From the Department of Pharmacology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854
Received for publication, December 16, 2002, and in revised form, January 31, 2003
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ABSTRACT |
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Inactivation of the budding yeast telomere
binding protein Cdc13 results in abnormal telomeres (exposed
long G-strands) and activation of the DNA damage checkpoint. In the
current study, we show that inactivation of Cdc13p induces apoptotic
signals in yeast, as evidenced by caspase activation, increased
reactive oxygen species production, and flipping of phosphatidylserine in the cytoplasmic membrane. These apoptotic signals were suppressed in
a mitochondrial ( Cdc13p is a budding yeast telomere binding protein (1-3). Cdc13p
appears to be a multifunctional protein involved in both telomere
replication and protection (1-4). Its role in telomere replication has
been suggested based on the findings that it interacts with the
catalytic subunit of DNA polymerase Yeast apoptosis is not well understood. Bax, Bcl-2, and other
established apoptotic proteins have not been found in yeast cells.
However, expression of human BAX in yeast induces cell death (7,
8), accompanied by mitochondrial alkalinization and cytosol
acidification (9). This process can be blocked by co-expression of
human anti-apoptosis protein Bcl-2 (8). Additionally, aged mother cells
and H2O2-treated cells have shown apoptosis
markers in yeast, including flipping of phosphatidylserine (PS)1 from the inner leaflet
to the outer leaflet of the cytoplasmic membrane, DNA damage (by
TUNEL assay), and reactive oxygen
species (ROS) induction (10, 11). More recently, a
caspase-like protease Yca1p has been identified in yeast and was found
to regulate yeast cell death induced by
H2O2 treatment (12). Moreover, a broad range
mammalian caspase inhibitor Z-VAD-fmk inhibits cell death in
yeast induced by H2O2 treatment (12). In
addition, like apoptosis in mammalian cells, yeast apoptosis is
inhibited by the protein synthesis inhibitor cycloheximide (11).
In the current study, we show that inactivation of Cdc13p activates
caspase activity, increases ROS production, and induces flipping of PS
in the cytoplasmic membrane. Caspase activation and ROS induction were
shown to be MEC1-dependent and antagonized by
MRE11, suggesting the involvement of DNA damage signaling. Caspase activation appears to involve mitochondria and is linked to
cell death because a mitochondrial mutant ( Yeast Strains--
YPH cdc13-1a (MATa cdc13-1
his7 leu2-3 ura3-52 trp1-289), YPH cdc13-1 PS Flipping--
PS flipping was measured by using the ApoAlert
annexin V-EGFP apoptosis kit from Clontech
with modifications as instructed by the manufacturer. Briefly, log
phase yeast cells (5 × 106) were harvested, washed in
a sorbitol buffer (1.2 M sorbitol, 0.5 mM
MgCl2, 35 mM potassium phosphate, pH 6.8), and
digested with 5.5% glusulase (Roche Molecular Biochemicals) and 15 units/ml lyticase (Sigma) in the sorbitol buffer for 2 h at
28 °C. Cells were washed with a binding buffer (10 mM
Hepes/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM
CaCl2, 1.2 M sorbitol). Annexin V-EGFP was
added to cells according to the manufacturer's instructions and
incubated for 20 min at room temperature. Cells were washed with the
binding buffer and resuspended in 0.5 ml of binding buffer for FACS analysis.
Caspase Activity--
Caspase activity was measured by using a
CaspACE FITC-VAD-fmk in situ marker from Promega according
to the manufacturer's instructions. In brief, 5 × 106 yeast cells were stained with 100 µM
FITC-VAD-fmk at room temperature in the dark for 20 min. Cells were
then washed and resuspended with 1 × phosphate-buffered saline
buffer. FACS analysis of cells was performed with excitation of 488 nm
and emission of 525-550 nm.
ROS Measurement--
ROS was measured by adding
dihydrorhodamine 123 to log phase cells (final concentration, 25 µg/ml). Cells continued to grow in the medium for 2 h. Cells
were then subjected to FACS analysis.
Identification of Mitochondrial Proteins as Multicopy Suppressors
of cdc13-1--
In an attempt to isolate cdc13-1
suppressors, we transformed cdc13-1 with a yeast genomic
library (15), which had inserts of about 1 kb in size linked to a Gal1
promoter. Two clones that suppressed the cdc13-1 temperature
sensitivity at 34 °C were isolated. These clones were identified as
fragments of YDR333C and antisense of CYC8 (Fig.
1). We also transformed
cdc13-1 with a human HeLa cDNA library. Expression of
cDNAs in this library is also under the control of the Gal1
promoter. One clone was isolated and identified as the mitochondrial
protein MTCO3 (Fig. 1).
All three proteins, Ydr333Cp, Cyc8p, and MTCO3, are involved in
mitochondrial functions. Ydr333Cp was found to interact with the
mitochondrial ribosomal protein Mrp51p (16), which is involved in
regulating the expression of COX2 and COX3 (17),
subunits 2 and 3 of yeast mitochondrial cytochrome c
oxidase, respectively. CYC8 encodes a protein that regulates
expression of COX3 and COX6 of mitochondrial
cytochrome c oxidase (18). MTCO3 is subunit 3 of human
mitochondrial cytochrome c oxidase. As mitochondrial functions are known to affect apoptotic cell death in mammalian cells
(14, 19), these results point to the possibility that cdc13-1 cells may die of apoptosis.
Inactivation of Cdc13p Triggers Apoptotic Signals in Yeast--
To
test whether cdc13-1 cells die of apoptosis, various known
apoptosis markers were measured at the non-permissive temperature. A
caspase inhibitory substrate conjugated with FITC (FITC-VAD-fmk) was
used to monitor caspase activation in cdc13-1 cells (12). This compound can freely enter yeast cells and binds only to the activated caspase (12). The caspase activity can be measured by the
intensity of the FITC fluorescent signal. As shown in Fig. 2B, after incubation of
cdc13-1 at 37 °C overnight, about 60% of the cell
population exhibited intense FITC fluorescence, indicative of caspase
activation. Interestingly, 43.5% of cells survived the overnight
incubation at 37 °C, which was monitored by plating the same samples
at the permissive temperature (23 °C) (Fig. 2A). The
caspase inhibitor, VAD-fmk, is a broad spectrum caspase inhibitor in
mammalian cells. Yca1p, being the only identified caspase in yeast, is
known to be inhibited by VAD-fmk (12). Consequently, caspase activation
in cdc13-1 cells revealed by the use of FITC-conjugated VAD-fmk is likely because of activation of Yca1p.
Another early event of apoptosis in mammalian cells is the increased
production of ROS in mitochondria (14). Early log phase yeast cells,
wild type and cdc13-1, incubates at room temperature or
37 °C were mixed with the compound dihydrorhodamine 123, which can be oxidized by ROS to become fluorescent chromophore rhodamine 123. Flow cytometric analysis of rhodamine 123 (Fig. 2C) showed an additional peak with more fluorescent intensity in
cdc13-1 at 37 °C but not in the wild type strain at
37 °C. This result indicates that ROS was induced upon inactivation
of Cdc13p.
We also checked the flipping of PS from the inner leaflet to the outer
leaflet of the cytoplasmic membrane. PS is normally present in the
inner leaflet of the bi-layer cell membrane in budding yeast (20), as
in mammalian cells (21). During apoptosis, PS is
transferred to the outer leaflet of the cell membrane in mammalian cells by an unknown mechanism, although the cell membrane structure is intact (21). PS has high affinity to a protein, annexin V
(22, 23), and therefore can be detected by annexin V-EGFP. As shown in
Fig. 2D, PS flipping, as revealed by surface fluorescence
because of binding of annexin V-EGFP to the flipped PS in the outer
leaflet of the membrane, occurred in cdc13-1
(bottom right panel) but not in the
wild type strain (top right panel) at
37 °C. As shown in Fig. 2E, the annexin V-EGFP
fluorescence signal revealed by FACS analysis was greatly enhanced upon
inactivation of the telomere binding protein Cdc13p at 37 °C in
cdc13-1 cells (bottom right
panel) compared with that in cdc13-1 cells at
room temperature (bottom left panel)
or wild type cells at 37 °C (top right
panel).
A Mitochondrial Mutant (
To test if the MEC1 Plays an Important Role in Mediating Apoptotic Signals in
cdc13-1 Cells--
The essential protein Mec1, an ATM-like
kinase (24), is a checkpoint protein and is involved in telomere
maintenance (25-28). Its essential function can be separated from its
checkpoint and telomere functions by deletion of Sml1p, which regulates
the nucleotide pools (26, 28-30). To test whether Mec1p mediates
apoptotic signals in cdc13-1 cells, we constructed the
mec1
We also tested the role of Tel1p in telomere-initiated apoptosis
signaling. Like Mec1p, Tel1p is another yeast ATM homologue (24). Tel1p
is known to be involved in telomere maintenance and checkpoint (32,
33). We analyzed multiple isolates of the tel1 MRE11 Deletion Inhibits Apoptosis in cdc13-1 Cells--
The
MRE11/RAD50/XRS2 (MRX) complex has
been shown to function in telomere length maintenance (32) and
checkpoint activation by DNA double strand breaks (33). The MRX complex
contains exonuclease activity and has been proposed to process double
strand breaks for repair (33, 34). Deletion of any gene in the MRX
complex abolishes the function of the complex and therefore exhibits
the same phenotypes (30, 32, 35). To study the roles of the MRX complex
in cdc13-1-induced apoptosis, we constructed cdc13-1 mre11
The cdc13-1 mre11 Inactivation of Cdc13p Triggers Apoptotic Signals in Yeast--
We
have shown that inactivation of Cdc13p in cdc13-1 yeast
cells leads to caspase activation, PS flipping, and increased ROS production (Fig. 2). These three events are major landmarks of apoptosis in mammalian cells (37) and have been demonstrated to occur
in yeast (10-12). These apoptotic signals probably reflect cell death
because caspase activation and cell death appear to be correlated
(e.g. about 60% of cells were shown to undergo both caspase
activation and cell death as shown in Fig. 2, A and
B). Moreover, we demonstrated that a mitochondria-deficient
The involvement of mitochondria in telomere-initiated apoptotic
signals was also supported by suppression analysis. We have identified
three multicopy suppressors of cdc13-1. Interestingly, these
suppressors are either directly or indirectly related to mitochondrial
cytochrome c oxidase. Inhibition of mitochondrial cytochrome
c oxidase is known to trigger apoptosis in mammalian cells (38, 39). In addition, apoptosis has been shown to be associated
with reduction of MTCO3 (40). Similarly, Bax-induced apoptosis in yeast
has been shown to result in reduction of cytochrome c
oxidase (8, 41). It seems possible that overexpression of cytochrome
c oxidase may suppress apoptosis. Indeed, overexpression of
subunit 3 of the human mitochondrial cytochrome c oxidase
(MTCO3) resulted in reduced apoptotic signals in human 293T
cells.2
We have noticed that none of the suppressors or the
mitochondria-deficient strains can fully rescue cdc13-1 at
37 °C. In addition, cell death as measured by colony formation in
mec1 MEC1 Promotes Apoptosis in cdc13-1 Cells--
Both MEC1
and TEL1 are yeast homologues of the human ATM kinase (24)
and are involved in telomere maintenance (25, 26, 30). Our studies have
shown that MEC1 deletion can dramatically reduce (from 52 to
8%) caspase activation and ROS production in cdc13-1 cells
(Fig. 4, A and B), suggesting that the apoptotic signaling in cdc13-1 cells is primarily mediated by
MEC1. Surprisingly, TEL1 did not significantly
affect caspase activation in cdc13-1 cells at the
non-permissive temperature (Fig. 4, C and D).
Telomere dysfunction in human cells by inactivation of TRF2, a
mammalian telomere binding protein (43), results in
ATM-dependent apoptosis in some human cells (44). It has
been suggested that MEC1 is more related to ATR
whereas TEL1 is more related to ATM (45). It is unclear why
telomere dysfunction in yeast triggers MEC1- rather than
TEL1-dependent caspase activation. One possibility is that
cdc13-1 inactivation and TRF2 inhibition produce different biochemical effects on telomeres. It is known that Cdc13p binds preferentially to single-stranded (ss) telomeric sequences (1), whereas
TRF2 prefers to bind to double-stranded (ds) telomeric sequences at the
ds/ss junctions and promotes t-loop formation (43, 46). Moreover,
inactivation Cdc13p results in long ss G-tails, whereas inactivation of
TRF2 results in loss of G-tails in apoptotic cells (44). Another
possibility is that TEL1 plays a dual role at telomeres:
damage repairing and checkpoint signaling, which would have opposing
effects on caspase activation. Further studies are necessary to
distinguish between these possibilities. In addition, it is worth
noting that MEC1, but not TEL1, is involved in a
telomere checkpoint pathway required for senescence in yeast (27).
The MRX Complex Plays a Role in Protecting
Telomeres--
Inactivation of Cdc13p has been shown to result in
activation of the RAD9 checkpoint and arrest of cells in
G2/M phase because of the generation of extended ss G-rich
strands (1). It has been reported that the MRX complex acts upstream of
RAD9 in activating checkpoint and DNA damage repair (33).
The MRX complex also functions in telomere maintenance (32). Our
results have shown that cdc13-1 mre11
In the aggregate, our results suggest a model that is schematically
shown in Fig. 5. In this model,
inactivation of Cdc13-1p results in extensive degradation of the
telomeric C-strands and hence the exposure of the G-tails. The exposed
G-tails then activate a MEC1-dependent nuclear
mitochondrial apoptotic pathway leading to caspase activation and other
apoptotic signals. The G-tails can also trigger
RAD9-dependent DNA damage signals under less severe conditions (1, 6). The DNA damage in cdc13-1 can be
repaired by the MRX complex, presumably through homologous and/or
non-homologous recombination. Extensive degradation of the telomeres
may also lead to essential gene deletion or mitotic catastrophe in
addition to apoptosis.
Our results suggest that apoptosis or apoptosis-like cell death occurs
in yeast upon exposure of the G-tails. Although it is not clear if the
initial signal is the exposed G-tails in mammalian cell, this signal
pathway appears to be conserved: initiated by abnormal telomeres,
mediated by ATM-like kinase in the nucleus and through mitochondria in
the cytosol. At present, the functional significance of this conserved
telomere-initiated, nuclear mitochondrial apoptotic pathway is still
unclear. It is conceivable, however, that the telomeres may play an
important role in cell death regulation.
o) mutant. Moreover, mitochondrial proteins
(e.g. MTCO3) were identified as multicopy
suppressors of cdc13-1, suggesting the involvement of mitochondrial functions in telomere-initiated apoptotic signaling. These telomere-initiated apoptotic signals were also shown to depend on
MEC1, but not TEL1, and were antagonized by
MRE11. Our results are consistent with a model in which
single-stranded G-tails in the cdc13-1 mutant trigger
MEC1-dependent apoptotic signaling in yeast.
INTRODUCTION
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ABSTRACT
INTRODUCTION
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DISCUSSION
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and an essential subunit of
telomerase, Est1p (4), and a mutation form of CDC13, cdc13-2, shows the telomerase negative phenotype (3). Its
essential role in protecting yeast telomeres is presumably related to
its binding to the exposed G-strand (1-3, 5). Cdc13-1p (P371S), a
temperature-sensitive mutant form of Cdc13p, is unable to protect telomeres at the restrictive temperature (1). At the non-permissive temperature, cells with the cdc13-1 allele exhibit extensive
degradation of telomeres from the 5'-ends, the C-rich strands
(C-strands). The C-strand degradation results in long single-stranded
G-tails (up to 20 kb) at the 3'-ends. The abnormal telomeres in
cdc13-1 at the non-permissive temperature lead to
RAD9-dependent cell cycle arrest at the
G2/M phase, as well as cell death (1, 6). However, the
mechanism of cell death triggered by Cdc13p inactivation is unclear. It
was assumed that essential gene deletion because of the C-strand
degradation is the cause of the cell death.
o) significantly
suppressed caspase activation and cell death. Moreover, mitochondrial
proteins have been identified as multicopy suppressors of cell death in
cdc13-1.
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(MAT
cdc13-1 his7 leu2-3 ura3-52 trp1-289) and
the isogenic wild type (his7 leu2-3 ura3-52 trp1-289),
W13
(MAT
cdc13-1 his7 leu2-3, 112 ura3-52 trp1-289)
and its isogenic wild type strain (MAT
his7 leu2-3, 112 ura3-52 trp1-289), YPH499
tel1::HIS3, YPH499
mre11::LEU2, and YPH499
mec1::URA3
slm1::TRP1 were obtained from the laboratory
of Dr. Virginia A. Zakian (Princeton University, New Jersey). The
haploid cdc13-1 mec1::URA3
sml1::TRP1 was generated by mating YPH499
mec1::URA3
slm1::TRP1 with W13
. The haploid cdc13-1 mre11::LEU2 was generated by
mating W13
with YPH499 mre11::LEU2. The haploid strain cdc13-1 tel1::HIS3
was generated by mating YPH499 cdc13-1 or W13a with YPH500
tel1::HIS3. The W13
o
strain was generated by growing W13
in a minimum medium containing 25 µg/ml ethidium bromide to saturation. The ethidium-treated culture
was inoculated in the same medium containing ethidium and grew to
saturation again, as described (13, 14). Under these conditions,
essentially all clones will be
o. These
o clones
are unable to grow in medium containing a non-fermentable carbon source
(e.g. 2% glycerol).
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Fig. 1.
Suppression of cdc13-1
temperature sensitivity. A, suppression of
cdc13-1 temperature sensitivity by overexpression of yeast
and human cDNAs. W13 cells were transformed with a HeLa cDNA
expression library or a yeast genomic library (all DNA inserts were
under the control of the yeast Gal1 promoter for expression). Colonies
at the non-permissive temperature (34 °C) were isolated and
characterized. One human cDNA clone SC1 (MTCO3) and two yeast
genomic DNA clones SC2 (YDR333C) and SC3 (antisense of
CYC8) were identified. The plasmids carrying these clones
were purified and re-transformed into yeast cells. Wild type
(wt) or cdc13-1 cells carrying indicated clones
were 10-fold serially diluted and spotted on plates. Plates were then
incubated at 34 °C. All of these clones partially rescued the growth
of cdc13-1 at the non-permissive temperature, 34 °C.
B, identity of the suppressor clones. All cloned cDNAs
were sequenced, and the identity of these clones was determined by gene
bank data search. aa, amino acid residues.
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Fig. 2.
Inactivation of Cdc13p results in cell death
and induction of apoptotic signals. A, loss of
viability of cdc13-1 at the non-permissive temperature. Log
phase wild type (wt) and cdc13-1 cells were
incubated at 37 °C overnight. Cells were examined for both cell
death (colony formation) and caspase activation (in B). For
cell death measurement, cells were plated on YEPD plates at 23 °C.
The number of colonies was counted. Cell survival (%) was expressed as
the number of colonies divided by the number of colonies after
overnight incubation at 23 °C. B, activation of caspase
activity in cdc13-1. Wild type and cdc13-1 cells
were incubated at either 23 °C (left panels)
or 37 °C (right panels), followed by
incubation with FITC-VAD-fmk, a FITC-conjugated inhibitory
substrate of yeast caspase Yca1p. RT, room temperature.
C, induction of ROS in cdc13-1 cells. Log phase
wild type and cdc13-1 cells were incubated at either
23 °C (left panels) or 37 °C
(right panels), followed by incubation with
dihydrorhodamine 123 for 2 h. Rhodamine generated by ROS oxidation
of dihydrorhodamine 123 was then analyzed by FACS. D,
inactivation of Cdc13p induces PS flipping. Wild type and
cdc13-1 cells were incubated at either 23 or 37 °C in
YEPD medium. Log phase cells were digested by zymolyase and stained
with annexin V-EGFP. Cells were then visualized by fluorescence
(right panels) or regular light (left
panel). E, FACS analysis of annexin V-EGFP
fluorescence signals of wild type cells at 23 °C (top
left) or 37 °C (top right) and
cdc13-1 cells at 23 °C (bottom
left) or 37 °C (bottom
right).
o) Rescues cdc13-1 and
Suppresses cdc13-1-induced Caspase Activation--
Mitochondria play a
pivotal role in apoptosis in mammalian cells (19). To test the
involvement of mitochondria in cdc13-1 death, we generated a
mitochondria-deficient mutant
o in both wild type and
cdc13-1 strain backgrounds using the ethidium method (13).
Because these mitochondria-deficient cells cannot respire, they cannot
utilize non-fermentable carbon sources such as glycerol. As shown in
Fig. 3A (top
four panels),
o mutants grew in Glc
but not Gly medium at 26 °C.
o rescued cdc13-1
partially at a non-permissive temperature, 30 °C (Fig.
3A, bottom two panels).
Suppression of cdc13-1 by
o was observed up to
34 °C but not at 37 °C (data not shown).
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Fig. 3.
A mitochondrial
o mutant suppresses cell death and caspase
activation in cdc13-1. A, partial
rescue of cdc13-1 temperature sensitivity by a mitochondrial
o mutant. Log phase cells were 10-fold serially diluted and
spotted on YEP plates with 2% Gly (top panel) or
2% Glc (middle and bottom panels).
Plates were incubated at indicated temperatures. wt, wild
type. B, suppression of caspase activation in
cdc13-1 by the
o mutant. Exponentially growing
cells at room temperature in YEPD medium were shifted to 30 °C and
incubated overnight. Cells were then stained with FITC-VAD-fmk,
an FTIC-conjugated inhibitory substrate of yeast caspase Yca1p, and
analyzed by FACS. wild type, top left; wild type
o, top right; cdc13-1,
bottom left; cdc13-1
o,
bottom right.
o mutant blocks cdc13-1-induced
caspase activation, we used the same caspase assay. As shown in Fig.
3B, the
o mutant exhibited greatly reduced
activation of caspase in cdc13-1 background
(bottom panels).
sml1
cdc13-1 mutant strain (also labeled as
mec1 cdc13-1 in Fig. 4). The
mutant mec1
sml1
cdc13-1 had a slight higher
permissive temperature (about 29 °C), as previously reported (31).
However, mec1
sml1
greatly reduced caspase activation
(Fig. 4A) and ROS production (Fig. 4B) in
cdc13-1. The reduction in apoptotic signals in the triple mutant is because of mec1
but not sml1
,
because sml1
did not reduce the apoptotic signals
(caspase activation and ROS induction) in cdc13-1 (data not
shown). Both sml1
and mec1
sml1
mutants were viable and did not generate apoptotic signals (data not shown). Taken together, these results suggest that the MEC1 mediates
the apoptotic signals generated by inactivation of Cdc13p.
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Fig. 4.
Effect of MEC1/MRE11/TEL1 on
telomere-initiated apoptosis signals in cdc13-1
cells. Log phase yeast cells were incubated at 23 °C
(labeled RT at the top of the panels) or 37 °C (labeled
37°C at the top of the panels) overnight, followed
by analysis for caspase activation (labeled Caspase at the
right) and ROS production (labeled ROS at the
right). A and B, wild type
(wt), cdc13-1, and cdc13-1 mec1
strains. C and D, wild type, cdc13-1,
and cdc13-1 tel1
strains. E and F,
wild type, cdc13-1, and cdc13-1 mre11
strains.
cdc13-1
double mutant for caspase activation and ROS production. As shown in
Fig. 4, C and D, unlike the mec1
cdc13-1 double mutant, the tel1
cdc13-1 double
mutant showed slightly reduced caspase activation (C) and
ROS production (D), suggesting that Tel1p is not
significantly involved in telomere-initiated apoptotic signaling.
double mutants.
double mutant had a lower maximum
permissive temperature (26 °C) than the cdc13-1 single
mutant (28 °C) (data not shown), in agreement with a previous report
(36). The cdc13-1 mre11
double mutant died more
extensively than the cdc13-1 single mutant after overnight
incubation at 37 °C as assayed by plating at the permissive
temperature (23 °C) (about 100-fold lower survival, data not shown).
Similarly, the cdc13-1 mre11
double mutant exhibited more
extensive caspase activation (Fig. 4E, right
panel) and ROS induction (Fig. 4F,
right panel) than the cdc13-1 single
mutant. The mre11
single mutant was viable and did not
activate caspase nor induce ROS (data not shown). These results suggest
that the MRX complex may function in repairing damaged telomeres
and thereby antagonizing telomere-initiated apoptotic signals.
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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REFERENCES
o mutant suppressed both cdc13-1-induced caspase
activation (Fig. 3B) and death of cdc13-1 cells
at the non-permissive temperature (Fig. 3A).
sml1
cdc13-1 cells was almost the same as in
cdc13-1 cells, although the apoptotic signals were
essentially abolished in mec1
sml1
cdc13-1 cells (Fig.
4, A and B). However, the cell number in
mec1
sml1
cdc13-1 did increase 3-5-fold compared with
cdc13-1 after overnight incubation at 37 °C in agreement
with a previous report (42). These results suggest that in addition to
apoptosis, cdc13-1 cells may die through another cell death
pathway(s). It is conceivable that essential gene deletion because of
extensive degradation from the telomere ends or mitotic
catastrophe because of MEC1 DNA damage checkpoint defect may
result in cell death by a pathway distinct from apoptosis in
cdc13-1 cells.
double mutants died
more extensively than the cdc13-1 single mutant and
exhibited more extensive caspase activation and ROS production (Fig. 4,
E and F), suggesting that the MRX complex
antagonizes the telomere-initiated apoptotic signals. The simplest
interpretation of this result is that the MRX complex may terminate the
telomere-initiated apoptotic signaling by promoting homologous
recombination or non-homologous end joining at the de-protected telomeres.
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Fig. 5.
A proposed model for apoptotic signaling
induced by Cdc13p inactivation. Inactivation of Cdc13p at the
non-permissive temperature results in extensive degradation of the
C-strands, followed by exposure of the single-stranded G strands
(G-tails). G-tails then trigger MEC1-dependent
nuclear mitochondrial signaling leading to caspase activation and other
apoptotic signals.
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ACKNOWLEDGEMENTS |
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We are grateful to Drs. Virginia A. Zakian, Andrew K. P. Taggart, and Yasumasa Tsukamoto and to Lara Goudsouzian for providing various yeast strains and helpful discussion.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants GM27731 and CA39662.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: Dept. of Pharmacology,
UMDNJ-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ
08854. Tel.: 732-235-5483; Fax: 732-235-4073; E-mail: qiha@umdnj.edu.
Published, JBC Papers in Press, February 3, 2003, DOI 10.1074/jbc.M212808200
2 T-K. Li, H. Qi, and L. F. Liu, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: PS, phosphatidylserine; ROS, reactive oxygen species; FACS, fluorescence-activated cell sorter; FITC, fluorescein isothiocyanate; ss, single-stranded; ds, double-stranded; ATM, ataxia-telangiectasia mutated gene; ATR, ataxia-telangiectasia and Rad3-related; VAD-fmk, valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone.
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