1 Center for Molecular Oncology, University of Chicago, Chicago IL 60637,
USA
2 Department of Molecular Genetics and Cell Biology, University of Chicago,
Chicago IL 60637, USA
Author for correspondence (e-mail: skron{at}midway.uchicago.edu )
Accepted 27 January 2002
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Summary |
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Key words: DNA damage checkpoint, DNA repair, G1 phase, Sacchromyces cerevisiae, Genetics, Metabolism, Gamma radiation, Cell cycle, RAD9
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Introduction |
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Recent data suggest that DNA damage is processed by endonucleases to reveal
single strands (Garvik et al.,
1995; Lee et al.,
1998
; Lydall and Weinert,
1995
). These strands are then detected by proteins implicated both
in repair and signaling, such as the tumor suppressors BRCA1 and Nibrin
(Scully et al., 1997
;
Zhong et al., 1999
). Complexes
of these and other factors that form upon damage induction are thought to mark
the sites of damage. The phosphoinositol-3 kinase homologues ATM and ATR, once
recruited to these foci, phosphorylate and upregulate downstream effectors,
including the dual-specificity kinase CHK2
(Cortez et al., 1999
;
Matsuoka et al., 2000
;
Tibbetts et al., 2000
).
Subsequent phosphorylation of the unstable transcription factor p53 promotes
its accumulation (Chehab et al.,
2000
; Hirao et al.,
2000
), leading to expression of the p21CIP1 CDK
inhibitor, preventing onset of replication
(Li et al., 1994
;
Waldman et al., 1995
). Cells
lacking ATM, p53 or p21 progress into S phase unabated by DNA damage.
The correspondence between bud morphology and cell cycle progression in
S. cerevisiae (Lew et al.,
1997) has made yeast a powerful system for genetic analysis of the
cell cycle (Nasmyth, 1996
) and
DNA damage checkpoints (Weinert,
1998b
). Passage through Start occurs before bud emergence, when
cells become irreversibly committed to cell division. Exit from G1 is also
shown by activation of the yeast C1b5,6-Cdc28 CDK complex, spindle pole body
duplication, onset of DNA synthesis and loss of sensitivity to the yeast
mating pheromone, alpha factor (
f). Subsequent bud growth continues
through S phase and mitosis. Return to G1 is marked by cytokinesis and bud
separation. Classic studies of DNA damage checkpoint arrest in S.
cerevisiae revealed that irradiated yeast cells accumulate with a
large-budded morphology (Weinert and
Hartwell, 1988
). This S phase or G2/M arrest depends on DNA damage
sensors that activate the ATM homologue MEC1 and the CHK2 homologue
RAD53 (Longhese et al.,
1998
; Weinert,
1998a
). Like other eukaryotes, yeast process DNA damage to reveal
single strands (Garvik et al.,
1995
) that are then bound by the RPA protein Rfa1p to initiate the
DNA damage signal (Pellicioli et al.,
2001
). The RFC homologue RAD24 and a PCNA-like complex
composed of MEC3, RAD17 and DDC1
(Kondo et al., 1999
) may
recognize these lesions (Durocher and
Jackson, 2001
). This RAD17 epistasis group is required
for all known DNA damage checkpoints in budding yeast. DNA damage in S phase
activates a checkpoint dependent upon the Pol
POL2 that
mediates delays in fork progression and origin firing. RAD9, though
required for the G2/M checkpoint, has not been shown to function in the DNA
replication checkpoint. The RAD17 pathway, in concert with
RAD9 or POL
(de la
Torre-Ruiz et al., 1998
; Navas
et al., 1996
), may recruit Mec1p, promoting its phosphorylation of
Rad53p (Sanchez et al.,
1996
).
En route to the large-budded arrest, yeast perform a RAD17- and
RAD9-dependent delay in bud emergence and DNA replication after
X-ray-, UV- and MMS-induced DNA damage (de
la Torre-Ruiz et al., 1998;
Siede et al., 1994
;
Siede et al., 1993
).
Stoichiometric inhibitors of Cdc28 exist in yeast; however there are no
sequence homologues to p53 or p21. Instead, MEC1-dependent activation
of Rad53p in G1 (Pellicioli et al.,
1999
) delays expression of G1 cyclins to slow activation of Cdc28p
(Sidorova and Breeden, 1997
).
Nonetheless, the existence of a bona fide G1 arrest in response to DNA damage
has recently come into question (Neecke et
al., 1999
). Neecke and colleagues observed that UV irradiation in
G1 induced a prolonged arrest in mutants lacking the excision repair gene
RAD14. However, although these rad14
cells maintain a
1N DNA content, mimicking a G1 arrest, they have entered S phase in the
presence of DNA damage as demonstrated by activation of early origins. This
arrest is RAD9 independent, consistent with an S-phase delay.
Progression into the cell cycle in the face of unrepaired DNA damage throws
doubt on the significance or existence of even the previously described
transient G1 checkpoint delay in budding yeast.
Using ionizing radiation, we confirmed that DNA damage in G1 delays both
bud emergence and spindle pole body duplication and extends the window of
sensitivity to f. This G1 arrest is dose dependent and requires
RAD9 but not POL
. This arrest is not transient.
Induced by lethal
or UV irradiation, the RAD9- and
RAD17-dependent G1 checkpoint arrest can be maintained over 18 hours.
In turn, at tolerable levels of
irradiation, haploid yeast
preferentially arrest in G1 without loss of viability. This G1 arrest after
irradiation is prolonged by defects in DSB (double-strand break)
repair. Surprisingly, rad14 mutants arrest in G1 after
irradiation but demonstrate a G1 checkpoint defect after UV. Since UV does not
directly produce breaks, failure to excise crosslinks may effectively mask DNA
damage until replication through the lesion creates a single strand and
initiates a DNA damage signal. This model reconciles the apparent conflicts in
previous studies of S. cerevisiae G1 DNA damage checkpoint
response.
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Materials and Methods |
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Biochemistry
Protein was extracted by glass bead disruption in PBS pH 7.4, 5% glycerol,
0.5% Nonidet P40, 1 mM PMSF and protease inhibitor cocktail (Boehringer). For
western analysis, 50 µg total protein was separated on 8% SDS-PAGE,
transferred to nitrocellulose (Osmonics), probed with monoclonal -Myc
epitope antibody (9E10, Babco) and horseradish-peroxidase-conjugated sheep
-mouse secondary antibody (Amersham) and developed with Pierce
SuperSignal using Hyperfilm ECL (Amersham). p13suc1-(CalBiochem)
associated histone H1 kinase assay was carried out as described
(Amon et al., 1992
), using 50
µg protein/assay.
f/nocodazole trap assay
5 ml of saturated overnight culture was diluted to 107
cells/ml in 50 ml YPD with
f. Cells were incubated (
3 hours) at
30°C until >95% displayed mating projections. 10 ml aliquots were
transferred to culture tubes, equilibrated on ice and irradiated with 0, 100,
200 or 400 Gy in the 60Co irradiator.
f was removed by
vacuum filtration with 100 ml water. Cells on filters were transferred to
culture tubes and resuspended in 22°C YPD by vortexing. At 10 minute
intervals, 0.5 ml aliquots were combined with 0.5 ml trapping media (15
µg/ml nocodazole, 15 µM
f in YPD), incubated for 90 minutes at
22°C then prepared for flow cytometry. A 2D gate (SSC versus FL2-A) on the
basis of
f-arrested controls was used to determine the percentage of
cells with 1N DNA content in test samples. 50,000 cells were analyzed from
each sample.
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Results |
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S. cerevisiae maintains a 1N DNA content after
irradiation
After release from f, yeast enter S phase within 45 minutes and
complete DNA synthesis by 90 minutes as monitored by the rise in DNA content
(Fig. 2A). Prior exposure to
irradiation results in a dose-dependent delay. Cells irradiated with
200 Gy (
10% lethality) begin to increase DNA content 30 minutes after
unirradiated controls. After 1000 Gy (
70 DSB/haploid genome,
95%
lethality), cells remain 1N for over 2 hours
(Fig. 2A).
|
These data are consistent with a regulated cell cycle delay before
replication. An alternative hypothesis is that loss of viability and/or DNA
damage itself may prevent timely replication. Nonetheless, both unirradiated
and irradiated (1000 Gy; >99% lethality in checkpoint deficient strains)
rad17 (Fig.
2B), rad24
, rad17
rad24
rad9
triple mutants and mecl
(not shown) mutants enter S phase
within 30 minutes and continue replication with similar kinetics. This
indicates that replication is possible, yet prevented by checkpoint
activation. In contrast, cells only lacking RAD9 remain 1N for over 2
hours after 1000 Gy irradiation (Fig.
1A). Since rad9
cells, unlike
rad17
or rad24
, are proficient for the
intra-S-phase checkpoint (Navas et al.,
1996
), the observed 1N delays cannot distinguish G1 arrest from
stalled replication (Neecke et al.,
1999
).
In light of this result, it is of note that although increased
irradiation dosage delays cell cycle progression in the early stages of the
cell cycle, this does not correlate with rapid progression through mitosis. If
G1/S DNA damage checkpoints allowed for completion of DNA repair, then it
might be expected that cells would not delay in a subsequent mitosis.
Unirradiated cells reach a 2N DNA content at 120 minutes after release from
f (Fig. 2A). A return to
1N DNA content is observed at 150 minutes, and the majority of cells are 1N at
180 minutes. Mitosis, defined in this assay as the length of time spent at 2N,
lasted 1 hour in unirradiated cells. After 200 Gy, which delayed replication
by 30 minutes, cells remained at 2N until 240 minutes after
f release.
In other experiments, this 2N delay after 200 Gy was found to persist for at
least 5 hours (data not shown). Similarly, no return to 1N DNA content was
observed at higher
irradiation doses. This again could argue for a
lack of G1 DNA damage checkpoint regulation or for an adaptive response to
irradiation in G1.
Early G1/S events are delayed after DNA damage
Although progression and completion of DNA synthesis are subject to
intra-S-phase checkpoint regulation
(Paulovich and Hartwell,
1995), completion of other Start events remains unaffected by
stalled replication (e.g. hydroxyurea treatment). We examined bud formation
and spindle pole body duplication (SPD) after G1 irradiation in cells carrying
fluorescently marked spindle poles via expression of a SPC42-GFP gene
fusion (Schutz and Winey,
1998
) (Fig. 3). In
unirradiated cells, DNA synthesis, as measured by flow cytometry, is >50%
complete by 20 minutes. Bud emergence is
50% complete by 40 minutes and
SPD is
50% complete by 75 minutes. After 200 Gy, 50% budding is only
reached after 60 minutes, whereas SPD is 50% complete at
120 minutes
after release. These delays increase with dose increases. After 400 Gy and 800
Gy, 50% budding is reached at 90 minutes, whereas >25% SPD is not reached
during the 120 minute time course.
|
We also assayed the appearance of active CDK as an independent marker for
passage through Start. We used the RAD53-13Myc-expressing strain to
simultaneously report DNA damage signaling
(Fig. 4A,B). After release from
f, Cdc28 kinase activity is detected at 30 minutes in unirradiated
wild-type cells, and Rad53-13Myc remains unshifted
(Fig. 4A). However, 200 Gy
delayed appearance of Cdc28p kinase activity to
45 minutes by which time
Rad53-13Myc had returned to its unshifted mobility. 400 Gy induced a
persistent shift of Rad53-13Myc and Cdc28 kinase activity did not appear until
60 minutes. For the rad9
rad17
RAD53-13Myc double
mutant, 400 Gy did not delay Cdc28 kinase activity nor shift Rad53-13Myc
(Fig. 4B).
|
Delayed budding, SPB duplication and Cdc28 activity all suggest G1 arrest,
but they are not positive indicators. By contrast, yeast cells are only
responsive to mating factor in G1. After release from f, cells remain
sensitive to re-arrest by
f for less than 30 minutes. After that, they
pass Start and become
f resistant. We tested whether DNA damage in G1
can extend this window of
f sensitivity. During the time course in
Fig. 4A,B, additional aliquots
were fixed for flow cytometry. Irradiated cells appeared to delay replication
after release from
f (Fig.
4C). To determine whether these cells remained in G1 or had
entered S phase, aliquots were incubated for 90 minutes in
f and
nocodazole (Fig. 4D). The
subsequent addition of
f arrested G1 cells with a 1N DNA content,
whereas nocodazole trapped cells that had passed Start
(Siede et al., 1996
).
Unirradiated RAD53-13Myc cells are no longer arrested by
f at
30 minutes and accumulate with 2N DNA content. 30 minutes after 200 Gy,
f continues to arrest >80% of cells. After 400 Gy,
60% of cells
can be arrested by
f at 45 minutes. However, in the rad9
rad17
RAD53-13Myc double mutant irradiated with 400 Gy, cells lost
f resistance by 45 minutes. Nonetheless, all cells were capable of
increasing DNA content. When incubated in nocodazole alone at the 0 minutes
timepoint, both the irradiated and unirradiated cells accumulate at 2N by 90
minutes (Fig. 4D, top
histograms).
Genetic characterization of -irradiation-induced G1
arrest
We used a quantitative version of this f/nocodazole trap assay to
examine genetic regulation of the G1 arrest. Unirradiated wild-type,
rad9
and rad17
cells rapidly lose
f
sensitivity, as less than 50% of cells remain in G1 20 minutes after release
(Fig. 5). Wild-type cells
display a
radiation dose-dependent delay in exit from G1
(Fig. 5A), whereas 50% of
rad9
(Fig. 5B)
and rad17
(Fig.
5C) mutants remain in G1 at 20 minutes regardless of the dose.
Mutants lacking RAD52 are defective in both recombinational and
end-joining DSB repair (Hegde and Klein,
2000
). Treated with only 50 Gy, rad52
mutants
maintain a 50% G1 population 18 minutes longer than unirradiated cells, which
is proportional to the 200 Gy delay in repair-proficient wild-type cells
(Fig. 5D).
|
The previous experiments establish a G1 arrest but can not rule out the
possibility that replication checkpoints play a role in the observed delays.
POL detects DNA damage encountered by the replication fork and
is required for the intra-S phase checkpoint
(Navas et al., 1995
). The
f/nocodazole trap assay demonstrates that checkpoint defective
pol
-12::kanR cells irradiated with 200 Gy remain
sensitive to
f at 50 minutes after release, whereas unirradiated
controls and irradiated or unirradiated rad9
pol
-12::kanR double mutants become
f resistant
after 20 minutes (Fig. 6).
|
The G1 checkpoint in continuous irradiation
The previous results used radiation doses that induce 10-100 DSB
per haploid yeast genome in 2-20 minutes, inducing 10-95% lethality
(Frankenberg-Schwager and Frankenberg,
1990
). At these high doses, checkpoint responses might be
complicated by stress and survival responses. To eliminate these effects and
assess checkpoint regulation at a tolerable radiation dose, we incubated cells
in rich media at 22°C in a 137Cs irradiator that induces
0.25 DSB/minute. Wild-type log-phase cells irradiated for 4 hours delay
cell division but remain viable as measured by plating efficiency
(Fig. 7A,B). rad9
rad17
double mutants show only slight delays in growth and rapidly
lose viability. Flow cytometry of the irradiated wild-type cells revealed that
the 1N population of cells steadily increases for 1 hour
(Fig. 7C). In turn, the
proportion of cells that have passed Start and display small to medium sized
buds decreases from 33% to 12% within 30 minutes. No G1 shift is observed in
the irradiated rad9
rad17
cells.
|
To test if this tolerable radiation maintains G1, f arrested cells
were released and incubated in the 137Cs irradiator for 90 minutes.
Without irradiation, cells enter the cell cycle promptly and complete
replication by 60 minutes (Fig.
8A). Irradiated cells remained unbudded, with 1N DNA content even
after 90 minutes. However, once removed from the irradiator, these cells began
replication without delay (Fig.
8B). Confirming a G1 arrest,
f restricted 57% of these
cells from completing S phase (Fig.
8C). Despite being arrested in G1, the irradiated cells do not
lose viability (not shown). This G1 arrest was also observed when the
synchronized G1 population was obtained through elutriation (not shown).
|
These data also provide a comparison with the results in
Fig. 2. 90 minutes in the
137Cs source results in 450 Gy accumulated irradiation. In
Fig. 2A, it was found that
cells receiving a similar dose, delivered in about 7 minutes, maintained a 1N
DNA content 60 minutes longer than unirradiated controls (400 Gy panel). In
contrast, cells removed from the 137Cs source increased DNA content
without delay as though no accumulated damage was retained. This argues for
ongoing repair in G1 during the 137Cs treatment, although these
results do not exclude the possibility that less strand breaks occur in the
low-level treatment.
G1 arrest can be maintained for over 18 hours
The G1 delay and replication checkpoints are described as transient delays
before a persistent G2/M arrest. Asynchronous wild-type cells in liquid media
treated with 3000 Gy, the minimum dose for 0% viability, were plated at low
dilution onto YPD to monitor bud emergence and division of individual cells
(Fig. 9A). Of the 38±5%
of unbudded cells at 0 hours, 23±4% remained unbudded at 18 hours. In a
rad9 rad17
double mutant, the unbudded cells dropped
from 43±4% to 9±3% after 18 hours. This loss was reflected in
48±3% of cells undergoing second and third divisions. Alternatively,
cells were first plated then exposed to 100 J/m2 UV irradiation, a
lethal dose. Here, the unbudded population in wild-type cells only dropped
from 45±7% to 42±2% after 18 hours
(Fig. 9B), whereas the unbudded
population in rad9
rad17
cells dropped from
44±3% to 21±7%. Even after 18 hours unbudded cells were both
alive and remained in G1 (Schrick et al.,
1997
). Over 30% of wild-type cells from the above experiments
formed mating projections after 3.5 hours incubation in
f after either
(Fig. 10A) or UV
irradiation (Fig. 10B).
|
|
UV radiation of a strain lacking the RAD14 excision repair gene
resulted in a drop in unbudded cells from 42±4% to 25±6% at 18
hours (Fig. 9B). Unlike
rad9 rad17
, these cells accumulated with large buds,
suggesting that the G1 checkpoint alone is compromised in the
rad14
background. By contrast,
irradiation of the
rad14
strain yielded a G1 arrest indistinguishable from wild
type (Fig. 9A).
The 50 minutes required to deliver 3000 Gy, compared with <10 seconds for 100 J/m2, may underlie the apparent superiority of UV irradiation in maintaining unbudded arrest. When wild-type cells were incubated for 20 minutes on plates before UV treatment (Fig. 9C), 53±5% of the cells remained unbudded immediately after irradiation. However, only 26±5% remained unbudded after 18 hours.
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Discussion |
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The recruitment of Rfa1p to damage foci and transient activation of Rad53p
demonstrate that G1 haploid yeast detect strand breaks and signal their
presence. We then used several criteria to determine the phase of the ensuing
arrest. Spindle pole duplication, bud emergence and activation of Cdc28 are
all delayed in a dose-dependent manner, which strongly argues for prolonged
G1. Finally, the f/nocodazole trap assay quantitatively determined the
proportion of G1 cells during a DNA damage response. We demonstrated that
wild-type cells remain
f sensitive after
irradiation and that
prolonged
f sensitivity absolutely depends on checkpoint signaling.
RAD17, RAD24 or MEC1 mutants become rapidly
f
resistant irrespective of the dose. Distinguishing this checkpoint from the
intra-S-phase delay, mutation of RAD9 completely abrogates G1 arrest.
In turn, G1 arrest is unaffected by a POL
mutation that voids
the replication checkpoint.
A key question is whether there is a link between repair and recovery from the G1 checkpoint. Clearly, the delay is proportional not only to the amount of damage but also to the extent of repair. In haploid cells, G1 arrest is prolonged by mutating RAD52 or YKU70, genes involved in non-homologous end-joining repair. Strikingly, diploid yeast do not arrest in G1 after 400 Gy, unless they lack recombinational repair genes RAD50 or RAD57 (J.N.F.G. and S.J.K., unpublished). Increased delay in DSB repair mutants suggests that the G1 arrest allows significant repair and prevents a potentially deleterious S phase and mitosis. However, it is not certain whether the G1 cell is capable of complete repair before passing Start or even whether cells in G1 are responsive only to certain types of lesions.
After 200 Gy, the phosphorylated form of Rad53p is lost as cells pass
Start. At 400 Gy, Start occurred even in the presence of phosphorylated
Rad53p. One interpretation is that competing pathways may promote recovery and
cell cycle progression. Cells may wait for repair to be completed but adapt
and proceed if repair is not possible. Alternatively, G1 cells may repair
lesions that would be deleterious to replication yet pass Start if the
remaining damage is better suited for replicative repair or homologous
recombinational repair. Indeed, it was found that a 200 Gy dose of
irradiation arrests cells in G1 for 15 minutes, but these same cells progress
to G2/M and remain with a 2N DNA content for several hours after unirradiated
cells have divided and returned to G1. This is consistent with unrepaired
lesions remaining after the G1 checkpoint.
Evidence that the G1 arrest does facilitate timely repair and therefore
protects viability was found when cells were incubated in a tolerable
low-level source. In these experiments, wild-type cells accumulate in
G1 and are able to survive a 3 hour exposure. Checkpoint mutants fail to
arrest and rapidly lose viability. Importantly, wild-type G1 cells released
from the 137Cs source initiate and complete replication as rapidly
as unirradiated cells. If ongoing repair had not occurred in G1, damage would
be expected to accumulate, leading to low viability and delayed replication,
as was found when similar doses were delivered in the 60Co source.
In fact, S phase only delayed when the G1 checkpoint was compromised in
rad9
mutants.
The duration of - and UV-induced arrest differs in repair-deficient
backgrounds. UV irradiation was clearly capable of inducing a G1 arrest, but
this was compromised in the absence of RAD14. Yet, in response to
irradiation, rad14
strains arrest in G1, as do
mismatch repair mutants (not shown). This argues that UV lesions, unlike
-radiation-induced DSBs, may require DNA metabolism to promote
checkpoint signaling. The excision activity of Rad14p may be a limiting factor
for the conversion of thymidine dimers and other UV damage to the
single-stranded gaps that activate checkpoint proteins. In cells lacking
Rad14p, Pol
-regulated stalling of the replication fork provides an
alternate, Rad9p-independent checkpoint mechanism. This is a very attractive
answer to the paradoxical results of Neecke et al.
(Neecke et al., 1999
).
In conclusion, our results substantiate an authentic G1 checkpoint arrest
in budding yeast that is dependent on the amount of DNA damage and the extent
of repair. This arrest is physiological, depends on checkpoint signaling
pathways and appears to be a primary response to low-level, tolerable
radiation and is a terminal arrest point after lethal UV or
irradiation.
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Acknowledgments |
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