, mixed with
the appropriate reaction, and then incubated for
an additional 60 min
at 30°C. Reactions were immunoprecipitated
with the anti-Flag M2
monoclonal antibody as described above.
The reciprocal complex made
with
35S-labeled GST-hTERT and trace labeled
FLAG-hTERT-FLAG was also
immunoprecipitated with 1 µg of the Z-5
anti-GST antibody (Santa
Cruz Biotechnology, Inc.). As controls,
HDAC1-FLAG and GST were
immunoprecipitated in the presence of
GST-hTERT.
 |
RESULTS |
Identification of functional domains in the N terminus of hTERT
by mutational analysis.
To define domains essential for
telomerase function, we generated a panel of 90 individual
tandem NAAIRS substitution mutations within the N terminus of
hTERT, beginning immediately after the initiating methionine and
terminating at the conserved T-motif (46). NAAIRS
substitution mutagenesis presumably has only minor effects on protein
structure, since substitutions do not alter protein length and the
NAAIRS sequence has the unique ability to adopt multiple structural
conformations (62). Moreover, this mutagenesis approach
has been successfully employed to map the pocket region of pRB
(57), as well as locate C-terminal domains within
hTERT (Banik et al., unpublished). The panel of NAAIRS substitution
mutants was introduced into telomerase-negative HA5 cells by
retroviral infection. HA5 cells are human embryonic kidney cells
transformed with the SV40 T-Ag gene, which lack hTERT expression and lose telomeric DNA every cell division until they reach crisis and
die (12, 59). The proliferative potential of these cells can therefore serve as a reliable indicator of the biological consequence of hTERT mutations, since stable expression of
biologically active versions of hTERT restores telomerase
activity, stabilizes telomere length, and immortalizes HA5 cells (see
reference 13 and also below). The resulting HA5 infected
cell lines were assayed for telomerase function in vitro
by assessing telomerase enzyme activity and telomerase
function in vivo by determining if the infected cells bypass crisis
induced by telomere shortening.
To verify expression of hTERT mutants, we used quantitative RT-PCR
to detect hTERT mRNA. This method was chosen because
overexpression
of hTERT by the retroviral promoter in HA5 cells
produces undetectable
levels of protein, as assessed by Western
blotting with an anti-FLAG
antibody (not shown). RNA was isolated from
all 90 stably infected
cell lines and RT-PCR amplified with primers
specific for hTERT
transcripts (Fig.
1A). Vector control-infected HA5 cells
were
found to have extremely low levels of hTERT mRNA,
corresponding
to ~1 transcript per 100 cells, which is >150-fold
lower than
that observed in tumor cell lines by quantitative SAGE
analysis
(
37). Consistent with low hTERT expression,
HA5 cells do not
have readily detectable levels of telomerase
activity, lose telomeric
DNA, and fail to immortalize
(
12). Cell lines stably infected
with FLAG-hTERT
mutant constructs expressed hTERT mRNA at variable
levels.
However, in every case the expression was several orders
of magnitude
higher than vector cell lines when normalized with
the housekeeping
gene PBGD and was comparable to that detected
in wild-type
FLAG-hTERT-infected cells (Fig.
1A and Table
1).

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FIG. 1.
Expression and telomerase activity of N-terminal
hTERT mutants. (A) Total RNA was isolated from HA5 cell lines
stably infected with vector ( ), FLAG-hTERT ( , or
FLAG-hTERT NAAIRS substitution mutants representative of
nonessential (+212: ), essential (+158, ), slow-growth (+110,
), and biologically essential (+128, ) and RT-PCR amplified with
primers specific for hTERT by quantitative, real-time RT-PCR. The
amount of transcript detected by fluorescence with FRET probes is
plotted in arbitrary units against each PCR cycle (top panel). The
housekeeping PBGD transcript was similarly measured to verify
equivalent RNA addition per reaction (bottom panel), while
H2O ( ) was assayed in both reactions as a
negative control. (B) A total of 0.2 µg of lysate prepared from the
described HA5 cell lines was assayed for telomerase activity by
TRAP assay. As a control, a portion of the lysate was heat treated (HT)
to inactivate telomerase prior to assaying. The internal
standard (IS) served as a positive control for PCR amplification.
Catalytic activity for each sample was normalized with the internal
standard and is expressed as a percentage of wild-type FLAG-hTERT
activity, indicated as follows: ++ (>60%), + (60 to 15%), +/
(<15%), and (extremely low or no detectable activity). Domain
refers to the location of the mutant, as described in the text. Life
span (M, mortal; I, immortal; S, slow growth) as defined in the text.
(C) Biologic activity of hTERT mutants was measured by serially
passaging HA5 cell lines to determine whther cells
entered crisis like vector or immortalized like wild-type hTERT.
Representative clones are shown: vector ( ), FLAG-hTERT ( ),
+212 ( ), +50 ( ), +14 ( ), and +128 ( ). (D) Telomere length
of representative HA5 cells infected with NAAIRS mutants that result in
an immortal, slow-growth, or finite life span was determined by
releasing the terminal restriction fragments of genomic DNA
isolated from the described cell lines at early passage (pd 2 to 3)
with the restriction enzymes HinfI and
RsaI. These fragments were resolved and detected by
Southern hybridization with a telomeric probe. , Sample +212
was underloaded. Domain refers to the location of the mutant, as
described in the text.
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TABLE 1.
hTERT expression and catalytic and biologic
telomerase activity for the panel of N-terminal hTERT
mutants
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|
Having confirmed that hTERT mutants were equivalently
overexpressed, we next characterized each mutant cell line for in vitro
telomerase activity and extended life span as a measure of in
vivo telomerase function. As described in detail below, mutant
hTERT proteins gave rise to four distinct phenotypes: nonessential
(catalytically and biologically active), essential (catalytically
and
biologically inactive), slow growth (catalytically active,
biologically
impaired), and biologically essential (catalytically
active,
biologically dead). Compilation of the different phenotypes
with the
respective mutation position revealed clustering along
the primary
amino acid sequence (Table
1; see also Fig.
3), implying
distinct
domains within the N terminus. Specifically, we defined
four domains
(I-A, I-B, II, and III) that are essential for catalytic
activity, two
nonessential or linker regions (L1 and L2), and
one biologically
essential domain (Fig.
3). As discussed below,
based on the ability of
mutations within the biologically essential
domain to separate in vivo
and in vitro telomerase function, we
have named this last
domain the "dissociates activities of telomerase"
(DAT)
domain.
Linker regions are dispensable for telomerase
activity.
A total of 39 separate hTERT mutants were found to
be phenotypically similar to wild-type hTERT, when expressed in HA5
cells. Lysates from the HA5 cells expressing these mutants contained high to moderate levels of catalytic activity, as measured by the
ability of these extracts to elongate a single-stranded oligonucleotide with telomeric repeats (Fig. 1B and Table 1). It is formally possible
that this activity was due to spurious activation of the endogenous
hTERT gene, which would not be distinguished with the hTERT
primers used to confirm FLAG-hTERT expression (Fig. 1A). To rule
out this possibility, mRNA was isolated from representative cell
lines containing NAAIRS substitutions in each of the nonessential regions at early passage, as well as at very late passage (a point after crisis of vector control cells), when the endogenous gene would
be expected to be activated. This RNA was then RT-PCR amplified with
primers specific for either endogenous or ectopic hTERT mRNA. Endogenous hTERT was found at neither early nor late (postcrisis) passage, despite clear expression of the ectopic TERT and a control housekeeping gene (Fig. 2), indicating
that the observed telomerase activity in these mutants was a
direct result of ectopic expression of the hTERT mutants.

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FIG. 2.
Absence of endogenous hTERT expression in
telomerase-positive HA5 cell lines. Total RNA collected from
HA5 cells at either early passage (pd 2 to 3) (A) or at late passage
(pd >39) (B) was analyzed by RT-PCR with primers specific for either
endogenous or ectopic hTERT or GAPDH (control for RNA content).
Results with nonessential (+212, +326, +422, and +524), slow-growth
(+14, +110, and +398), and biologically essential (+128) mutants are
shown. CWR and LNCaP are prostate cancer cell lines expressing
endogenous hTERT and serve as positive controls for endogenous and
a negative control for ectopic hTERT expression. A water sample is
used to control for contaminating DNA in the reaction mix.
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Consistent with the high levels of activity, HA5 cell lines stably
expressing each of these 39 mutants were, like wild-type
hTERT-infected cells, able to bypass crisis and continued to
proliferate
in culture (Fig.
1C and Table
1). Additionally, cell lines
expressing
representative mutants had larger telomeres (~6 kbp)
compared
to vector control HA5 cells (~4.5 kbp) at early passage
(Fig.
1D). The two regions mapped by these mutants may serve as
linkers,
since these regions are, by all known biological criteria,
nonessential
for telomerase function and have little predicted
secondary structure
(Fig.
3).

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FIG. 3.
Domain structure of the N terminus of hTERT.
Secondary structure of the N terminus of hTERT as predicted by the
Jprep2 program (http://jura.ebi.ac.uk:8888/) is shown, cylinders
represent -helices or -sheets. Essential domains I-A, I-B, II,
and III, as well as the T-motif, are denoted above structure
prediction. Shaded regions denote the DAT domain; L1 and L2 define the
nonessential linker regions. Two structured regions, outside of defined
domains, are indicated by dashed lines. Essential domains I, II, and
III found in the N terminus of Est2p (19) or conserved
regions GQ, CP, and QFP identified in TERT proteins by alignment
(63) are shown below structure prediction.
|
|
N-terminal essential domains.
Cell lysates from 34 independent
hTERT mutants had extremely low or no detectable catalytic activity
(Fig. 1B and Table 1). Like vector-infected cells, HA5 cells stably
expressing these hTERT mutants lost telomeric DNA (Fig. 1D) and
succumbed to crisis after undergoing a limited number of cell divisions
(Fig. 1C and Table 1). Thus, loss of enzyme activity rendered these
mutants biologically inactive, and hence these mutants define regions of hTERT that are essential. Mapping these essential mutants to hTERT sequence revealed a clustering in four regions (Fig. 3), which align with essential domains I, II, and III defined by mutational analysis in yeast (19), or homology blocks GQ, CP, and QFP
(63). We note that, in humans, domain I is actually
separated into two halves, which we term I-A and I-B, by a novel domain
dispensable for telomerase in vitro enzyme activity (see below).
Since hTERT and hTR reconstitute a fully active enzyme in vitro, we
reasoned that the absence of activity in essential domain
mutants could
be due to protein instability or loss in hTR interaction.
To address
the first possibility, we transiently overexpressed
NAAIRS mutants from
the four different essential domains in 293T
cells to determine whether
these mutants were produced at levels
comparable to the wild type.
Western blots indicated that there
were no substantial differences in
protein levels between the
wild type and essential hTERT mutants
(Fig.
4A) or noticeable
degradation
products (data not shown). Based on these findings,
we propose that
poor protein expression is not a major factor
for reduction in
catalytic activity of essential domain hTERT
mutants. However,
since hTERT protein is ectopically expressed
at far higher levels
transiently in 293T cells compared to stably
in HA5 cells, we cannot
rule out the possibility that a slight
reduction in protein levels, not
detected in 293T cells, could
have an impact on telomerase
activity when expressed in HA5 cells.
The absence of telomerase
activity in the described cell lines
could also be argued to be due to
low hTR levels. We discount
this possibility since both
telomerase-positive and -negative
cell lines were derived from
the same cells and because we assayed
for telomerase activity
in polyclonal populations, which are unlikely
to have uniformly lower
hTR expression in telomerase-negative
cells compared to the
similarly derived telomerase-positive cells.

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FIG. 4.
Protein stability and hTR binding of mutants
within essential domains of hTERT. (A) Lysates from 293T cells
transiently transfected with FLAG-hTERT-FLAG, wild type, or the
indicated NAAIRS mutants were resolved by SDS-PAGE and examined
by anti-FLAG Western blotting. An anti-actin Western blot was used
to ensure equal protein loading. (B) Binding of hTR with
hTERT was examined in vitro by coimmunoprecipitating
35S-labeled FLAG-hTERT-FLAG (F-hTERT-F)
with purified 32P-labeled hTR by using anti-FLAG
antibodies. Immunoprecipitates were separated by SDS-PAGE and exposed
to autoradiograph. Input hTR was diluted 1/1,000 and hTERT 1/10 for
visualization. (C) Binding of hTR with FLAG-hTERT-FLAG protein
containing NAAIRS substitutions (+50, +152, +386, and +512) in
essential domains I-A, I-B, II, and III, respectively, was
similarly examined. As a control for nonspecific interactions,
HDAC1-FLAG (HDAC1-F) was immunoprecipitated in the presence of labeled
hTR. The positions of F-hTERT-F, HDAC1-F, and hTR are indicated
left of gel.
|
|
Since a large deletion of the first 350 amino acids of hTERT
abolishes both hTR binding and telomerase activity in vitro
(
7),
we next addressed whether N-terminal essential
domains are involved
in hTR-binding.
32P-radiolabeled hTR was incubated with
35S-labeled double FLAG epitope-tagged
hTERT generated in vitro
and immunoprecipitated with an anti-FLAG
antibody to assess the
hTR association (Fig.
4B). In this in vitro
system, hTERT specifically
interacted with hTR. The FLAG-tagged
hTERT protein, but not the
irrelevant FLAG-tagged protein
HDAC1, coimmunoprecipitated hTR,
despite the fact that HDAC1 was
readily immunoprecipitated with
the same antibody. Similarly, hTERT
containing representative
NAAIRS substitution in essential
domains I-A and I-B (+50 and
+152, respectively) also interacted with
hTR, although these mutants
are telomerase negative. However,
immunoprecipitates of hTERT
containing representative NAAIRS
mutants within essential domains
II and III (+386 and +512,
respectively), which are essential
for telomerase activity,
showed a clearly visible two- to fourfold
reduction in hTR binding.
Since these mutants were expressed at
levels equivalent to that of
wild-type hTERT, we propose that
domains II and III are critical
for stable interaction between
hTERT and hTR and that disruption of
this interaction resulted
in loss of enzyme
activity.
hTERT mutants that only partially restore telomerase
function.
HA5 cells expressing nine different hTERT mutants
were found to have impaired growth dynamics but nevertheless a greatly
extended life span (Fig. 1C and Table 1). All but two (mutants +350 and +362) of these slow-growth mutant cell lines contained comparable levels of in vitro catalytic activity to cells infected with wild type
or with nonessential mutants that had a similarly extended, if not
immortal, life span (Fig. 1B and Table 1). Representative HA5 cell
lines expressing these mutants did not have detectable levels of
endogenous hTERT at early or late passage, indicating that enzyme
activity was not due to activation of the endogenous hTERT gene
(Fig. 2). Each of these mutant proteins could also be transiently
expressed in 293T cells at levels equivalent to wild-type hTERT
with no apparent proteolysis (Fig. 5A and
data not shown). Although we cannot rule out that the proteins would behave identically when expressed in HA5 cells, the fact that most of
these mutant are also highly telomerase positive argues against
a loss of protein expression underlying the phenotypes of these
mutants. Lastly, this slow-growth phenotype was reproducible. We
infected HA5 cells again with three randomly chosen hTERT mutants that gave rise to the slow-growth phenotype (+32, +86, and +116); all
had slow-growth, of which two continued to proliferate beyond crisis of vector control cells (not shown). Taken together, these data
indicate that the slow-growth phenotype is directly related to
the mutations in hTERT.

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FIG. 5.
Expression and cell viability of slow-growth hTERT
mutants. (A) Anti-FLAG Western blot of slow-growth (+14 and +110) and
nonessential (+212 and +422) hTERT mutants transiently expressed in
293T cells. Equal loading is shown by the anti-actin blot. (B)
Viability of slow-growth and immortal HA5 cells was determined by flow
cytometry of annexin V and propidium iodide double-stained cells. The
percentages shown are averages for three independent experiments.
|
|
Slow-growth mutants lost substantial amounts of telomeric DNA at early
passage; closely resembling telomere sizes found in
telomerase-negative cells rather than in wild-type- or
nonessential
hTERT mutant-infected cell lines (Fig.
1D).
The presence of shortened
telomeres at early passage suggests the
possibility that these
cell populations teeter on the edge of
extinction, with only a
small fraction of cells having functional
telomeres at any one
time. One prediction of such a model is that the
slow growth would
result from increased cell death in the population.
To test this
prediction, we double stained late-passage HA5 cell
cultures stably
expressing three independent slow-growth mutants or, as
controls,
wild-type or nonessential mutants of hTERT, with annexin
V, a
marker of early apoptosis, and propidium iodide, an indicator
of
late-stage apoptosis (Fig.
5B). Two slow-growth mutants (+14
and +110)
showed a >3-fold increase in the amount of double-stained,
late-stage
apoptotic cells, while the remaining mutant cell line
(+398) exhibited
higher levels of annexin V staining compared
to normal immortalized HA5
cells. In every case, HA5 cells infected
with slow-growth mutants had a
lower amount of unstained, nonapoptotic
cells than those infected with
wild type or with nonessential
hTERT mutants. Therefore, the
apparent slow growth found in HA5
cells infected with the described
hTERT mutants was a result of
reduced viability of cells within the
population.
The novel DAT domain is essential for in vivo telomerase
function.
A region of hTERT comprised of a series of eight
NAAIRS substitution mutants were discovered to be dispensable for in
vitro enzyme catalysis but essential for biological activity. Lysates from HA5 cells containing these mutants had high to moderate levels of
in vitro telomerase activity (Fig. 1B and Table 1) and yet lost
significant amounts of telomeric DNA (Fig. 1D) and were mortal (Fig. 1C
and Table 1). Molecular characterization revealed that all of these
mutants were overexpressed in HA5 cells (Fig. 1A) and representative
mutants generated stable protein, at least when transiently expressed
in 293T cells (Fig. 6A). Although it is
formally possible that the stability of the same mutants may be much
lower in HA5 cells, the fact that DAT domain mutations bestow high
levels of telomerase activity in HA5 cells argues against this
possibility. The biologically essential phenotype was also
reproducible, since HA5 cells reinfected with three independent DAT
domain mutants (+68, +92, and +128) demonstrated the same phenotype
(not shown). Thus, mutations in the DAT domain disrupt functions
distinct from those we have so far characterized by NAAIRS substitution
analysis.

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FIG. 6.
Protein stability and nuclear localization of hTERT
with mutations in the DAT domain. (A) Anti-FLAG Western blot of
lysates from 293T cells transiently transfected with biologically
essential hTERT mutants +92, +122, wild-type hTERT, or control
vector. The anti-actin blot shows equal protein loading. (B)
Subcellular localization of DAT domain mutants transiently expressed in
U2OS cells by indirect immunofluorescence. Localization of
FLAG-hTERT-FLAG was visualized with an anti-FLAG antibody
recognized by a fluorescein isothiocyanate-conjugated secondary
antibody (green). Hoechst was used to stain nuclei (blue).
|
|
In vitro telomerase activity can be detected in lysates derived
from human and yeast cells regardless of cell cycle progression
(
15,
30). However, telomeres are elongated in a cell
cycle-dependent
fashion in
Saccharomyces cerevisiae
(
15), implying a regulation
of the biological activity of
telomerase. Indeed, binding of 14-3-3
proteins to hTERT has
recently been reported to influence the
subcellular localization of
hTERT (
56), raising the possibility
that
cytosolic-nuclear shuttling may be a regulatory mechanism
for
telomerase function in vivo. Loss of nuclear localization
could
leave a protein catalytically active but unable to reach
its biological
substrate. To therefore test whether the DAT domain
is required for
nuclear localization, an empty vector or one encoding
double
FLAG-tagged hTERT or representative DAT domain mutants
NAAIRS
+92 and +122 was transiently transfected into human U2OS
cells and the
resulting protein detected by indirect immunofluorescence
with an
anti-FLAG antibody. U2OS cells were chosen because they
have a clearly
defined nucleus and cytoplasm, which is ideal for
monitoring nuclear
localization. Wild-type hTERT was found predominantly
in the
nucleus of U2OS cells (Fig.
6B), although we did observe
rare cells in
which the signal was dispersed throughout the cell
or localized to the
cytosol (data not shown). Both DAT domain
mutants displayed the same
localization as the wild-type protein,
being found predominantly in the
nucleus, with some cells exhibiting
cytosolic signals (Fig.
6B). We
thus conclude that the biological
dysfunction of the DAT domain mutants
cannot be attributed to
a failure in nuclear
localization.
Finally, based on mounting evidence that hTERT may form homomeric
complexes (
7,
60), we investigated whether the defects
in
the DAT or essential domain I-A, I-B, II, and III mutants could
be
explained by the inability of these mutants to form higher-order
complexes. We found that hTERT can indeed form homomeric
complexes
in vitro when expressed in rabbit reticulocyte
lysates. This was
determined by the ability to coimmunoprecipitate
GST-hTERT and
FLAG-hTERT-FLAG by using either an
anti-FLAG antibody or an anti-GST
antibody. This reaction could occur
in the absence or presence
of DNA substrate or the hTR subunit,
indicating that multimerization
is independent of these two
parameters (Fig.
7A). The specificity
of
this interaction was demonstrated by the lack of an association
of
GST-hTERT with immunoprecipitated HDAC1-FLAG protein, as well
as
the inability of the FLAG-hTERT-FLAG protein to bind to
immunoprecipitated
GST (Fig.
7). We next tested whether mutations to
any of the essential
or DAT domains affected this interaction. GST- and
FLAG-tagged
hTERT containing representative mutants in the
described domains
were incubated and immunoprecipitated with an
anti-FLAG antibody.
In each case, both the GST- and FLAG-tagged protein
were coimmunoprecipitated,
arguing that the mutations did not affect
multimerization, when
expressed in rabbit reticulocyte lysates (Fig.
7B). Based on these
in vitro experiments, the catalytic and biological
defects of
these mutants do not appear to be related to an impaired
ability
to form multimers, although we cannot exclude the possibility
that the mutants may not multimerize in vivo.

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FIG. 7.
Homomeric complex formation of essential and DAT
domain hTERT mutants. (A) Immunoprecipitation of
35S-labeled FLAG-hTERT-FLAG (F-hTERT-F)
with either 35S-labeled GST-hTERT or GST in the
presence or absence of hTR and Ts oligonucleotide substrate with
anti-GST or anti-FLAG antibodies as indicated. (B)
35S-labeled GST-hTERT and F-hTERT-F, wild-type,
or NAAIRS substitution in domains I-A, DAT, I-B, II, and III (+50, +92,
+152, +386, and +512 mutants, respectively) were incubated together and
immunoprecipitated with an anti-FLAG antibody to monitor protein
association. As a control, an irrelevant FLAG-tagged protein (HDAC1-F)
failed to coimmunoprecipitate GST-hTERT.
|
|
 |
DISCUSSION |
Functional conservation of N-terminal domains in evolutionarily
diverse TERT proteins.
We stably expressed a panel of tandem
NAAIRS substitution mutants in telomerase-negative cells
and screened for telomerase activity to identify functional
domains in the N terminus of hTERT. NAAIRS substitution allows a
large region of hTERT to be mutated with a reasonable number of
changes, without altering protein length or causing large changes to
secondary structure. The use of a cell-based screen allows mutants to
be produced in a cellular environment containing factors that may be
absent in vitro and allows each mutant to be simultaneously
characterized for both in vitro and in vivo activity. The screen
revealed four phenotypes for N-terminal mutants: essential
(catalytically and biologically inactive), nonessential (catalytically
and biologically active), biologically essential (catalytically active,
but biologically dead), or slow growth (catalytically active, but
biologically impaired). Mutants that give rise to the essential
phenotype reside in one of four domains (I-A, I-B, II, and III) which
were usually preceded by linker regions in which NAAIRS substitutions
had minimal effects on the in vitro and in vivo functions of hTERT.
We find that all four domains correspond to the yeast essential domains I, II, and III (19), as well as the similar domains GQ,
CP, and QFP (63), defined by alignments of TERT proteins
from evolutionarily diverse organisms (Fig. 3).
The domains that we defined as being essential in humans also appear to
be conserved at the functional level with those of
lower eukaryotes.
Domain I in yeast (
63) and humans (Fig.
3)
is essential
for long-term viability. Specific mutations in this
region in these
organisms, or in the ciliate
Tetrahymena (
36,
42), have little effect on RNA binding but result in a partial
or complete loss of telomerase activity. In humans, the region
defined as domain I is divided by the DAT domain and hence was
termed
domains I-A and I-B. Intriguingly, N-terminal hTERT mutants
lacking
the first 200 amino acids (including essential domains
I-A and I-B and
the DAT domain) are nonprocessive in vitro (
7)
, whereas specific NAAIRS substitutions in the same region abrogate
catalytic activity (Fig.
1B and Table
1). Perhaps the large deletion
removes a portion of hTERT, which functions in an inhibitory manner
when mutated by NAAIRS substitution. Nevertheless, both types
of
analysis define domains I-A and I-B as critical for proper
enzyme
function, as observed in lower
eukaryotes.
Mutations in domains II and III were found to abolish
telomerase activity and hTR binding and failed to rescue
transformed
human cells from crisis. In yeast, these domains are
required
for enzyme activity, telomere elongation, proliferation and,
in
the case of two mutants in domain III, RNA binding
(
19). Similarly,
large fragments of TERT minimally
encompassing these domains and
the conserved T-motif can bind the RNA
subunit in humans and
Tetrahymena (
7,
36). Like
domain I, domains II and III appear to be functionally
conserved.
We also found that, in addition to the sequence and functional
conservation of the N-terminal domains of TERT, the most structured
regions of this portion of hTERT mapped to the biologically defined
domains of the protein, delineating these regions as important
structural domains (Fig.
3). Therefore, with the exception of
motifs
unique to ciliates (
9,
11,
42), the organization
and
function of the N-terminal domains appears to have remained
intact
throughout evolution. Thus, despite low sequence homology,
the N
terminus of TERT proteins do contain evolutionarily conserved
functional
domains.
The DAT domain in cellular regulation of telomerase.
We identified a region termed the DAT domain defined by 11 contiguous
mutants in which in vitro telomerase catalytic activity was
dissociated from the ability of telomerase to function
efficiently in vivo. Cells expressing DAT domain mutants either entered
a period analogous to crisis observed in vector control cells or displayed less dramatic cell death, possibly representing a partial crisis. The latter phenotype, which we termed slow growth, was also
found in cells expressing hTERT containing NAAIRS mutations in
domain I-A (three-quarters of the slow-growth mutants mapped to either
the I-A or the DAT domain). In one case, we found that a mutation in
the DAT domain (mutant +86) could give rise to either a slow-growth or
a mortal phenotype. Domains I-A, DAT, and I-B also form one continuous
structured region that appears to have no obvious role in hTR binding
or multimerization. Taken together, we speculate that the function of
domains I-A and I-B may therefore be related to that of the DAT domain
but that mutations to these two domains are more intrusive to
biochemical activity.
Mutations in the DAT domain caused cell death, which was accompanied by
a large decrease in telomere length, clearly demonstrating
the loss of
a novel in vivo telomerase function that is dispensable
for
biochemical enzyme catalysis in vitro. One aspect of
telomere
elongation that would not be represented in an in vitro assay
for catalytic activity is posttranscriptional regulation of
telomerase
(
15,
29,
39,
56). Recently, mutants
have been isolated
that affect hTERT entry into the nucleus,
suggesting that cellular
localization may be involved in coordinating
hTERT-mediated telomere
elongation (
56). Although the
DAT domain does not appear to
contain any obvious nuclear localization
sequence (NLS), nuclear
localization could be mediated through a
noncanonical NLS or a
binding partner. However, we ruled out this
possibility, since
there were no noticeable defects in cellular
localization of hTERT
containing mutations in the DAT
domain.
Although in vitro assayed telomerase activity purified from
Euplotes consists of a single catalytic subunit
(
38), experiments
from both yeast and human systems
support the notion that the
enzyme may function biologically in a
complex containing more
than one TERT and RNA subunit (
7,
51,
52,
60). Disruption
of this interaction could underlie the
defect we observed in the
DAT domain mutants. However, we find
biochemically hTERT forms
a homomeric complex in vitro,
irrespective of mutations to the
DAT domain. Thus, the biologically
essential phenotype of the
DAT domain cannot be ascribed to a failure
of hTERT to
multimerize.
Since mutations in the DAT domain neither grossly altered hTERT
localization patterns nor homomultimerization, we speculate
that
this domain could instead be involved in recruitment of
telomerase
to telomeres. We note that mutations mapping to the
corresponding
DAT domain region in yeast Est2p had a similar
biologically essential
phenotype and that the proteins Est1p, Est3p,
Cdc13p, and Ku are
necessary for biological telomerase function
and have been implicated
in recruiting telomerase to telomeres
in yeast (
17,
22,
31,
50,
53). This raises the possibility
that the hTERT DAT domain
may interact with orthologs of these
proteins. Alternatively,
the DAT domain may participate in the
coordination of 3'G-rich
single-strand elongation by telomerase
and lagging-strand synthesis
of the C-rich strand (
1,
15,
53).
Functional domains of hTERT.
The ability of hTERT to
elongate telomeres undoubtedly requires complex and precise regulation
involving nuclear import, substrate recognition, and coordinated
synthesis of the C strand. Since it is not feasible to reconstitute
this complex process in vitro, we employed intact human cells to scan
hTERT for regions that will further our understanding of these
important biologically defined functions. The identification of the
biologically essential DAT domain has clearly demonstrated the utility
of this approach and represents a definitive step in elucidating the
regulation of telomerase function in vivo. Lastly, since
the inhibition of telomerase has been shown to prevent
cancer cell lines from forming tumors in vivo, all of the essential
domains that we identified in hTERT may represent suitable
pharmacological targets for the treatment of human cancers.
 |
ACKNOWLEDGMENTS |
We thank members of the Counter, Wang, Pendergast, and Yao
laboratories for help and advice, Tso-Pang Yao for plasmid
pCMV-HDAC1-FLAG, and Sally Kornbluth for critical review of the
manuscript. We thank L. A. Cleveland for technical assistance.
This work was supported by grants from the National Institute of
Health, administered through the National Cancer Institute (CA82481-01), and the V-Foundation. C.M.C. is a Kimmel Scholar, and
B.N.A. and S.S.R.B. are supported by Department of Defense Breast
Cancer Research Predoctoral Fellowships.
 |
FOOTNOTES |
*
Corresponding author. Mailing address: LSRC Bldg., Rm.
C225, Research Dr., DUMC, Durham, NC 27710. Phone: (919) 684-9890. Fax:
(919) 684-8958. E-mail: count004{at}mc.duke.edu.
 |
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Molecular and Cellular Biology, November 2001, p. 7775-7786, Vol. 21, No. 22
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.22.7775-7786.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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