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RESEARCH COMMUNICATION
Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, New York 10016
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Abstract |
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 Top Abstract Results and DiscussionMaterials and methodsAcknowledgmentsReferences |
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[Keywords: Telomere; ubiquitin; TRF1; tankyrase 1; PARP]
Received January 22, 2003; revised version accepted March 28, 2003.
). Telomere length is tightly controlled in tumor cells, where
despite high levels of telomerase, telomeres are maintained at a constant
length setting (Counter et al.
1992). Cells employ a complex set of positive and negative
regulatory mechanisms to modulate telomere length, including regulating access
of telomerase to chromosome termini (Evans
and Lundblad 2000).
Mammalian telomeres consist of tandem arrays of TTAGGG repeats bound by the
sequence-specific, double-stranded, DNA-binding proteins TRF1
(Chong et al. 1995) and TRF2
(Bilaud et al. 1997;
Broccoli et al. 1997). TRF2 is
required to protect chromosome ends (van
Steensel et al. 1998; de Lange
2002), possibly through its ability to assemble t-loops,
higher-order structures at telomeres
(Griffith et al. 1999). In
addition, TRF2 can influence telomere length through a telomerase-independent
mechanism (Ancelin et al. 2002;
Karlseder et al. 2002). TRF1,
on the other hand, is a negative regulator of telomerase-mediated telomere
length, acting in cis at chromosome ends to repress telomere elongation
(van Steensel and de Lange
1997; Ancelin et al.
2002).
Tankyrase 1 is a telomeric poly(ADP-ribose) polymerase (PARP) that binds
and modifies TRF1 (Smith et al.
1998). Like other PARP family members
(Smith 2001), tankyrase 1 uses
NAD+ as a substrate to catalyze formation of ADP-ribose polymers
onto specific protein acceptors, including itself and TRF1
(Smith et al. 1998;
Rippmann et al. 2002).
ADP-ribosylation of TRF1 by tankyrase 1 inhibits TRF1 binding to telomeric DNA
in vitro (Smith et al. 1998).
Overexpression of tankyrase 1 in human tumor cells releases TRF1 from
telomeres and induces telomere elongation
(Smith and de Lange 2000;
Cook et al. 2002). Both
tankyrase 1-induced activities (loss of TRF1 and telomere elongation) require
the catalytic PARP activity of tankyrase 1
(Smith and de Lange 2000;
Cook et al. 2002). These
findings suggest that tankyrase 1-mediated removal of TRF1 from telomeres by
ADP-ribosylation could allow access of telomerase to chromosome termini.
Here, we show that tankyrase 1 induces proteasome-mediated degradation of TRF1. We demonstrate that TRF1 is ubiquitinated in vivo and in vitro. We present evidence to suggest that it is not ADP-ribosylation per se, but rather, release of TRF1 from telomeres that serves as a signal for ubiquitination and subsequent degradation.
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Results and Discussion |
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TopAbstractResults and DiscussionMaterials and methodsAcknowledgmentsReferences |
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Previous results indicated that tankyrase 1 could induce telomere
elongation in telomerase-positive human tumor cells (HTC75), but not
telomerase-negative primary cells (WI38;
Smith and de Lange 2000;
Cook et al. 2002). To determine
if telomerase is required for tankyrase 1-induced telomere elongation, we
rendered the WI38 cells telomerase-positive by infection with a retrovirus
carrying the human telomerase reverse transcriptase (TERT). As expected, WI38
cells expressing TERT (Fig. 1A)
displayed telomerase activity (Fig.
1B) and showed elongated telomeres
(Fig. 1C). Coexpression of
wild-type tankyrase 1 (FN-tankyrase1.WT) in these, now telomerase-positive
WI38-TERT cells, resulted in additional telomere elongation
(Fig. 1F), indicating that
telomerase is required for tankyrase 1-mediated telomere elongation.
FN-tankyrase1.WT had no effect on telomerase protein levels
(Fig. 1D) or telomerase
activity (Fig. 1E), consistent
with an indirect effect on telomerase. However, FN-tankyrase1. WT expression
resulted in a dramatic reduction in TRF1 levels
(Fig. 1D). Expression of a
PARP-dead tankyrase 1 mutant (FN-tankyrase1.HE/A; containing a double-point
mutation in the catalytic PARP domain; Cook
et al. 2002) had no effect on telomere length
(Fig. 1F) or TRF1 levels
(Fig. 1D) and was similar to
the vector control. Together, these data demonstrate that tankyrase 1
regulates telomere elongation by telomerase, and as shown previously in other
cell types (Smith and de Lange
2000; Cook et al.
2002), telomere lengthening correlates with loss of TRF1 and
requires the PARP activity of tankyrase 1.
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TRF1 is ubiquitinated in vivo and degraded by the proteasome
TRF1 loss could be due to regulated degradation of the protein. To address
this question, we used a previously characterized HTC75 cell line (FN-30)
expressing an inducible allele of tankyrase 1
(Smith and de Lange 2000). As
shown in Figure 2A, immunoblot
analysis indicates that upon induction of tankyrase 1, TRF1 is lost (cf. lanes
1 and 2). Incubation of cells with MG132 (a specific inhibitor of the
proteasome) rescued the tankyrase 1-induced loss of TRF1
(Fig. 2A, cf. lanes 2 and 4).
Whereas, incubation of cells with E-64 (an inhibitor of lysosomal proteolysis)
had no effect (Fig. 2A, cf.
lanes 2 and 6). TRF2 was unaffected by tankyrase 1 overexpression or
proteasome inhibition. These results indicate that the tankyrase 1-induced
loss of TRF1 is due to TRF1 degradation and that this degradation is mediated
by the proteasome pathway.
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Degradation of proteins by the proteasome depends upon conjugation of ubiquitin to the target protein. To determine if TRF1 is ubiquitinated in vivo, HeLa cells were transfected with a HA-tagged ubiquitin construct (HA-ubiquitin) and ubiquitinated proteins were isolated on an anti-HA antibody affinity matrix. As shown in Figure 2B, slower migrating ubiquitin conjugates of TRF1 could be detected specifically in the HA-ubiquitin transfected cells. This modification was slightly stimulated by proteasome inhibitor. In an alternative approach, HTC75 cells stably expressing a mycpitope tagged allele of TRF1 (myc-TRF1) were transfected with HA-ubiquitin, isolated on an anti-myc antibody affinity matrix, and immunoblotted with anti-HA antibodies. As shown in Figure 2C, lane 2, slower migrating ubiquitin conjugates were detected specifically in the myc-TRF1, HA-ubiquitin transfected cells. Notably, the presence of multiple slower migrating forms indicates that TRF1 is polyubiquitinated in vivo.
Finally, a common feature of proteins targeted for degradation by
ubiquitin-mediated proteolysis is a relatively short half-life. To determine
the half-life of TRF1, cells were incubated with cycloheximide and analyzed by
immunoblotting with anti-TRF1 antibody. As shown in
Figure 2D, and graphically in
Figure 2E, TRF1 (in contrast to
the long-lived 
-tubulin) was turned over with a half-life of <1 h,
indicating that TRF1 is a short-lived protein.
TRF1 is ubiquitinated in vitro, independent of ADP-ribosylation
To establish a cell-free system for ubiquitination of TRF1, 35S-methionine-labeled TRF1 was generated by coupled in vitro transcription/translation (Fig. 3A, lane 1) and then diluted into a ubiquitination reaction mix [retic(+)] containing reticulocyte lysate supplemented with methyl ubiquitin, ubiquitin aldehyde, and an energy source. As shown in Figure 3A, lane 2, upon incubation with retic(+), TRF1 distributed to slower migrating forms, which likely represent TRF1 conjugated with one to several ubiquitin chains. Immunoblot analysis with anti-TRF1 antibodies confirmed that these slower migrating bands comprise TRF1 (Fig. 3B, lane 2). To confirm that these bands represented ubiquitinated forms of TRF1, exogenous excess ubiquitin was added to the reaction. As shown in Figure 3A, lane 3, addition of ubiquitin (which dilutes out the chain limiting methyl ubiquitin in the reaction) shifted the bands to much higher molecular weight species, indicating longer ubiquitin chains.
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As shown in Figure 1D and
previously (Cook et al. 2002),
tankyrase 1-induced loss of TRF1 requires a catalytically active form of
tankyrase 1, suggesting that ADP-ribosylation of TRF1 may be required for
ubiquitination and subsequent degradation. Since immunoblot analysis indicated
that the reticulocyte lysate used in the in vitro reactions contained
tankyrase 1 (data not shown), we sought to determine if tankyrase 1 was
required for ubiquitination. To address this question, we first asked if
interaction between TRF1 and tankyrase 1 was required for ubiquitination of
TRF1. A TRF1 construct lacking the N-terminal, tankyrase 1-binding
(Smith et al. 1998), acidic
domain of TRF1 was generated. As shown in
Figure 3C, 
acidic TRF1
was efficiently ubiquitinated in vitro, although there was a shift in the
distribution of ubiquitinated species from low to higher molecular weight
forms in wild type versus acidic TRF1. These results indicate that TRF1
binding to tankyrase 1 is not required for ubiquitination of TRF1 in
vitro.
In a second approach, we asked if PARP activity was required for
ubiquitination of TRF1. In vitro translation and subsequent ubiquitination of
TRF1 was performed in the presence of the PARP inhibitor 3-aminobenzamide
(3AB). Previous results indicated that 1 mM 3AB was sufficient to inhibit ADP
ribosylation of TRF1 by tankyrase 1 in vitro
(Smith et al. 1998). As shown
in Figure 3D, inclusion of 1 or
10 mM 3AB in the reactions did not inhibit ubiquitination of TRF1, indicating
that ADP-ribosylation of TRF1 is not required for ubiquitination in vitro.
Telomere-bound TRF1 is protected from ubiquitination
Our studies thus far indicate that while catalytic activity of tankyrase 1 is required for TRF1 degradation in vivo, ADP-ribosylation of TRF1, per se, is not a prerequisite for ubiquitination of TRF1 in vitro. What then is the role of ADP-ribosylation? One possibility is that this modification is required solely to remove TRF1 from telomeres in order to render TRF1 accessible to the ubiquitination machinery; that is, perhaps telomere bound TRF1 is protected from ubiquitination. To address this question, increasing amounts of double-stranded TTAGGG repeat DNA were included in the in vitro reaction. As shown in Figure 4A, ubiquitination of TRF1 was inhibited by double-stranded TTAGGG repeats, but not by nonvertebrate, double-stranded telomeric TTAGGC repeats nor by single-stranded vertebrate telomeric repeats TTAGGG or CCCTAA.
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To rule out the possibility that the TTAGGG DNA had a nonspecific effect on
ubiquitination, we generated a point mutation in TRF1 (conversion of R to V at
position 425 in the myb domain), which abolishes DNA binding
(Fairall et al. 2001). As
shown in Figure 4B, TRF1.RV
(like TRF1) was efficiently ubiquitinated; however, ubiquitination of TRF1.RV
(unlike that of TRF1) was not inhibited by double-stranded TTAGGG telomeric
repeat DNA. These data show that TRF1 is protected from ubiquitination when
bound to telomeric DNA and suggest the possibility that the DNA-binding, myb
domain of TRF1 might serve as a site for recognition or modification by the
ubiquitinating machinery. Indeed, as shown in
Figure 4C, ubiquitination of
TRF1 lacking the myb domain (mybTRF1) was dramatically reduced compared
to wild-type TRF1.
Our findings are consistent with the notion that TRF1 is subjected to a set
of sequential, posttranslational modifications. First, tankyrase 1
ADP-ribosylates TRF1 to release it from telomeres. Second, the
telomere-dissociated form of TRF1 is then ubiquitinated and targeted for
degradation. We wondered why degradation of TRF1 was necessary; that is, was
it not sufficient to release TRF1 from telomeres? Studies indicate that
because of rapid hydrolysis by the glycohydrolase PARG, the intracellular
half-life of ADP-ribose polymers is 
1 min
(Davidovic et al. 2001). Thus,
one possibility is that while tankyrase 1-mediated ADP-ribosylation of TRF1 is
sufficient to release TRF1 from telomeres, rapid degradation of the polymer by
PARG would allow immediate reassociation of TRF1 with telomeres. Indeed, the
abundant repetitive sequences at telomeres could act as a sink for rapid
rebinding of TRF1 to telomeres. Thus, degradation of TRF1 may be necessary to
keep TRF1 off telomeres for an extended period of time. This question was
addressed by determining the fate of TRF1 localization when it is released
from telomeres by ADP-ribosylation, but not degraded by the proteasome. As
shown in Figure 4D, and
previously (Smith and de Lange
2000; Cook et al.
2002), overexpression of tankyrase 1 in the nucleus results in
release of TRF1 from telomeres. However, when proteasome-mediated degradation
is inhibited, TRF1 is found relocalized to telomeres
(Fig. 4E). These results
suggest that tankyrase 1-mediated ADP-ribosylation of TRF1 is not sufficient
to keep TRF1 off telomeres; that is, if TRF1 is not degraded it reassociates
with telomeres, even in the presence of excess tankyrase 1.
A model for telomere length regulation
Our results are presented in terms of a model for telomere length regulation. As shown in Figure 5, TRF1-bound telomeres are in a configuration that blocks access to telomerase. Poly(ADP-ribosyl)ation of TRF1 by tankyrase 1 releases TRF1 from telomeres. The telomere-unbound form of TRF1 is then ubiquitinated and degraded by the proteasome, thereby preventing its rapid reassociation with telomeres. Telomerase can then gain access to the TRF1-free telomere to add telomeric repeats. The telomere is reassembled using newly synthesized TRF1 into a configuration that once again blocks access to telomerase.
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This model offers a number of interesting features. First, it provides a
rapid mechanism for release of TRF1 from telomeres. Since tankyrase 1 contains
five binding sites for TRF1 (Seimiya and
Smith 2002), it has a large capacity to bind, modify, and remove
TRF1 from telomeres. Second, since poly(ADP-ribosyl)ation reactions are
tightly controlled in vivo, activation of tankyrase 1's PARP activity could be
regulated to coordinate release of TRF1 and access to telomerase with the cell
cycle. And third, because of the rapid turnover of TRF1, reassembly of
telomeres with TRF1 into a telomerase-inaccessible state would occur with
newly synthesized TRF1. This could provide an opportunity for TRF1 to recruit
regulatory proteins to chromosome termini. Indeed, previous results indicate
that TRF1 can recruit tankyrase 1 to telomeres when the proteins are
coexpressed (Smith and de Lange
1999).
How does loss of TRF1 change telomere configuration to allow telomerase
access? Telomeres exist in a protective configuration that requires TRF2 and
likely involves a higher-order structure, such as the t-loop
(de Lange 2002). Studies
indicate that when TRF1 is lost from telomeres (either through a
dominant-negative allele of TRF1 or through ADP-ribosylation by tankyrase 1)
TRF2 remains on telomeres and telomeres remain protected (as indicated by
unimpeded cell growth), yet they undergo telomere elongation
(van Steensel and de Lange
1997; Smith and de Lange
2000; Cook et al.
2002). Thus, loss of TRF1 may induce an intermediate state where
telomeres remain in a protected configuration, but still allow access to
telomerase for telomere elongation. Whether TRF1 loss influences higher-order
structure at telomeres and/or the proteins that bind and recruit telomerase,
will be important questions for the future.
While poly(ADP-ribosyl)ation of TRF1 is a very dramatic reaction,
sufficient to release TRF1 from telomeres, because of its transient nature, a
second modification (ubiquitination and subsequent degradation) appears to be
required to keep TRF1 off telomeres. The ubiquitin-proteasome degradation
pathway consists of a series of enzymatic reactions involving a
ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2) and
ubiquitin ligase (E3; Hershko and
Ciechanover 1998). Of particular interest are the E3s, a growing
family of proteins that determine the timing and specificity of
ubiquitination. Undoubtedly, identification of the E3 that recognizes TRF1
will be informative. Our studies suggest that the putative E3 does not
recognize telomere-bound TRF1. E3 recognition may occur through the myb-type
DNA-binding domain of TRF1. Consistent with this notion, in vitro
ubiquitination of TRF1 lacking a myb domain is severely reduced
(Fig. 4C). Thus, when TRF1 is
bound to telomeres, the myb domain may not be accessible to the E3 and/or
lysine residues (that serve as ubiquitination sites) may be masked by DNA
interactions.
Although a role for ubiquitination in telomere function has not been
directly demonstrated, a number of studies suggest the possibility. Mutations
in Drosophila UbcD1, a ubiquitin conjugating (E2) enzyme, induce
transient, resolvable telomere-telomere associations in mitosis and meiosis,
suggesting that a telomere-associated protein could be a target for
ubiquitination (Cenci et al.
1997). More recently, the fission yeast F-box protein Pof3 was
found to be required for genomic integrity and telomere function
(Katayama et al. 2002). F-box
proteins are members of a large family of proteins that provide substrate
specificity for the SCF ubiquitin ligase (E3) complexes
(Kipreos and Pagano 2000).
Yeast cells lacking Pof3 displayed shortened telomeres and were defective in
telomeric silencing, suggesting again that a telomere-associated protein could
be a target for ubiquitination. In this report, we have identified a telomeric
target for ubiquitination, TRF1. While we have yet to identify the ubiquitin
machinery responsible for this reaction in human cells, our studies, along
with those in other divergent organisms, suggest the possibility of a
conserved role for ubiquitin-mediated proteolysis in telomere function.
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Materials and methods |
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TopAbstractResults and DiscussionMaterials and methodsAcknowledgmentsReferences |
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TRF1 constructs were cloned into the retroviral vector pLPC
(Serrano et al. 1997) and
contain an N-terminal myc-epitope tag followed by amino acids 2–439
(pLPCTRF1), amino acids 66–439 (acidicTRF1), or amino acids
2–378 (mybTRF1). pLPC-TRF1.RV was generated using the Stratagene
quickchange site directed mutagenesis kit. FN-tankyrase1.WT and HE/A contain
full-length tankyrase 1 (amino acids 2–1327) with an N-terminal
FLAG-epitope tag and nuclear localization signal in pLPC
(Cook et al. 2002).
Retroviruses and cell lines
Retroviruses were generated and used to infect cells as described
previously (Cook et al. 2002).
WI38 cells (ATCC), human primary fibroblasts at population doubling (PD) 30
were infected with pBABE-hygro or pBABE-hygro-TERT
(Counter et al. 1998) and
selected in 90 µg/mL hygromycin. WI38-TERT cells at PD 5 were infected with
pLPC, pLPC-FN-Tankyrase1.WT, or pLPC-FN-Tankyrase1.HE/A and selected with 2
µg/mL puromycin. On day 3 of retroviral infection, cells were subcultured
1:2 and upon confluence designated PD 0.
HT1080 (ATCC) is a human fibrosarcoma cell line. HTC75 is a HT1080-derived
clonal cell line that stably expresses the tetracycline(tet)-controlled
transactivator (van Steensel and de Lange
1997). FN30 is a HTC75-derived clonal cell line that stably
expresses doxycylin-inducible FN-tankyrase1.WT
(Smith and de Lange 2000).
Stable HTC75 cell lines expressing myc-TRF1 or vector control were generated
by retroviral infection using pLPCTRF1 or pLPC as described
(Cook et al. 2002).
Genomic blotting and TRAP assays
Southern blotting for telomere-length analysis was performed as described
previously (Cook et al. 2002).
TRAP assays (Kim et al. 1994)
contained 1 µg CHAPS (Pierce) extract with or without 10 µg/mL RNase
A.
Immunoblotting
Immunoblots were incubated with the following primary antibodies: rabbit
anti-poly(ADP-ribose) serum (1:1000; Alexis Biochemicals), rabbit anti-TRF1
415 (0.2 µg/mL; Cook et al.
2002), rabbit anti-tankyrase 1 376 (0.1 µg/mL;
Cook et al. 2002), mouse
anti--tubulin ascites (1:500,000; Sigma), rabbit anti-TERT 374 (0.8
µg/mL; raised and affinity purified against Escherichia
coli-derived fusion protein containing hTERT amino acids 561–698),
or mouse monoclonal anti-TRF2 (1.0 µg/mL; Imgenex Clone 4A794), followed by
horseradish peroxidase-conjugated donkey anti-rabbit or anti-mouse IgG
(Amersham; 1:2500). Bound antibody was detected using the Enhanced
Chemiluminescence (Amersham), Super-Signal West Dura, or Femto (Pierce)
kits.
Cell extracts and immunoprecipitation
For immunoblot analysis, whole-cell extracts were prepared as described
(Cook et al. 2002) and 25 µg
was fractionated by SDS-PAGE.
For immunoprecipitation, HA-ubiquitin transfected cell extracts were prepared in buffer C [20 mM Hepes-KOH at pH 7.9, 420 mM KCl, 25% glycerol, 0.1 mM EDTA, 5 mM MgCl2, 0.2% NP-40, 1 mM dithiothreitol, and 2.5% protease inhibitor cocktail (Sigma)] containing 10 mM N-ethylmaleimide (Sigma), and then incubated with anti-HA (Roche) or anti-myc (Sigma) affinity matrix for 3 h with shaking at 4°C. HA-matrix-bound proteins were washed three times in buffer D (20 mM Hepes at pH 7.9, 100 mM KCl, 20% glycerol, 0.2 mM EDTA, 0.2 mM EGTA) and myc-matrix bound proteins were washed four times in buffer C (without NP40).
Transient transfections and indirect immunofluorescence
HelaI.2.11 cells (van Steensel et al.
1998) were transfected for 24 h with pMT123 (encoding
HA-ubiquitin; Treier et al.
1994) or pcDNA3-FN-tankyrase1.WT, full-length tankyrase 1
containing an N-terminal FLAG tag and nuclear localization signal in the
expression vector pcDNA3 (Invitrogen; B. Houghtaling and S. Smith, unpubl.),
using Lipofectamine 2000 regeant (Invitrogen). Cells were processed for
immunofluorescence as described previously
(Cook et al. 2002) using mouse
monoclonal anti-FLAG M2 (1 µg/mL; Sigma) and rabbit anti-TRF1 415 (0.1
µg/mL; Cook et al. 2002) as
primary antibodies.
In vitro ubiquitination reactions
The ubiquitination reaction mix [retic(+)] contained 5 µL
rabbit reticulocyte lysate (Promega), 1 µM ubiquitin aldehyde (Boston
Biochem), 121 µM methyl ubiquitin (Boston Biochem), 1x energy
solution (Boston Biochem), and 150 µM additional ubiquitin (Boston Biochem)
in 10 µL total reaction mix. The reaction indicated in
Figure 3A and B, lane 2, lacked
additional ubiquitin. The ubiquitin reaction mix was preincubated at 37°C
for 5 min to allow ubiquitin aldehyde to inhibit deubiquitinating enzymes. To
generate 35S-labeled TRF1, pLPC-TRF1, pLPC-TRF1.RV, or
pLPC-acidic TRF1, plasmids were in vitro transcribed and translated
with S35 methionine in a 12.5 µL reaction using Promega TNT
coupled-reticulocyte lysate system. Then, 2.5 µL was incubated with the
ubiquitination reaction mix at 37°C for 30 min. Reactions were stopped by
addition of 2x sample buffer.
For telomere inhibition studies, oligonucleotides containing (TTAGGG)6 and (CCCTAA)6 for DS (TTAGGG) or (TTAGGC)6 and (GCCTAA)6 for DS (TTAGGC) or (TTAGGG)6 for SS (G) or (CCCTAA)6 for SS (C) were used. In vitro translated TRF1 was incubated with DS or SS oligonucleotides at 37°C for 30 min prior to addition to the ubiquitination reaction.
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Acknowledgments |
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TopAbstractResults and DiscussionMaterials and methodsAcknowledgmentsReferences |
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The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
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Footnotes |
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1 E-MAIL
smithsu{at}saturn.med.nyu.edu;
FAX (212) 263-5711. 
Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/gad.1077103
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