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MOLECULAR AND CELLULAR BIOLOGY, Oct. 2007, p. 7007–7017 Vol. 27, No. 20
0270-7306/07/$08.00⫹0 doi:10.1128/MCB.00290-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
TOR Signaling Is a Determinant of Cell Survival in Response to
DNA Damage
䌤
†
Changxian Shen, Cynthia S. Lancaster, Bin Shi,‡ Hong Guo,
Padma Thimmaiah, and Mary-Ann Bjornsti*
Department of Molecular Pharmacology, St. Jude Children’s Research Hospital, 332 N. Lauderdale, Memphis, Tennessee 38105
Received 16 February 2007/Returned for modification 1 April 2007/Accepted 31 July 2007
The conserved TOR (target ofrapamycin) kinase is part of a TORC1 complex that regulates cellular
responses to environmental stress, such as amino acid starvation and hypoxia. Dysregulation of Akt-TOR
signaling has also been linked to the genesis of cancer, and thus, this pathway presents potential targets for
cancer chemotherapeutics. Here we report that rapamycin-sensitive TORC1 signaling is required for the
S-phase progression and viability of yeast cells in response to genotoxic stress. In the presence of the
DNA-damaging agent methyl methanesulfonate (MMS), TOR-dependent cell survival required a functional
S-phase checkpoint. Rapamycin inhibition of TORC1 signaling suppressed the Rad53 checkpoint-mediated
induction of ribonucleotide reductase subunits Rnr1 and Rnr3, thereby abrogating MMS-induced mutagenesis
and enhancing cell lethality. Moreover, cells deleted for RNR3 were hypersensitive to rapamycin plus MMS,
providing the first demonstration that Rnr3 contributes to the survival of cells exposed to DNA damage. Our
findings support a model whereby TORC1 acts as a survival pathway in response to genotoxic stress by
maintaining the deoxynucleoside triphosphate pools necessary for error-prone translesion DNA polymerases.
Thus, TOR-dependent cell survival in response to DNA-damaging agents coincides with increased mutation
rates, which may contribute to the acquisition of chemotherapeutic drug resistance.
TOR (target ofrapamycin) is a phosphatidylinositol 3-ki-
nase-related kinase family member that regulates cellular re-
sponses to wide-ranging environmental stresses, including nu-
trient starvation, growth factor deprivation, and hypoxia (8, 20,
45). These diverse environmental cues are transmitted by mul-
tiprotein TOR complexes through a variety of downstream
pathways to regulate cap-dependent mRNA translation, tran-
scriptional stress responses, cell cycle progression from G
1
to S
phase, and cell survival. Rapamycin (RAP) is a macrocyclic
lactone antibiotic that, in complex with FKBP12 (the Saccha-
romyces cerevisiae homolog is Fpr1), specifically targets TOR.
Since dysregulation of Akt-TOR signaling has been associated
with tumorigenesis (7, 8), this pathway provides potential tar-
gets for cancer chemotherapy, as evidenced by the develop-
ment of RAP analogs in clinical trials. Yet despite intense
investigation of RAP action and the phenotypic consequences
of TOR inhibition, the mechanistic basis for the antitumor
activity of RAP in preclinical and clinical studies remains un-
clear.
The TOR kinase was initially identified in the yeast S. cer-
evisiae, in a genetic screen for mutants conferring resistance to
RAP. S. cerevisiae carries two closely related Tor1 and Tor2
kinases, while other eukaryotic genomes encode a single ki-
nase, typified by mammalian mTOR (45). As in mammalian
cells, TOR signaling in yeast regulates cell growth through the
function of distinct multiprotein complexes (29, 39, 45, 46). In
yeast, Tor1 or Tor2 participates in the formation of a RAP-
sensitive TOR complex 1 (TORC1) that consists of Kog1, Lst8,
and Tco89. Mammalian TORC1 consists of mTOR, raptor (an
ortholog of Kog1), and mLst8. Under favorable environmental
conditions, TORC1 regulates the accumulation of cell mass, in
part by controlling the translation initiation of a limited subset
of capped mRNAs, nutrient uptake, and ribosome biogenesis.
RAP treatment or depriving cells of nutrients or essential
growth factors induces a starvation response characterized by
decreased protein synthesis, macroautophagy, and the induc-
tion of stress response transcription factors (45).
A second multiprotein complex, TORC2, is RAP “insensi-
tive” and provides an essential function in regulating actin
cytoskeletal organization during cell growth (45). Yeast
TORC2 composition is restricted to Tor2 in association with
other conserved proteins, including Avo3 (rictor in mammalian
cells). TORC2 also functions in endocytosis and in regulating
calcineurin and sphingolipid signaling (34, 42, 45), while recent
studies suggest the functional interplay of TORC1 and mem-
brane trafficking (3).
The phosphatidylinositol 3-kinase-related kinase family
members ATM, ATR, and DNA-PK and the yeast Mec1 and
Tel1 kinases play important roles in DNA repair and check-
point responses to DNA damage (1). Although a direct role for
TOR signaling in S phase has not been defined, several obser-
vations prompted us to investigate a requirement for TORC1
function in response to DNA replication stress. First, when
p53
⫺/⫺
mouse embryo fibroblasts or mutant p53 cancer cells
are cultured under serum-free conditions, RAP treatment in-
duces apoptosis, which coincides with entry into S phase (22,
* Corresponding author. Mailing address: Department of Molecular
Pharmacology, St. Jude Children’s Research Hospital, 332 N. Lauder-
dale, Memphis, TN 38105. Phone: (901) 495-2315. Fax: (901) 495-4290.
E-mail: mary-ann.bjornsti@stjude.org.
† Supplemental material for this article may be found at http://mcb
.asm.org/.
‡ Present address: Department of Molecular and Cellular Oncology,
The University of Texas M. D. Anderson Cancer Center, 1515 Hol-
combe Boulevard, Houston, TX 77030.
䌤
Published ahead of print on 13 August 2007.
7007
23). Second, the inhibition of mTOR signaling by RAP en-
hances the cytotoxic activity of the DNA-damaging agent cis-
platin (5, 41). However, the underlying mechanisms affecting
cell survival in S phase remain unclear. Third, we previously
reported the isolation of conditional yeast mutants exhibiting
enhanced sensitivity to the DNA topoisomerase I (Top1) poi-
son camptothecin (17, 25, 37). Several of these mutants, in-
cluding mutants in hypomorphic alleles of CDC45 and DPB11,
exhibit alterations in DNA replication, which exacerbate the
S-phase-dependent toxicity of camptothecin. Surprisingly,
many of these mutants are also hypersensitive to RAP (see
Table S1 in the supplemental material).
The TOR pathway regulates cellular responses to a variety
of environmental stresses; however, these considerations fur-
ther implicate TOR signaling as a determinant of cell survival
in response to aberrations in DNA replication. To directly
address this hypothesis, we examined the effects of RAP inhi-
bition of TORC1 on S-phase progression and the viability of
synchronized cells in response to genotoxic stress. Here we
report that TORC1 signaling is required for replication fork
progression and to maintain the elevated levels of ribonucle-
otide reductase (RNR) subunits Rnr1 and Rnr3 induced by
Rad53 checkpoint activation, which contribute to cell survival
in response to DNA damage. The RAP-induced decrease in
Rnr1/3 suppressed the mutagenic activity of methyl methane-
sulfonate (MMS). These findings establish TORC1 as a sur-
vival pathway in response to genotoxic stress and provide a
mechanistic basis for the antitumor activity of RAP analogs
used in combination with cytotoxic agents in clinical studies.
MATERIALS AND METHODS
Chemicals, yeast strains, and media. Hydroxyurea (HU) was purchased from
U.S. Biological (Swampscott, MA). RAP, obtained from the NCI drug reposi-
tory, was dissolved in dimethyl sulfoxide, and stock solutions of 1 mg/ml were
stored at ⫺20°C. The mating pheromone ␣-factor, from Diagnostic Chemicals
Ltd. (Oxford, CT), was stored at ⫺20°C at 1 mg/ml in methanol and used at a
final concentration of 5 g/ml. MMS, cycloheximide (CHX), and canavanine
were purchased from Sigma (St. Louis, MO).
S. cerevisiae strains, cultured under standard conditions, were derived from
strain FY251 (MATaura3-52 his3⌬200 leu2⌬1 trp1⌬63) and carried TRP1,
GAL1-RNR1-3HA,RNR1-3HA,RNR2-3HA,RNR3-3HA,RNR4-3HA,HTA2-
3HA,TOR1
RR
(TOR1S
1972
R), TOR2
RR
(TOR2S
1975
R), mrc1⌬,rad9⌬,rnr3⌬,
tof1⌬,rfx1⌬,tor1⌬, and sml1⌬or a combination thereof. Gene disruptions and
hemagglutinin (HA) tagging were accomplished with PCR-amplified selectable
markers and confirmed by PCR, using primers that flank the sites of integration.
TOR1
RR
and TOR2
RR
mutant alleles were obtained by PCR-based mutagenesis
and selection on RAP and were confirmed by DNA sequencing. In GAL1-RNR1-
3HA cells, the GAL1 promoter was 100 bp upstream of the RNR1 start codon.
YCp-T-RAD53-HA was kindly provided by K. Sugimoto (University of Medicine
and Dentistry of New Jersey).
Cell cycle analysis and viability assays. MATacells, ␣-factor arrested in G
1
phase of the cell cycle or arrested in early S phase with 10 mg/ml HU, were
washed by filtration and released into medium alone containing 200 ng/ml RAP,
0.05% MMS, 0.05% MMS plus 200 ng/ml RAP (MMS⫹RAP), 100 g/ml CHX,
or 100 g/ml CHX plus 0.05% MMS (CHX⫹MMS). In experiments with tor1⌬
strains, a lower concentration of RAP (50 ng/ml) was also used. At the times
indicated, aliquots of cells were washed by centrifugation to remove the drugs,
serially 10-fold diluted, and plated on yeast-peptone-dextrose (YPD) agar plates
to assess cell viability or on synthetic medium without Arg and with canavanine
to assess the frequency of canavanine resistance among surviving colonies. Ali-
quots of cells were also fixed with 70% ethanol and stored at 4°C for subsequent
fluorescence-activated cell sorting (FACS) analysis or microscopy. For DAPI
(4⬘,6⬘-diamidino-2-phenylindole) staining of DNA, 3 to 5 l of cells was spotted
on polylysine-coated Teflon slides, followed by 5 lof1g/ml DAPI and 2 l
Prolong antifade solution (Molecular Probes, Inc.). Cells were viewed with a
Zeiss Axioskop 2 microscope equipped with differential interference contrast
(DIC) and epifluorescence, and images were acquired with a Micromax
charge-coupled device camera (Princeton Instruments, Inc.) and IP lab soft-
ware (Scanalytics).
Western and Northern blot analyses. To assess epitope-tagged protein levels,
NaOH-trichloroacetic acid (TCA) cell extracts were prepared following release
from ␣-factor or HU as described previously (25). HA-tagged proteins were
detected by immunoblotting with a monoclonal HA antibody (12CA5; Roche,
Indianapolis, IN) and by chemiluminescence (Pierce, Rockford, IL). RNAs,
purified using a Ribopure-yeast kit (Ambion, Austin, TX), were subjected to
Northern blot analysis using a NorthernMax-Gly kit (Ambion) and gene-specific
probes PCR amplified from yeast genomic DNA and radiolabeled by random
priming using a DECAprime II kit (Ambion). Bands were visualized by phos-
phorimage analysis.
2-D gel analysis of replication intermediates. Replication intermediates were
purified 10 min, 30 min, 1 h, or 3 h following release of cells from ␣-factor into
YPD or YPD plus RAP, MMS, or MMS⫹RAP, as described previously (31). To
analyze ARS305 replication intermediates, purified DNAs, restricted with
EcoRV and HindIII, were resolved by two-dimensional (2-D) gel electrophore-
sis, transferred to nylon membranes, hybridized with a
32
P-labeled probe span-
ning ARS305, and visualized by phosphorimage analysis.
RESULTS
RAP-sensitive TORC1 signaling maintains cell viability and
promotes S-phase progression in response to DNA damage.
RAP inhibition of TOR signaling induces yeast cell cycle arrest
in early G
1
phase, which precedes the G
1
block induced by the
␣-factor mating pheromone (4). Thus, we could assess TOR
signaling in S phase by releasing cells from ␣-factor arrest in
late G
1
phase into YPD medium containing RAP, in the pres-
ence or absence of the DNA-damaging agent MMS. As shown
in Fig. 1A, wild-type cells released into medium (control) syn-
chronously transited S phase and acquired a 2C (2N) DNA
content by 40 min. When the cells were released into RAP, a
subpopulation of ␣-factor-arrested cells failed to transit S
phase. As previously reported (4), only 82% of the cells had
entered S phase (as defined by cells forming buds), relative to
the number of cells released into medium alone, 20 min fol-
lowing release from ␣-factor into RAP. Nevertheless, the ki-
netics of S-phase transit for these cells mirrored those of the
untreated control cells, with RAP-treated cells accumulating in
the next G
1
phase. As expected, S-phase transit was decreased
in the presence of MMS due to activation of the Rad53 check-
point (30, 35) (Fig. 1A, MMS panel). Surprisingly, however,
RAP treatment further delayed the slow S-phase transit in-
duced by MMS (Fig. 1A, MMS⫹RAP panel). For instance,
220 min following ␣-factor release, the majority of MMS-
treated cells had a DNA content approaching 2C, while cells
released into MMS⫹RAP had a considerably reduced DNA
content. The persistent accumulation of MMS⫹RAP-treated
cells in early S phase relative to the late S-G
2
DNA content of
MMS-treated cells is highlighted by the superposition of the
220-min FACS profiles in Fig. S1 in the supplemental material.
However, during this time course of drug exposure, the
discrepancy in S-phase transit between MMS- versus
MMS⫹RAP-treated cells became apparent from 100 min on,
coinciding with a more pronounced reduction in cell viability in
MMS⫹RAP-treated cells than that for MMS-treated cells
(Fig. 1B). RAP treatment alone was growth inhibitory, not
cytotoxic, with only a slight increase in the number of colonies
from time zero to 220 min. In contrast, the cytotoxic activity of
MMS or MMS⫹RAP was reflected in the decrease in colony
7008 SHEN ET AL. MOL.CELL.BIOL.
formation over time following removal of the drugs and plating
of cells on YPD agar.
To ensure that these effects were restricted to S phase and
not due to RAP-induced alterations in cell cycle transit from
late G
1
to S phase, several independent experimental strategies
were pursued. First, cells were arrested in early S phase with
HU and then treated as described above. HU inhibition of
RNR induces the activation of the Rad53 S-phase checkpoint
as a consequence of alterations in replication fork progression.
Consequently, the cell cycle arrest induced by HU occurs in
early S phase. In these experiments, similar results to those for
cells synchronized with ␣-factor were obtained: RAP alone was
cytostatic, while cotreatment with MMS⫹RAP further slowed
S-phase progression and increased cell killing induced by MMS
(Fig. 1C; also see Fig. 3). Thus, independent of the mechanism
of cell synchronization (␣-factor in G
1
phase or HU in early S
phase), RAP induced the same effects on the S-phase transit
and viability of cells exposed to MMS.
A second approach involved exposing cells that express high
levels of Top1 to RAP and camptothecin. Since camptothecin
cytotoxicity requires ongoing DNA replication to induce rep-
lication-dependent DNA lesions (6), any alteration in cell vi-
ability induced by RAP cotreatment would be attributable to
S-phase-dependent events. Indeed, a similar increase in cyto-
toxicity was observed when ␣-factor-arrested cells were re-
leased into medium containing RAP and camptothecin (data
not shown). In addition, in time course experiments where
MMS and RAP were added at 10-minute intervals following
release from G
1
arrest, the effects of TOR inhibition appeared
to be restricted to early S phase. RAP-induced phenotypes,
FIG. 1. RAP inhibition of TOR signaling decreases S-phase transit and cell viability in response to MMS treatment. (A) Wild-type cells
released from ␣-factor into YPD containing no drug (control), MMS, RAP, or MMS⫹RAP were processed for flow cytometry at the times
indicated. (B) Serial dilutions of cells treated as described for panel A were spotted onto YPD plates. Colony formation was assessed at 30°C.
(C) Cells released from HU arrest into YPD containing no drug (control), MMS, RAP, or MMS⫹RAP were collected and serially diluted at the
times indicated. The number of viable cells forming colonies on YPD plates following incubation at 30°C was plotted relative to that at time zero
(release from HU) (n⫽3).
VOL. 27, 2007 RAPAMYCIN INHIBITION OF TORC1 FUNCTION IN S PHASE 7009
evident in cells treated at 10 min, disappeared when cells
where treated at 20 min, when the majority of cells had
acquired a close-to-2C DNA content. Thus, in cells exposed
to sufficient DNA damage to induce the intra-S-phase
checkpoint, TOR signaling enhanced cell survival and S-
phase transit.
We next asked if the alterations in S-phase transit sug-
gested by FACS profiles and morphological examinations of
MMS⫹RAP- versus MMS-treated cells (Fig. 1 and data not
shown) might reflect diminished replication origin firing rather
than a decrease in fork progression. To assess origin firing and
fork stability in cells treated with MMS, with or without RAP,
replication intermediates were purified, resolved in 2-D gels,
and probed with sequences corresponding to an early origin of
replication (ARS305) on chromosome III (Fig. 2). ARS305
efficiently fires early in S phase, while origins more proximal to
the left telomere end of chromosome III, ARS301 to ARS304,
are normally dormant. In these gels, a bubble arc reflects
bidirectional origin firing and Y arcs result from the asymmet-
ric movement of replication forks through the restriction frag-
ment being probed. X spikes accompany origin firing and de-
crease in intensity as forks migrate (31).
After release of cells into S phase, firing of ARS305 was
unaffected by RAP, as evidenced by a robust bubble arc at 10
min (Fig. 2, RAP panel). Untreated cells continued to cycle:
the bubble arc apparent after release from ␣-factor was not
detected at 60 min and reappeared at 180 min as cells entered
subsequent cell cycles. The decrease in replication intermedi-
ates after 60 min of RAP treatment coincided with accumula-
tion of cells in G
1
phase, as evidenced by FACS analysis and
cell morphology (Fig. 1 and data not shown). With MMS, the
accumulation of a strong Y arc and X spike indicates slow fork
progression at 30 min. The decrease in intermediates at 60 min
coincided with fork progression and the accumulation of cells
with a 2C DNA content (as in Fig. 1A). A similar pattern of
robust ARS305 firing was also obtained with MMS⫹RAP-
treated cells at 10 min, although a slightly less intense pattern
of replication intermediates was obtained at 30 min. However,
the decrease in replication intermediates at 60 and 180 min
(Fig. 2), relative to the levels with MMS alone, did not corre-
spond with increased DNA content (Fig. 1A). A persistent cell
cycle arrest in early S phase due to Rad53 checkpoint activa-
tion would yield stable replication intermediates over the
course of these experiments. In contrast, these data suggest
that MMS⫹RAP induces a decrease in fork stability that co-
incides with a failure to transit S phase and with a decrease in
cell viability.
One downstream pathway regulated by RAP-sensitive
TORC1 is the initiation of translation of a subset of mRNAs by
eukaryotic initiation factor 4E and eukaryotic initiation factor
4G (8). To ensure that these observations were not simply an
artifact of translation inhibition by RAP, we asked if the global
inhibition of protein translation by CHX in the presence of
MMS induced similar effects on cell cycle and viability to those
induced by RAP. Because protein synthesis is required for cells
to transit from G
1
into S phase (16), these experiments were
performed with cells synchronized in early S phase. When
HU-arrested cells were released into YPD medium containing
CHX, cells progressed through S phase, albeit at a lower rate
than those of control and RAP-treated cells alone (Fig. 3A).
However, these cells failed to enter the next cell cycle, as
protein synthesis is required for nuclear division (9). A
more pronounced delay in S-phase transit was induced by
MMS⫹CHX (compare the 220-min profiles in Fig. 3A). Yet,
in contrast to the increased lethality of MMS⫹RAP-treated
cells, CHX treatment did not affect the viability of MMS-
treated cells. Thus, these data refute the simple notion that
global effects of RAP on protein translation decrease cell via-
bility in response to DNA damage in S phase.
Several lines of investigation were next pursued to ensure
that these phenotypes derived from RAP inhibition of TORC1
signaling. First, efficient inhibition of TOR signaling by RAP
was demonstrated by the induction of a starvation response
(Fig. 4A). Following the same experimental strategy as that
described for Fig. 1A, RAP-induced autophagy (26) was ap-
parent in the majority of cells 220 min after release from
␣-factor in either the presence or absence of MMS. Moreover,
a persistent autophagic phenotype was evident in the termi-
FIG. 2. TORC1 signaling maintains replication fork stability in the presence of MMS. At the indicated times following ␣-factor release into
YPD, MMS, RAP, or MMS⫹RAP, replication intermediates were resolved in 2-D gels. The distribution of bubble arcs (circles), Y arcs (Y
diagram), and X spikes (triple arrows), which indicate origin firing, passive DNA replication, and slow fork progression, respectively, was
determined in Southern blots with a probe for ARS305, an early firing origin of replication on the left arm of chromosome III.
7010 SHEN ET AL. MOL.CELL.BIOL.
nal S-phase-arrested cells following a 24-h exposure to
MMS⫹RAP (data not shown). In contrast, no autophagic
bodies were observed in MMS-treated cells.
Second, a series of isogenic yeast strains were engineered to
assess specific alterations in TOR signaling. In these experi-
ments, the effects of RAP were evident from 100 min on (data
not shown). However, to simplify the presentation, cell viabil-
ities 220 min following release from ␣-factor arrest are pre-
sented, as these data distinguish the cytotoxic and protective
effects of TOR inhibition in the presence of MMS. Relative to
that of wild-type cells, deletion of TOR1 had no effect on MMS
cytotoxicity (Fig. 4B). However, deletion of TOR1 increased
the RAP sensitivity of MMS-treated cells ⬎10-fold (Fig. 4B).
In yeast, RAP inhibits the multiprotein complex TORC1, com-
prised of either Tor1 or Tor2 (29). Thus, tor1⌬would reduce
the cellular complement of RAP-sensitive TORC1 to those
complexes containing Tor2, thereby enhancing cell sensitivity
to RAP. Indeed, consistent with this interpretation, higher
concentrations of RAP were required to induce wild-type cell
sensitivity to MMS than those needed for isogenic tor1⌬cells
following release into S phase (compare MMS⫹50 ng/ml RAP
patterns in Fig. 4C). We then asked if TORC1 comprised of
Tor2 preferentially modulates cell sensitivity to DNA damage.
A RAP-resistant TOR
RR
strain, where the Ser1972 codon was
mutated to an Arg codon in TOR1S
1972
R(10), showed no
effect of RAP on MMS cytotoxicity (Fig. 4B). Similar results
were obtained with a RAP-resistant TOR2S
1975
Rmutant (data
not shown). Since the RAP resistance conferred by these single
amino acid substitutions in either Tor1 or Tor2 is dominant,
these findings establish that RAP-sensitive signaling through
TORC1 containing Tor1 or Tor2 modulates the survival of
cells exposed to DNA damage in S phase.
S-phase checkpoint activation is required for the protective
function of TOR. In response to genotoxic stress, activation of
the Rad53 checkpoint maintains cell survival by regulating
events that enhance the stability and repair of stalled replica-
tion forks. The checkpoint also minimizes the potential for
increased DNA damage by coordinating origin firing, DNA
polymerization, and histone synthesis (13, 30, 32, 35, 43). We
next asked if RAP inhibition of TORC1 affected S-phase
checkpoint signaling in isogenic strains defective for Rad53
checkpoint activation or function. For instance, Mrc1 pro-
motes replication fork progression and acts as a mediator to
enhance Rad53 phosphorylation in response to replication
stress (2, 27, 44). Indeed, the protective function of TORC1 in
MMS-treated cells required Mrc1 (Fig. 4D). The kinetics of
MMS-induced lethality of mrc1⌬cells were increased relative
to those of wild-type cells and were unaffected by cotreatment
with RAP. Moreover, this pattern of cell lethality coincided
with a failure to slow S-phase progression (see Fig. S2 in the
supplemental material). Because mrc1⌬cells exhibit check-
point-independent defects in S phase, additional studies were
carried out with strains deleted for Rad9, which promotes
Rad53 phosphorylation in the DNA damage checkpoint (18);
Tof1, which functions with Csm3 and Mrc1 to stabilize stalled
forks (11); or the Rad53 checkpoint kinase. Similar results
were obtained in each case, as follows: the enhanced sensitiv-
ities of rad9⌬,tof1⌬, and rad53⌬strains to MMS were not
increased by cotreatment with RAP, and TORC1 signaling did
not affect S-phase progression (Fig. 4B and data not shown).
Thus, diminished activation or abrogation of the S-phase
checkpoint eliminates the ability of TOR signaling to maintain
cell survival and promote S-phase transit.
Inhibition of TORC1 increases MMS-induced Rad53 phos-
phorylation. We then asked if TORC1 inhibition affected
Rad53 signaling. Rad53 phosphorylation by Mec1 or Tel1 re-
flects the extent and duration of checkpoint activation (32).
Following release of cells into S phase, RAP treatment alone
failed to induce Rad53 phosphorylation or to affect Rad53
protein levels (Fig. 5A). In contrast, MMS treatment induced
a shift in Rad53 mobility, which indicates Rad53 phos-
phorylation and checkpoint activation. Cotreatment with
MMS⫹RAP produced an even more pronounced shift in
Rad53 mobility and the downregulation of Rad53 protein lev-
els. These data suggest that the inhibition of TORC1 signaling
might increase the amount of DNA damage induced by MMS
and, ergo, the hyperphosphorylated state of Rad53. However,
the downregulation of Rad53 protein levels in the absence of
TORC1 signaling might also impair cell viability in response to
genotoxic stress.
To address these distinct possibilities, we considered the
mechanism of Rad53 protein downregulation. First, cotreat-
ment of cells with MMS⫹RAP and the proteasome inhibitor
MG132 failed to stabilize Rad53 or any higher-molecular-
weight forms of the protein (data not shown). Thus, increased
protein turnover by ubiquitin-mediated proteolysis is unlikely.
FIG. 3. CHX and RAP induce distinct alterations in MMS-treated cell viability in S phase. (A) Cells released from HU arrest into YPD
containing no drug (control), MMS, RAP, MMS⫹RAP, CHX, or MMS⫹CHX were collected at the indicated times and processed for flow
cytometry. (B) Serial dilutions of cells treated as described for panel A were assayed for cell viability as described in the legend to Fig. 1B.
VOL. 27, 2007 RAPAMYCIN INHIBITION OF TORC1 FUNCTION IN S PHASE 7011
Indeed, MG132 cotreatment slightly increased the extent of
Rad53 downregulation induced by MMS⫹RAP. However,
since the cytotoxic activity of MMS⫹RAP was unaltered by
MG132 (data not shown), these data suggested that Rad53
FIG. 5. RAP inhibition of TORC1 enhances MMS-induced
Rad53 checkpoint activation. (A) Cells transformed with YCpT-
RAD53-HA were released from ␣-factor into selective medium
(control), RAP, MMS, or MMS⫹RAP. At the indicated times, TCA
cell extracts were immunoblotted with HA and tubulin antibodies.
Unphosphorylated (arrow) and phosphorylated (line) Rad53 pro-
teins are indicated. (B) Cells transformed with YCpT-RAD53-HA
were released from HU into selective medium (control), RAP,
MMS, MMS⫹RAP, CHX, or MMS⫹CHX. At the times indicated,
TCA cell extracts were immunoblotted with HA antibodies. (C) Iso-
genic wild-type and tor1⌬cells transformed with YCpT-RAD53-HA
were released from ␣-factor into selective medium containing
MMS⫹RAP (200 ng/ml) or MMS plus a low concentration of RAP
(50 ng/ml). At the times indicated, TCA cell extracts were immu-
noblotted with HA and tubulin antibodies. The patterns of Rad53
mobility for the control, RAP (50 or 200 ng/ml), and MMS alone
mirrored those shown in panel A. (D) As in panel A, TCA extracts
of wild-type cells expressing histone H2A-HA (HTA2-HA) were
immunoblotted with HA and tubulin antibodies.
FIG. 4. TORC1-dependent cell viability and S-phase transit in the
presence of MMS requires a functional S-phase checkpoint. (A) DIC and
DAPI images of cells treated with MMS, RAP, or MMS⫹RAP for 220
min following release from ␣-factor. Arrows indicate autophagic bodies,
visible in DIC images as bumps in enlarged vacuoles. (B) Isogenic wild-
type, tor1⌬,TOR1
RR
, and rad9⌬strains were released from ␣-factor into
YPD alone (white bars), MMS (gray bars), or MMS⫹RAP (black bars).
The number of colonies formed at 220 min was plotted relative to the
number at time zero (n⫽3). Values of ⬎1 indicate cell proliferation, and
values of ⬍1 indicate cytotoxicity. RAP alone did not affect TOR
RR
cell
growth and allowed an ⬃2-fold increase in cell number (relative to that at
time zero) for other strains (data not shown). The number is the ratio of
colonies obtained with MMS⫹RAP to that obtained with MMS. (C) Iso-
genic wild-type and tor1⌬cells were released from ␣-factor arrest into
YPD alone (control), RAP, MMS, MMS⫹RAP (200 ng/ml), or MMS
plus a low concentration of RAP (50 ng/ml). At 0 and 220 min, aliquots
were serially diluted and spotted onto YPD agar. Similar numbers of
colonies to those shown for wild-type cells were obtained for tor1⌬cells at
time zero. (D) Isogenic wild-type and mrc1⌬cells released from ␣-factor
into YPD alone (control), MMS, RAP, or MMS⫹RAP were serially
diluted at the indicated times and plated onto YPD agar. The number of
colonies was plotted relative to the number obtained at time zero (n⫽3).
7012 SHEN ET AL. MOL.CELL.BIOL.
protein levels did not predict cell viability. Indeed, when cells
were released into S phase in the presence of CHX (as shown
in Fig. 3), Rad53 protein levels decreased, independent of
phosphorylation status (see Fig. S3 in the supplemental mate-
rial). While MMS⫹CHX induced a similar pattern of Rad53
downregulation, this drug combination failed to yield the in-
creased phosphorylation of Rad53 observed with MMS⫹RAP
(see Fig. S3 in the supplemental material). Moreover, CHX
cotreatment also failed to enhance cell sensitivity to MMS
(Fig. 3B). Taken together, these data suggest the downregula-
tion of Rad53 protein at the level of translation in S phase.
However, these data further indicate that it is the increased
phosphorylation of Rad53, not relative Rad53 protein levels,
which is relevant to the cytotoxic phenotype induced by
MMS⫹RAP.
Several lines of evidence also suggest that the extent of
Rad53 phosphorylation could not be attributed to RAP-in-
duced alterations in the cell’s ability to recover from check-
point activation. First, cells treated with HU accumulate in
early S phase due to Rad53 checkpoint activation. However, as
shown in Fig. 5B, when cells were released from HU into
medium containing RAP, the kinetics of cell recovery from
checkpoint arrest, as assessed by increased Rad53 mobility
relative to that at time zero, mirrored those of the untreated
control. Yet the pattern of Rad53 phosphorylation in the
presence of MMS or hyperphosphorylation induced by
MMS⫹RAP resembled that observed following release from
␣-factor arrest. Second, cells deleted for ESC4 are more sen-
sitive to MMS-induced DNA damage as a consequence of a
failure to recover from checkpoint-induced S-phase arrest
(38). However, these cells exhibited the same relative increase
in cell death in the presence of MMS⫹RAP as did isogenic
wild-type cells (data not shown).
The dependence of these events on RAP inhibition of
TORC1 signaling is further supported by studies of wild-type
and tor1⌬strains. The increased sensitivity of tor1⌬cells to
MMS and lower concentrations of RAP, as shown in Fig. 4C,
corresponds with an increase in Rad53 phosphorylation at
lower drug concentrations in the tor1⌬, but not isogenic wild-
type, strain (Fig. 5C). For both strains, the relative mobilities
of Rad53 were similar in extracts of control and RAP- and
MMS-treated cells (data not shown).
We then reasoned that any increase in checkpoint signaling
induced by RAP treatment might be evident in downstream
pathways. For instance, the Rad53 checkpoint coordinates his-
tone biosynthesis with DNA synthesis, as excess free histones
are toxic (19). When cells were released into S phase, histone
H2A protein levels decreased in response to MMS-induced
DNA damage; however, this effect was enhanced in the pres-
ence of MMS⫹RAP (Fig. 4C). RAP alone did not alter his-
tone H2A protein levels. Thus, a direct effect of TOR signaling
on histone H2A translation is unlikely. Rather, these data
support a model of enhanced Rad53 checkpoint activation in
response to the increase in MMS-induced DNA damage
caused by TORC1 inhibition.
TOR signaling maintains Rad53 checkpoint-induced ex-
pression of RNR subunits Rnr1 and Rnr3, but not Rnr2 and
Rnr4. Perhaps the best-understood effector of the Rad53
checkpoint is RNR, which catalyzes the rate-limiting step in
the production of deoxynucleoside triphosphates (dNTPs), the
precursors of DNA synthesis (21, 28, 48). Eukaryotic RNR
comprises an ␣
2

2
tetramer. In yeast, the large subunit is
typically a homodimer of Rnr1 (␣
1
␣
1
), while the small subunit
is a heterodimer of Rnr2 and Rnr4 (⬘) (28). A second large
subunit, Rnr3, is highly induced by DNA damage and can
complement an rnr1⌬mutation when overexpressed (16).
However, in contrast to other RNR genes, RNR3 is nonessen-
tial, and no phenotype has been ascribed to rnr3⌬strains (12,
14).
In response to DNA damage, a six- to eightfold increase in
intracellular dNTP pools is achieved by several mechanisms,
including relaxed dATP feedback inhibition of RNR (12),
Rad53-mediated transcriptional upregulation of RNR genes
(21), degradation of the RNR inhibitor Sml1 (48), and subcel-
lular relocalization of RNA small subunits (47). To ask if the
phenotypes induced by TORC1 inhibition in MMS-treated
cells resulted from alterations in Rad53 checkpoint regulation
of RNR, we first determined whether deletion of the gene
encoding the Sml1 inhibitor of RNR altered the cytotoxic
activity of MMS⫹RAP in S phase. The viabilities of isogenic
wild-type and sml1⌬cells exposed to MMS⫹RAP following
␣-factor release were identical (data not shown). Thus, the
protective function of TORC1 signaling following MMS-in-
duced checkpoint activation is not mediated by a direct effect
on Sml1 regulation of RNR.
We next assessed the levels of individual RNR subunits in
response to MMS⫹RAP. As shown in Fig. 6A, release from
␣-factor into RAP induced a progressive downregulation of
Rnr1 protein levels relative to those in untreated controls, in
part due to the accumulation of cells in the subsequent G
1
phase (as shown in Fig. 1A). As previously reported (14),
MMS-induced checkpoint activation produced a twofold in-
crease in Rnr1 levels. However, this DNA damage-induced
response was suppressed by RAP (MMS⫹RAP) (Fig. 6A),
despite the fact that the majority of cells remained in S phase
(Fig. 1A). This effect was even more pronounced with Rnr3.
Although this RNR subunit is barely detectable in the absence
of DNA damage, cotreatment with RAP dramatically sup-
pressed the high levels of Rnr3 induced by exposure to MMS
during S phase (Fig. 6A). Rnr2 and Rnr4 levels, on the other
hand, were unaltered by RAP treatment (Fig. 6B; see Fig. S4
in the supplemental material).
The initial increases of both Rnr1 and Rnr3 subunits in the
presence of MMS⫹RAP (compare time zero with 0.5 and 1 h
for Rnr1 and with 1 to 2 h for Rnr3) (Fig. 6A) coincide with
checkpoint activation and Rad53 phosphorylation (as shown in
Fig. 5A). At later times in MMS⫹RAP-treated cells, however,
Rnr1 and Rnr3 decreases also parallel the temporal pattern of
Rad53 downregulation (3 to5hinFig. 6A and 5A, respec-
tively). This transient accumulation of Rnr1 and Rnr3 suggests
that the phenotypic consequences of RAP treatment are not
restricted to translation inhibition. However, since DNA dam-
age-induced RNR3 transcription is achieved by Rad53 inacti-
vation of the Rfx1 (or Crt1) transcriptional repressor (21), we
also considered that the decrease in Rad53 levels could alle-
viate Rfx1 inactivation to suppress RNR3 transcription. If this
were the case, then an rfx1⌬mutation would abolish the RAP-
induced downregulation of Rnr3 in MMS-treated cells. How-
ever, as shown in Fig. 6A, this was not the case. As reported
(21), rfx1⌬cells exhibited little alteration in Rnr1 protein lev-
VOL. 27, 2007 RAPAMYCIN INHIBITION OF TORC1 FUNCTION IN S PHASE 7013
els. In contrast, Rnr3 was overexpressed in rfx1⌬cells, inde-
pendent of DNA damage. Although the absolute levels of
Rnr3 differed substantially between wild-type and rfx1⌬strains,
the kinetics of Rnr3 downregulation in the presence of
MMS⫹RAP were comparable. Thus, the decreases in Rnr1
and Rnr3 induced by RAP inhibition of TORC1 were not due
to alterations in Rad53 checkpoint regulation of Rfx1.
TORC1 signaling was also required to maintain the elevated
RNR1 and RNR3 mRNA levels induced by Rad53 checkpoint
activation. RNR1 expression is tightly cell cycle regulated (16).
After release of cells from ␣-factor into medium (Fig. 6C,
control panel), RNR1 mRNA levels increased by 15 min, when
cells entered S phase; decreased at 30 min, when cells were in
G
2
-M phase; and then increased again at 60 and 180 min as
cells entered subsequent cell cycles. When cells were released
into RAP, RNR1 transcript levels mirrored those of untreated
controls as the cells progressed through S phase and then
remained low as cells accumulated in the next G
1
phase. In
contrast, TOR signaling was required to sustain the increased
expression of RNR1 and RNR3 in response to long-term MMS
exposure. The increases in RNR1 and RNR3 transcripts in-
duced by exposure to MMS were suppressed at 180 min in cells
cotreated with RAP (Fig. 6C). This effect was more pro-
nounced with RNR3 mRNA, consistent with the transient in-
crease and then decrease in Rnr3 levels under the same con-
ditions. These data indicate that TORC1 signaling is required
to sustain checkpoint-induced expression of RNR1 and RNR3
transcripts and proteins.
Rnr3 protects cells from DNA damage in S phase. We then
considered the phenotypic consequences of RNR large-sub-
unit downregulation in the face of persistent DNA damage.
When TORC1 signaling is intact, RNR comprised of the Rnr1
large subunit (␣
1
␣
1
⬘) apparently constitutes the major cel-
lular complement of this essential enzyme, as deletion of RNR3
has no effect on cell viability in the presence or absence of
MMS. For example, as shown in Fig. 6D, the viability of rnr3⌬
cells exposed to MMS for 220 min following ␣-factor release is
the same as that of wild-type cells. However, relative to wild-
type RNR3 cells, rnr3⌬cells exhibited increased sensitivity to
MMS when TORC1 signaling was inhibited. Thus, in RAP-
FIG. 6. TORC1 signaling maintains DNA damage-induced expression of Rnr1 and Rnr3. TCA extracts of wild-type or rfx1⌬cells expressing
HA-tagged Rnr1 or Rnr3 (A) or of wild-type cells expressing HA-tagged Rnr4 (B), treated as described in the legend to Fig. 5A, were
immunoblotted with HA or tubulin antibodies. (C) RNAs isolated from wild-type cells treated as described for panel A were subjected to Northern
blot analysis with radiolabeled probes for RNR3,RNR1, and ACT1. (D) As described in the legend to Fig. 4B, rnr3⌬,rfx1⌬, and clb5⌬cells were
released from ␣-factor into YPD alone (light gray bars), MMS (gray bars), or MMS⫹RAP (black bars), and the number of viable cells forming
colonies at 220 min was plotted relative to that at time zero (n⫽3). For comparison, wild-type cell viability data were reproduced from Fig. 4B
(dashed lines). (E) RNR1-HA and GAL1 promoter-driven RNR1-HA strains were released from ␣-factor into galactose medium with no drug
(control), RAP, MMS, or MMS⫹RAP. At the times indicated, cells were serially 10-fold diluted and spotted onto galactose plates. Colony
formation was assessed at 30°C.
7014 SHEN ET AL. MOL.CELL.BIOL.
treated cells, Rnr3 functions to increase the survival of cells
treated with MMS. This surprising finding constitutes the first
reported phenotype for rnr3⌬cells.
rfx1⌬cells exhibited a slight increase in MMS resistance
(Fig. 6D) and higher basal levels of Rnr3 than that induced by
MMS in wild-type cells (Fig. 6A). However, rfx1⌬cells were no
more resistant to MMS⫹RAP than were isogenic wild-type
strains. These data suggest that in the absence of a coordinate
increase in Rnr1, increased levels of Rnr3 do not provide a
survival advantage to cells exposed to MMS⫹RAP in S phase.
To address the effect of Rnr1 overexpression, the galactose-
inducible GAL1 promoter was inserted upstream of RNR1 by
PCR-based homologous recombination. Since RNR1 is essen-
tial for cell viability, this approach necessitated culturing the
cells in medium containing galactose, where the levels of Rnr1
in S phase exceed that of the wild-type control (see Fig. S5 in
the supplemental material). This construct retained the 5⬘un-
translated region of the endogenous RNR1 gene, and thus a
partial downregulation of Rnr1 by RAP was still evident. Nev-
ertheless, as shown in Fig. 6E, the increased levels of Rnr1
partially suppressed the cytotoxic effects induced by RAP in-
hibition of TORC1 in MMS-treated cells.
Taken together, these findings suggest that the hyperphos-
phorylation of Rad53 in MMS⫹RAP-treated cells results from
increased replication stress induced by decreased Rnr1 and
Rnr3 levels. The analysis of replication intermediates in Fig. 2
indicates that early origins of replication fire efficiently in the
presence of MMS⫹RAP but that the forks are unstable upon
prolonged exposure to the drugs. A concomitant decrease in
Rnr1/3 levels induced by MMS⫹RAP would also impact fork
progression and stability. One prediction of these consider-
ations is that strains exhibiting a prolonged S phase should be
even more sensitive to MMS⫹RAP. In complex with Cdc28,
Clb5-Cdk activates the firing of early and late origins in S
phase, while Clb6-Cdk supports the firing of early origins only
(15). As shown in Fig. 6D, deletion of the B-type cyclin Clb5
had no effect on cell sensitivity to MMS yet enhanced the
cytotoxic activity of MMS in the presence of RAP. Restricting
the initiation of DNA replication to early origins, and thereby
decreasing the number of replication forks in the cell, in-
creased the requirement for TORC1 signaling as a survival
pathway following MMS activation of the S-phase checkpoint.
TORC1 signaling promotes MMS-induced mutagenesis. In-
creased dNTP levels produced in response to DNA damage
promote cell survival at the cost of increased mutation levels
(12). Chabes et al. (12) posited that higher levels of dNTPs
promote translesion DNA synthesis to bypass DNA lesions, as
polymerases generally have a higher K
m
for binding nucleo-
tides opposite a damaged base (36). We reasoned that the
converse would also apply—that RAP- induced decreases in
Rnr1/3 levels would fail to yield sufficient dNTP concentrations
to promote translesion DNA synthesis, thereby decreasing cell
viability and suppressing MMS-induced mutations. One mea-
sure of mutation frequency is the acquisition of canavanine
resistance due to mutation of the arginine permease encoded
by CAN1. Indeed, treatment with RAP completely suppressed
the acquisition of canavanine resistance induced by MMS as
well as reducing the frequency of spontaneous CAN1 mutants
in the absence of MMS (Fig. 7). As shown in Fig. 3, this effect
was not due to global effects on translation, as treating cells
with CHX under the same conditions induced the downregu-
lation of all RNR subunits yet failed to suppress the frequency
of spontaneous or MMS-induced CAN1 mutants (Fig. 7 and
data not shown). Thus, MMS-induced mutagenesis was selec-
tively suppressed by RAP inhibition of TORC1.
DISCUSSION
TOR is a highly conserved serine/threonine kinase that func-
tions as a central regulator of eukaryotic cell responses to
environmental stresses, such as amino acid starvation, hypoxia,
and growth factor deprivation (7, 8, 24, 33). Subsequent sig-
naling through distinct downstream pathways regulates the
cap-dependent translation of a subset of mRNAs as well as
transcriptional stress responses, progression from G
1
to S
phase of the cell cycle, and cell survival. TOR is also a member
of the family of phosphatidylinositol 3-kinase-related kinases,
which include DNA-PK, ATM, ATR, and the yeast Mec1 and
Tel1 kinases, all of which play important roles in DNA repair
and/or checkpoint responses to DNA damage. The macrocyclic
antibiotic RAP, in complex with an immunophilin (FKBP12),
specifically targets TOR (8, 40), and several RAP analogs are
currently in phase I to III oncology clinical trials (8).
Using yeast as a genetically tractable model, our studies
reveal a novel role for TORC1 signaling as a determinant of
cell survival in response to aberrations in DNA replication.
RAP-sensitive TORC1 functions to promote S-phase transit
and fork stability and to maintain cell viability following Rad53
checkpoint activation by DNA damage. Our studies further
establish a critical function for Rnr3 in maintaining the viabil-
ity of cells exposed to DNA damage in the absence of TORC1
signaling: while RAP-induced downregulation of Rnr1 and
Rnr3 levels enhances MMS cytotoxicity, this effect is exacer-
bated in cells deleted for RNR3.
FIG. 7. TORC1 signaling promotes MMS-induced mutagenesis.
Aliquots of wild-type cells released from ␣-factor into YPD medium
(control), MMS, RAP, or MMS⫹RAP or released from HU into YPD
medium (control), MMS, CHX, or MMS⫹CHX were plated onto
YPD agar or onto synthetic medium without Arg and with canavanine
at the indicated times. The number of canavanine-resistant colonies
per total number of viable cells forming colonies on YPD was plotted
relative to the spontaneous frequency of 2 ⫻10
⫺6
canavanine-resistant
mutants obtained at time zero.
VOL. 27, 2007 RAPAMYCIN INHIBITION OF TORC1 FUNCTION IN S PHASE 7015
Taken together, our data support the model shown in Fig. 8.
In S phase, RNR activity is regulated to a significant extent by
cell cycle-dependent expression of RNR1, such that Rnr1 levels
are maximal in S phase to produce sufficient dNTPs for DNA
replication (16) (Fig. 8A). In the presence of DNA damage,
Rad53 checkpoint activation enhances RNR activity, in part by
increased RNR subunit gene transcription (21, 28, 48) (Fig. 6C
and 8B). RNR3 is highly induced, yet rnr3⌬strains do not
exhibit increased sensitivity to DNA-damaging agents. How-
ever, Rnr3 levels only reach 1/10 the Rnr1 levels, and the
activity of ␣
3
␣
3
⬘ RNR is ⬍1% that of ␣
1
␣
1
⬘ RNR (14).
Thus, even though RNR comprised of Rnr1 and Rnr3
(␣
1
␣
3
⬘) exhibits synergistic activity in vitro (14), the contri-
bution of this complex to the net increase in dNTPs would be
negligible. Instead, RNR composed of ␣
1
␣
1
⬘ would predom-
inate, with relaxed feedback inhibition generating sufficient
dNTPs for cell survival (12).
This situation contrasts with the downregulation of both
Rnr1 and Rnr3 when TORC1 signaling is inhibited in the
presence of MMS-induced DNA damage (Fig. 8C). In this
case, diminished RNR (␣
1
␣
1
⬘) levels would adversely im-
pact dNTP production, such that the synergistic activity of even
limiting amounts of RNR comprised of ␣
1
␣
3
⬘ might pro-
mote cell survival. This model predicts that limiting dNTPs in
the presence of MMS⫹RAP would preclude efficient transle-
sion DNA synthesis, thereby suppressing MMS-induced muta-
tions. Indeed, RAP inhibition of TORC1 suppressed the fre-
quencies of spontaneous and MMS-induced CAN1 mutations,
as shown in Fig. 7.
Another prediction of this model is that deletion of RNR3
would selectively enhance MMS⫹RAP cytotoxicity. However,
alterations in Rnr3 levels should not affect cell survival when
TORC1 signaling is intact, since the normal complement of
Rnr1 would suffice to maintain dNTP levels. Indeed, while
rnr3⌬cells were no more sensitive to MMS than wild-type
cells in S phase, cells lacking Rnr3 were hypersensitive to
MMS⫹RAP (Fig. 6D). This constitutes a novel phenotype for
rnr3⌬cells and demonstrates that in the absence of TORC1
signaling, Rnr3 function is necessary to maintain the viability
of MMS-treated cells.
This model also explains the phenotypes of rfx1⌬cells. Since
the basal Rnr3 level in rfx1⌬cells exceeded that induced by
MMS in wild-type cells (Fig. 6A), the slight increase in MMS
resistance of rfx1⌬cells (Fig. 6D) may result from increased
levels of RNR composed of ␣
1
␣
3
⬘. However, the model
holds that unless Rnr1 levels are also sustained, Rnr3 over-
expression simply increases ␣
3
␣
3
⬘ RNR levels, which
would fail to suppress the increased cytotoxicity induced by
MMS⫹RAP. Indeed, Rnr1 levels were similar in wild-type and
rfx1⌬cells (Fig. 6A), as were wild-type and rfx1⌬cell sensitiv-
ities to MMS⫹RAP (Fig. 6D). Thus, in the absence of TORC1
signaling, Rnr1 and Rnr3 both function to promote cell viabil-
ity in the presence of DNA damage.
In summary, we have shown that TOR functions in S phase
to regulate cell survival in response to a variety of genotoxic
stresses. Our data support a model whereby the TOR pathway
is required to sustain the DNA damage-mediated induction of
RNR1 and RNR3, thereby ensuring sufficient RNR activity to
generate the high levels of dNTPs necessary for translesion
DNA synthesis to bypass MMS-induced DNA lesions. The
regulation of translesion synthesis of DNA adducts by this
mechanism could also explain the increased sensitivity of mam-
malian cells to cisplatin induced by RAP (5). One implication
of our work is that clinically acquired drug resistance may be a
consequence of TOR-dependent mutations resulting from S-
phase checkpoint activation. We consider this unique finding
pertinent at a time when molecularly targeted agents are being
combined with classical cytotoxic drugs. The potential to block
drug-induced mutations that confer resistance represents a
unique application of RAP analogs with potential clinical im-
portance for the treatment of adult and pediatric malignancies.
ACKNOWLEDGMENTS
We thank Peter Houghton and members of the Bjornsti lab for
helpful discussions and Carol Newlon and Jim Theis for help with 2-D
gel methodology.
FIG. 8. Model for RAP-induced alterations in RNR, cell survival, and mutation rates in response to Rad53 checkpoint activation (see the text
for details).
7016 SHEN ET AL. MOL.CELL.BIOL.
This work was supported by Public Health Service grant CA23099
(to M.-A.B) from the National Cancer Institute, by NCI Cancer Center
Core grant CA21765, and by the American Lebanese Syrian Associ-
ated Charities.
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