From the Institute of Biopharmaceutical Science, National Yang-Ming University, Shih-Pai, 112, Taipei, Taiwan, Republic of China
Received for publication, February 21, 2001, and in revised form, April 13, 2001
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ABSTRACT |
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Cdc13p is a single strand telomere-binding
protein of Saccharomyces cerevisiae; its telomere-binding
region is within amino acids 451-693, Cdc13(451-693)p. In this study,
we used purified Cdc13p and Cdc13(451-693)p to characterize their
telomere binding activity. We found that the binding specificity of
single-stranded TG1-3 DNA by these two proteins is
similar. However, the affinity of Cdc13(451-693)p to DNA was slightly
lower than that of Cdc13p. The binding of telomeric DNA by these two
proteins was disrupted at NaCl concentrations higher than 0.3 M, indicating that electrostatic interaction
contributed significantly to the binding process. Because both proteins
bound to strand TG1-3 DNA positioned at the 3' end, the 5'
end, or in the middle of the oligonucleotide substrates, our results
indicated that the location of TG1-3 in single-stranded
DNA does not appear to be important for Cdc13p binding. Moreover, using
DNase I footprint analysis, the structure of the telomeric DNA
complexes of Cdc13p and Cdc13(451-693)p was analyzed. The DNase I
footprints of these two proteins to three different telomeric DNA
substrates were virtually identical, indicating that the telomere
contact region of Cdc13p is within Cdc13(451-693)p. Together, the
binding properties of Cdc13p and its binding domain support the theory
that the specific binding of Cdc13p to telomeres is an important
feature of telomeres that regulate telomerase access and/or
differentiate natural telomeres from broken ends.
Single-stranded guanosine-rich DNA tail is a common structural
feature in most of the eukaryotic telomeres (1-7). For example, ciliated protozoa telomeres are extended to form a 12-16-base single-stranded G-tail (1, 3). In Saccharomyces cerevisiae, transient single-stranded TG1-3 tails with lengths larger than 30 bases are detected late in the S phase (4). This presence of
single-stranded G-tails was postulated as an intermediate for telomere
replication (8). Single-stranded G-tails could form a DNA quadriplex
in vitro known as the G-quartet (9, 10), although it remains
to be determined whether such a structure indeed exists in cells.
Protein factors that bind to the single-stranded telomeric DNA have
been identified in several organisms (11-19). Among these protein
factors, Oxytricha telomere-binding protein has been well characterized. It is heterodimeric and is composed of an The binding of Cdc13p to telomeric DNA is essential for its function in
telomeres (24), and it appears to have multiple functions in cells. For
example, Cdc13p is involved in cell cycle control since a
temperature-sensitive allele of CDC13, cdc13-1, causes cell cycles to arrest in the G2/M phase at non-permissive temperatures (25). The binding of Cdc13p to telomeres might cause yeast
cells to differentiate whether the ends of linear DNA are telomeres or
broken ends (26). In addition, Cdc13p appears to be a key factor in
telomere replication. It interacts with Est1p that is associated with
telomerase RNA (27-30) to recruit telomerase to telomeres for
replication. This was evidenced in part by the presence of a mutant
allele of CDC13, cdc13est, which causes a
gradual loss of the telomere (13). Moreover, Cdc13p could interact with
the catalytic subunit of DNA polymerase CDC13 is an essential gene that encodes a 924-amino acid
protein with a molecular mass of 104,895 Da (25). The Cdc13p fragment ranging from amino acids 451 to 693, Cdc13(451-693)p, contains the
telomere-binding region of Cdc13p; it is sufficient to bind single-stranded telomeric DNA in vitro and interacts with
telomeres in vivo (24, 31). However, a sequence comparison
among Cdc13(451-693)p and known DNA- or RNA-binding proteins did not
provide any information on Cdc13(451-693)p responsible for binding to
telomeres (24). Thus, Cdc13(451-693)p contains a novel motif for
telomere binding. To understand how Cdc13p interacts with telomeres, we
used purified Cdc13p and Cdc13(451-693)p to analyze their binding
properties. The results of both
EMSA2 and DNase I footprint
analysis revealed the specific binding of telomeric DNA by these two proteins.
Expression and Purification of 6xHis-tagged Cdc13p and
Cdc13(451-693)p--
A baculovirus system was used to purify Cdc13p.
An insect cell line sf21 was used as the host for virus
propagation and protein purification. Escherichia coli
DH5
To purify 6xHis-tagged Cdc13p, ~5 × 107 sf21
cells were infected with recombinant virus for 4 days. Cells were
washed with phosphate-buffered saline and then lysed by the addition of
Nonidet P-40 lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 200 µM
phenylmethylsulfonyl fluoride). The suspensions were incubated on ice
for 40 min and then sonicated. Total cell extracts were then collected
by centrifugation. Ni-NTA-agarose (Qiagen) was used to purify the
6xHis-tagged Cdc13p. Batch purification protocol was used according to
the manufacturer's recommendations. The bound 6xHis-tagged Cdc13p was
eluted by buffer containing 50 mM
NaH2PO4, pH 8.0, 250 mM imidazole,
20% glycerol. Purified protein was aliquoted and frozen by a dry
ice-ethanol bath. The yield of Cdc13p was ~0.5 mg/107 cells.
To purify 6xHis-tagged Cdc13(451-693)p, a 1-liter culture of E. coli harboring pET6H-CDC13(451-693) was grown at 25 °C to A600 to 0.5 and induced with the addition
of 1 mM
isopropyl-1-thio- Electrophoretic Mobility Shift Assay
(EMSA)--
Oligonucleotides (Table I)
were labeled with [
The apparent binding constant of Cdc13p and Cdc13(451-693)p to
telomeric DNA was determined using EMSA and quantified by a PhosphorImager. The DNA substrates used were at 5 nM in all
experiments. Values presented in Table II
were determined from interpolation on a Hill plot. Each value was the
average of 2-3 experiments.
DNase I Footprint--
Telomeric DNA strands were 5'-labeled and
purified as described above. The DNA was mixed with Cdc13p or
Cdc13(451-693)p in 40 µl of Buffer B (40 mM Tris-HCl, pH
8.0, 10 mM MgSO4, 1 mM
CaCl2) and incubated at 25 °C for 10 min. 1 unit of
DNase I was added and incubated at 37 °C for another 10 min. The
reaction was stopped with 10 µl of 250 mM EGTA. The DNA
was then precipitated by adding 1 µl of 10 mg/ml oyster glycogen and
150 µl of ethanol. The precipitant was collected by centrifugation,
dried, and analyzed by electrophoresis using a 12% polyacrylamide
sequencing gel.
Isolation of Recombinant Cdc13p and Its Specific Binding to
Single-stranded TG1-3DNA--
The baculovirus expression
system was used to purify Cdc13p. In this study, Cdc13p with six
histidines tagged at the N terminus was expressed in insect sf21
cells, and the protein was purified to homogeneity using a
Ni-NTA-agarose column (Fig. 1, lane
2). The purified protein has an apparent mass of 105 kDa, which is in agreement with the predicted size of Cdc13p. Western blotting analysis further confirmed that it is Cdc13p (data not shown).
The binding specificity of Cdc13p to telomeric DNA was determined using
EMSA analysis. Purified protein was mixed with 32P-labeled
single-stranded TG1-3 with various amounts of unlabeled
nucleic acid competitors before subjecting to the EMSA analysis. As
shown in Fig. 2, unlabeled TG15 competed
efficiently with 32P-labeled TG15. The binding was reduced
by ~50% when the competitor was presented at equal concentrations
(Fig. 2A, lanes 3-6, and Fig. 2B).
Vertebrate (T2AG3) telomeric DNA also competed
for Cdc13p binding (Fig. 2A, lanes 7-9),
although a 10-fold molar excess of
(T2AG3)3 was needed to obtain the
same level of competition as that from TG15 (Fig. 2B). In
contrast, Oxytricha (T4G4) and Tetrahymena (T2G4) telomeric DNA did
not compete for the binding of TG15 to Cdc13p (Fig. 2A,
lanes 7-12). Total yeast RNA, single-stranded C1-3A DNA, or duplex
TG1-3/C1-3A DNA did not compete for Cdc13p
binding (Fig. 3A, lanes
16-18) (data not shown). Interestingly, Cdc13p formed two
complexes with TG15, although the nature of these multiple complexes is
unclear. Nevertheless, these results indicated that Cdc13p bound
specifically to single-stranded TG1-3 telomeric DNA.
DNA encoding Cdc13(451-693)p with 6xHis tag was expressed in E. coli (BL21(DE3)pLysS). Although Cdc13(451-693)p formed insoluble aggregates at 37 °C (24), a sufficient amount of soluble
Cdc13(451-693)p, however, could be obtained at 25 °C (Fig. 1,
lane 3). We then investigated the binding properties of the
purified Cdc13(451-693)p. Similar to Cdc13p, purified Cdc13(451-693)p
bound specifically to single-stranded TG1-3 telomeric DNA
(Fig. 3). However, only one distinct complex was observed. This result
further confirmed our conclusion that the telomeric DNA-binding domain
of Cdc13p is located within amino acids 451-693. Identical results
were previously obtained with Cdc13p fusion protein and chemically renatured Cdc13(451-693)p (14, 24). Previously, E. coli
extracts containing Cdc13p fused to glutathione
S-transferase was used to show that Cdc13p bound
specifically to single-stranded TG1-3 DNA in
vitro (14), and the renatured Cdc13(451-693)p was used to
demonstrate that this region contained the telomere-binding domain of
Cdc13p, and its binding to telomeres was specific (24).
Binding of Cdc13p and Cdc13(451-693)p to Long Telomeric
DNA--
To determine whether Cdc13p and Cdc13(451-693)p could bind
to long single-stranded TG1-3 telomeric DNA, Cdc13p and
Cdc13(451-693)p were mixed with telomeric DNA substrates with
different lengths, and the complexes were analyzed. The results shown
in Fig. 4 demonstrated that both Cdc13p
and Cdc13(451-693)p were capable of forming complexes with long
telomeric DNA. Multiple protein-DNA complexes were apparent in long DNA
substrates, and the patterns of the complexes suggested that more than
one protein could bind to a single DNA molecule. We also determined the
binding affinity of Cdc13p and Cdc13(451-693)p to these DNA
substrates. An apparent binding constant was determined from the Hill
plot. As shown in Table II, Cdc13(451-693)p appears to bind to
telomeric DNA with an affinity similar to that of Cdc13p. Cdc13p
required telomeric sequences longer than 13 bases for proper binding,
whereas Cdc13(451-693)p required 15 bases for proper binding.
Telomeric DNA Binding Properties of Cdc13p and
Cdc13(451-693)p--
The telomeric DNA sequences of S. cerevisiae are combinations of TG, TGG, and TGGG repeats. To test
if Cdc13p and Cdc13(451-693)p have a sequence preference, the binding
affinities of these two proteins for (TG)12,
(TGG)8, or (TGGG)6 were determined. As shown in
Table II, the apparent binding constants of Cdc13p to these three
substrates were similar to other telomeric DNA substrates. This result
suggested that Cdc13p did not favor either repeat for binding. However,
Cdc13(451-693)p bound to these three substrates with affinities
significantly lower than binding to other telomeric DNA substrates,
suggesting that the telomeric DNA-binding domain alone preferred
TG1-3 sequences for binding.
Oxytricha telomere-binding proteins were shown to bind to
T4G4 telomeric DNA at a high concentration of
salt (22, 32, 33). We then investigated whether the affinity of Cdc13p
or Cdc13(451-693)p for TG15 may depend on NaCl concentrations. As shown in Fig. 5, both proteins
dissociated from TG15 at a NaCl concentration higher than 0.3 M. However, Cdc13(451-693)p appeared to tolerate NaCl
better than Cdc13p.
To evaluate the stability of the protein-DNA complex, the dissociation
rate of protein-DNA complex was measured. Cdc13p or Cdc13(451-693)p
was first bound to 32P-labeled TG15, and then an excess
amount of unlabeled TG15 was added to prevent a re-association of
protein to labeled DNA. The dissociation rate was estimated as the time
required for half of the protein-DNA complex to dissociate. As shown in
Fig. 6, both Cdc13p and Cdc13(451-693)p
had dissociation rates ~30 min, suggesting that these two proteins
bound to telomeric DNA with similar stability.
Cdc13p Does Not Require a 3' end for Binding--
To determine if
single-stranded TG1-3 has to locate at the 3' end for
Cdc13p binding, we synthesized oligonucleotides, 5'-Tel, Int-Tel, and
3'-Tel (Table I), of identical size harboring telomeric sequences at
various locations. They were tested for Cdc13p binding by EMSA. Because
all three substrates bound to Cdc13p or Cdc13(451-693)p to the same
extent (Fig. 7), the location of
TG1-3 in a single-stranded DNA does not appear significant for Cdc13p binding.
In Fig. 7, the three oligonucleotides migrated differently on a
polyacrylamide gel even though they have the same size and were
heat-denatured and quick-cooled on ice before the binding assay. This
result suggested an intrinsic position-dependent structure of these oligonucleotides. Also, although Int-Tel DNA gave the slowest
mobility on the polyacrylamide gel, it had a slightly faster mobility
upon binding to Cdc13(451-693)p. This migration behavior suggested
that Cdc13(451-693)p bound to telomeric DNA and minimized the
position-dependent structure of the DNA substrates. Moreover, faster mobility caused by protein binding to the middle of
the DNA fragment would imply a protein-induced structural alteration of
the DNA.
DNase I Footprint Analysis of Cdc13p- and
Cdc13(451-693)p-telomeric DNA Complexes--
To investigate the
effect of Cdc13p binding to telomeric DNA in detail, the structure of
protein-DNA complexes was subjected to DNase I footprint analysis.
Autoradiograms of Cdc13p and Cdc13(451-693)p footprints are shown in
Figs. 8 and
9, respectively, and the results are
summarized in Fig. 10. Both Cdc13p and
Cdc13(451-693)p selectively protected telomeric sequences from DNase I
digestion. The footprints of these two proteins with various DNA were
nearly identical, suggesting that amino acid 451-693 of Cdc13p was the
region that made contact with telomeres. It is interesting to note that
the binding of Cdc13p produced several DNase I hypersensitive sites right next to the protection area, although the nature of these hypersensitive sites is unclear.
Cdc13p is an essential sequence-specific DNA-binding protein
involved in a wide range of telomere functions including telomere length maintenance (13, 27, 28), telomere position effect (14), and
cell cycle regulation (25). Therefore, it is important to elucidate the
interaction of this protein with telomeric DNA. In this study, we
purified Cdc13p in native form and its telomere-binding domain,
Cdc13(451-693)p. Using these purified proteins, the telomeric DNA
binding properties were characterized. Both Cdc13p and Cdc13(451-693)p bound to telomeric DNA with similar specificity and stability. Furthermore, DNase I footprints showed virtually identical binding of
telomeric DNA by both Cdc13p and Cdc13(451-693)p, indicating that
amino acid 451-693 is the region within Cdc13p that contacts telomere DNA.
A dissociation constant of ~10 In Fig. 7, EMSA analysis implicated a structure alteration of telomeric
DNA induced by Cdc13(451-693)p. Similarly, DNase I footprint analysis
also supports the presence of protein-induced alteration on DNA (Figs.
8 and 9). DNase I hypersensitive sites are positions of DNA that are
easily digested by DNase I. These positions usually represent DNA with
aberrant structure. For example, DNA bends on double-stranded DNA
usually cause hypersensitive sites by DNase I digestion. Although we do
not know the exact nature of the structural change in the
single-stranded telomeric DNA induced by Cdc13p, it appeared that
Cdc13p indeed caused structural alteration in telomeric DNA. Such
alteration caused by Cdc13p may be an important feature of telomeres.
Protein-induced DNA distortion at specific sites is considered to be an
important mechanism for promoting the multiprotein interactions
involved in regulation of gene activity (35, 36), initiation of
replication (37-39), site-specific recombination (40, 41), or
recognition of DNA damage (42). Cdc13p was shown to interact with Stn1p (43), the catalytic subunit of polymerase
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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and
subunit (20-22). The
subunit is a single-stranded DNA-binding protein that binds to the G4T4 single-stranded
end of a telomere. Although the
subunit is not directly involved in
binding, it is required for making the terminus-specific binding.
Cdc13p is a single-stranded TG1-3-binding protein that
interacts with telomeres in S. cerevisiae (13, 14,
23). However, although Cdc13p in yeast is the functional
equivalent of Oxytricha
- and
-binding proteins, it
shares no sequence similarity with Oxytricha telomere-binding proteins (13, 14, 23).
, suggesting that it might
be involved in a C-strand synthesis of telomeres
(28).1 It might also prevent
end-to-end fusion of chromosomes and protect chromosome from
degradation by nucleases.
MATERIALS AND METHODS
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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
was used as a host for plasmid construction and propagation, and
BL21(DE3)pLysS was used as the host for Cdc13(451-693)p purification.
Plasmid pBac6His-CDC13 was constructed by inserting a 3.0-kilobase pair
NcoI-SalI fragment of CDC13 to
NcoI- and SalI-digested pBac6His (modified from
pBlueBac4 by J.-J. Lin, Invitrogen). This plasmid enabled the
expression of Cdc13p with 6xHis tagged at the N terminus. A recombinant
virus that expressed 6xHis-tagged Cdc13p was generated by co-infection of plasmid pBac6His-CDC13 and Bac-N-Blue DNA to sf21 cells
(Invitrogen). Plasmid pET6H-CDC13(451-693), which was used to purify
the Cdc13(451-693)p, was constructed by inserting the
NcoI-NruI fragment of pTHA-NLS-CDC13(451-924) into NcoI-SmaI-digested pET6H (donated by C.-H.
Hu, National Marine University, Taipei, Taiwan). The resulting plasmid
was used to express 6xHis-tagged Cdc13(451-693)p under the control of
the T7 promoter (24).
-D-galactopyranoside. The cells were
grown at 25 °C for another 4 h before harvesting by
centrifugation. Cells were resuspended in 10 ml of sonication buffer
(50 mM NaH2PO4, pH 7.8, 300 mM NaCl, 5 mM
-mercapto ethanol, 1 × protease inhibitors (Calbiochem)) and sonicated to release the cell contents. The sonicated cells were centrifuged at 13,000 g for 15 min at 4 °C to obtain total cell free extracts.
0.5 ml of Ni-NTA-agarose (Qiagen) was added to the total cell free
extracts and incubated at 4 °C for 1 h. The resin was washed
and eluted with 2 ml of buffer containing 50 mM
NaH2PO4, pH 8.0, 250 mM imidazole, 20% glycerol. Purified protein was aliquoted and frozen by the dry
ice-ethanol bath. The yield of Cdc13(451-693)p was ~4.2 mg from 1 liter of E. coli culture.
-32P]ATP (3000 mCi/mM, PerkinElmer
Life Sciences) using T4 polynucleotide kinase (New England
Biolabs) and subsequently purified from a 10% sequencing gel after
electrophoresis. To perform the assays (12), Cdc13p or Cdc13(451-693)p
in Buffer A (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 50 mM NaCl, 1 mM dithiothreitol, 1 µg of
heat-denatured poly[dI-dC]) was mixed with 5 nM
32P-labeled TG15 DNA with a total volume of 15 µl. The
reaction mixture was incubated at room temperature for 10 min. Then,
the mixtures were loaded directly onto an 8% nondenaturing
polyacrylamide gel, which was prerun at 125 V for 10 min.
Electrophoresis was carried out in TBE (89 mM Tris
borate, 2 mM EDTA) at 125 V for 105 min. The gels were
dried, autoradiographed, and the amounts of oligonucleotides bound to
the proteins were quantified with a PhosphorImager (Molecular
Dynamics).
Oligonucleotides used in this study
Binding affinity of Cdc13p and Cdc13(451-693)p to DNA substrates
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Fig. 1.
Purification of Cdc13p and
Cdc13(451-693)p. Cdc13p and Cdc13(451-693)p with 6xHis tag were
purified from sf21 and E. coli using a Ni-NTA-agarose
column, respectively (see under "Materials and Methods"). A
Coomassie Blue-stained 10% SDS-polyacrylamide gel is given. Lane
1 shows the molecular mass markers. Lanes 2 and
3 were 2 µg each of purified Cdc13p and Cdc13(451-693)p,
respectively.
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Fig. 2.
Specific binding of Cdc13p to single-stranded
TG1-3 DNA. Competition analysis with various
telomeric DNA was used to determine binding specificity. A,
20 nM 32P-labeled TG15 were mixed with several
concentrations of different competitors before incubating with 100 nM purified Cdc13p. Competitors were yeast TG15, vertebrate
(T2AG3)3, Oxytricha
(T4G4)3, Tetrahymena
(T2G4)3, and total yeast RNA
(Ysc RNA). The gel shift assay was then carried out as shown
in autoradiogram. B, quantification of the Cdc13p binding
activity. The amount of 32P-labeled TG15 bound to the
protein was quantified by a PhosphorImager, and binding without any
competitor was taken as 100% (A, lane 2). Data are the
average of three experiments.
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Fig. 3.
Specific binding of Cdc13(451-693)p to
single-stranded TG1-3 DNA. The binding of
Cdc13(451-693)p to different telomeric DNA was accessed by competition
assays. A, 5 nM 32P-labeled TG15
were mixed with several concentrations of competitors before the
addition of 110 nM purified Cdc13(451-693)p. Competitors
were TG15, (T2AG3)3,
(T4G4)3,
(T2G4)3, and total yeast RNA
(Ysc RNA). The mixtures were subjected to gel shift assay
and were subsequently autoradiographed. B, quantification of
the Cdc13(451-693)p binding activity. The binding was quantified by a
PhosphorImager, and the value obtained in the absence of competitor was
taken as 100% (A, lane 2). The data show the average of
three experiments.
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Fig. 4.
Binding of Cdc13p and Cdc13(451-693)p to
single-stranded TG1-3 DNA with a different length. 5 nM each of 32P-labeled TG10, TG15, TG20, TG25,
TG30, and TG35 were mixed with several concentrations of the purified
Cdc13p (A) or Cdc13(451-693)p (B) before
subjecting to gel shift assay. The concentrations of Cdc13p or
Cdc13(451-693)p used in each set of experiments were 0, 31, 63, 125, 250, and 500 nM. Autoradiograms are shown here.
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Fig. 5.
Both Cdc13p and Cdc13(451-693)p prefer low
salt for binding. 5 nM 32P-labeled TG15
were incubated with 50 nM of Cdc13p or Cdc13(451-693)p and
0, 0.05, 0.1, 0.15, 0.2, 0.3, or 0.5 M NaCl at room
temperature for 10 min. 1 µM unlabeled TG15 was added to
the reaction mixtures, and gel shift assay then was performed. The
binding was quantified by a PhosphorImager, and the activity without
NaCl was taken as 100%.
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Fig. 6.
Slow dissociation of Cdc13p and
Cdc13(451-693)p from single-stranded TG1-3DNA. 15 nM 32P-labeled TG15 were incubated with 50 nM Cdc13p or Cdc13(451-693)p at room temperature for 10 min. 1 µM unlabeled TG15 was then added
(t = 0). Aliquots of the mixtures were withdrawn at
indicated time points and were loaded onto a running gel. After
electrophoresis, the amount of 32P-labeled TG15 that
remained bound was quantified by a PhosphorImager, and the binding at
t = 0 was taken as 100%.
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Fig. 7.
Cdc13p does not require a 3' end for
binding. The binding of 5'-Tel, Int-Tel, or 3'-Tel by Cdc13p
(A) and Cdc13(451-693)p (B) is shown. 5 nM each of 32P-labeled DNA were mixed with
several concentrations of the purified Cdc13p or Cdc13(451-693)p, and
gel shift assay was then carried out. The concentrations of Cdc13p and
Cdc13(451-693)p used in each set of experiments were 0, 10, 40, and
160 nM. An autoradiogram is presented.
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Fig. 8.
DNase I footprints of Cdc13p to
single-stranded TG1-3 DNA. The footprint was done
using telomeric sequences located at the 5' end (5'-Tel),
internal (Int-Tel), or 3' end (3'-Tel) of the
oligonucleotides. 5 nM each of DNA substrates were used in
the reactions. The Cdc13p concentration was 12.5 (lane 3)
and 50 nM (lane 4), respectively. The protected
regions are bracketed, and the asterisks indicate
the positions of the hypersensitive sites.
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Fig. 9.
DNase I footprints of Cdc13(451-693)p to
single-stranded TG1-3 DNA. The footprint assay was
done using 5 nM each of 5'-Tel, Int-Tel, or 3'-Tel. The
concentrations of Cdc13(451-693)p were 40 nM (lane
3) and 160 nM (lane 4), respectively. The
protected regions are bracketed, and the
asterisks indicate the positions of the hypersensitive
sites.
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Fig. 10.
DNA sequences, protected regions, and DNase
I hypersensitive sites of 5'-Tel, Int-Tel, and 3'-Tel. The DNA
sequences of oligonucleotide 5'-Tel, Int-Tel, and 3'-Tel are shown with
telomeric sequences in bold. The regions protected from
DNase I digestion (see Figs. 7 and 8) are bracketed above
(Cdc13p) and below (Cdc13(451-693)p). The DNase I hypersensitive sites
are indicated by asterisks.
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
7 M is
corresponding to ~10 kcal/mol of binding energy. Binding energies of
this magnitude are certainly common among macromolecule-macromolecule
interactions. We proposed that the binding of Cdc13p to telomeric DNA
was the result of at least two factors, specific bond formation to
nucleic acid bases and nonspecific electrostatic interaction. The
binding to yeast telomeric DNA was not reduced by competition with
telomeric DNA from other species, suggesting that the binding is
sequence-specific, and specific interaction between Cdc13p and the
bases of nucleic acids should be critical for telomere binding.
Moreover, because the binding is sensitive to ionic strength,
electrostatic interaction must contribute significantly to the binding.
This property is significantly different from that of
Oxytricha telomere-binding protein, which used a series of
aromatic amino acid residues to interact with the extended bases of
single-stranded T4G4 DNA (34). In
Oxytricha, the phosphodiester-sugar backbone that
contributes to electrostatic interaction was largely solvent-exposed.
It could be worthwhile in future work to gain more precise information on this protein-DNA interaction.
, and Est1p (28). Moreover, Cdc13p was proposed to mediate telomerase access to telomeres
(27). Thus, the alteration of telomeric DNA by Cdc13p might facilitate
the formation of a multiprotein complex on telomeres. Alternatively,
the single-stranded telomeric tail was shown to loop back to the
double-stranded region of telomeres to form a "t-loop" structure in
mammalian telomeres (44). In yeast, telomeres are folded back to form a
looped structure (45, 46). By cooperating with the double-stranded
telomeric DNA bends induced by Rap1p (47, 48), the alteration of
single-stranded telomeric DNA by Cdc13p might facilitate the formation
of such a structure in yeast.
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ACKNOWLEDGEMENTS |
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We thank the laboratory members of J-J. Lin for help. We also thank Dr. C. Wang for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by NSC Grants 88-2314-010-070, 89-2314-B-010-008, and in part by Grant 89-B-FA22-2-4 (Program for Promoting Academic Excellence of Universities).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 886-2-2826-7258;
Fax: 886-2-2820-0067; E-mail: jjlin@ym.edu.tw.
Published, JBC Papers in Press, April 17, 2001, DOI 10.1074/jbc.M101642200
1 C.-L. Hsu and J.-J. Lin, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: EMSA, electrophoretic mobility shift assay; Ni-NTA, nickel-nitrilotriacetic acid.
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