DNA damage checkpoint triggers autophagy
to regulate the initiation of anaphase
Farokh Dotiwala1,2, Vinay V. Eapen2, Jacob C. Harrison2,3, Ayelet Arbel-Eden4, Vikram Ranade5, Satoshi Yoshida,
and James E. Haber6
Rosenstiel Center and Department of Biology, Brandeis University, Waltham, MA 02445
Contributed by James E. Haber, October 18, 2012 (sent for review September 24, 2012)
Budding yeast cells suffering a single unrepaired double-strand
break (DSB) trigger the Mec1 (ATR)-dependent DNA damage re-
sponse that causes them to arrest before anaphase for 12–15 h.
Here we find that hyperactivation of the cytoplasm-to-vacuole
(CVT) autophagy pathway causes the permanent G2/M arrest of
cells with a single DSB that is reflected in the nuclear exclusion of
both Esp1 and Pds1. Transient relocalization of Pds1 is also seen in
wild-type cells lacking vacuolar protease activity after induction of
a DSB. Arrest persists even as the DNA damage-dependent phos-
phorylation of Rad53 diminishes. Permanent arrest can be over-
come by blocking autophagy, by deleting the vacuolar protease
Prb1, or by driving Esp1 into the nucleus with a SV40 nuclear
localization signal. Autophagy in response to DNA damage can be
induced in three different ways: by deleting the Golgi-associated
retrograde protein complex (GARP), by adding rapamycin, or by
overexpression of a dominant ATG13-8SA mutation.
adaptation|separase|securin|cell cycle arrest
checkpoint kinase-dependent cell cycle arrest, but cells eventu-
ally escape from the arrest and reenter the cell cycle in a process
termed adaptation (1–6). Arrest before adaptation typically lasts
12–15 h, a time equivalent to five to six normal cell cycles. Adap-
tation is accompanied by the loss of checkpoint-induced hyper-
phosphorylation of checkpoint kinases Rad53 and Chk1 and the
loss of association with damaged DNA of the Mec1 (ATR) kinase-
associated Ddc2 (ATRIP), despite the persistence of DNA dam-
age (7, 8). Several proteins are required for adaptation, including
those with known roles in DNA repair (Yku70, Yku80, Rdh54/
Tid1, Rad51, Srs2, Sae2, Fun30, and Sgs1), as well as proteins
that are required to turn the checkpoint off (the PP2C phos-
phatases Ptc2 and Ptc3 and casein kinase II) and the Polo kinase
Cdc5, which plays several roles in regulating mitosis (5, 6, 9–14).
When DSB repair is relatively rapid, as during HO endonu-
clease-induced mating-type (MAT) switching, there is no cell
cycle arrest or Rad53 phosphorylation (7), although Mec1 and
Tel1 kinases rapidly phosphorylate histone H2A (15, 16). How-
ever, when DSB repair is slower, e.g., during ectopic homologous
recombination or long-distance single-strand annealing, the DNA
damage checkpoint is fully activated and G2/M arrest is main-
tained for several hours until repair is effected and cells reenter
the cell cycle in a process termed recovery (12, 17). Recovery is
an active process that requires the functions of the Srs2 helicase
and also the casein kinase II and PP2C phosphatases to dephos-
phorylate checkpoint kinases (11–13). None of the other adap-
tation mutants tested (yku70Δ, rdh54Δ, rad51Δ, sae2Δ, cdc5-ad,
and fun30Δ) is deficient in recovery (12, 14) although a number of
double mutants are recovery defective (3). Analysis of checkpoint
recovery in human cells has shown a requirement for the Plk1
polo kinase homologous to yeast Cdc5 and the mitotic phos-
phatase Cdc25 (17).
Although the DNA damage response leading to cell cycle
arrest appears to be nuclear autonomous (18), recent evidence
has suggested that the checkpoint response involves alterations
n the presence of a single unrepairable DNA double-strand
break (DSB), Saccharomyces cerevisiae arrest is due to Mec1
of cytoplasmic processes (19–24). Most notably, inhibition of
several yeast histone deacetylases in cells suffering DNA damage
results in the destruction of Sae2, a key DNA repair protein, by
autophagy (25). Very little is understood about how DNA damage
triggers cytoplasmic responses and how autophagy might play a
role in the regulation of cell cycle progression in the face of DNA
damage. Here we report that mutations in the Golgi-associated
retrograde protein (GARP) complex unexpectedly block check-
point adaptation and recovery. We show that this defect is as-
sociated with an increase in autophagy—specifically in the selective
cytoplasm-to-vacuole targeting (CVT) pathway. Increased auto-
phagy after DNA damage causes the partial destruction of the
chaperone/inhibitor securin (Pds1) that controls entry of sepa-
rase (Esp1) into the nucleus to facilitate anaphase. The exclusion
of Pds1 from the nucleus after DSB induction can be observed
transiently in cells lacking vacuolar protease activity, but this
state is maintained by increasing autophagy either by rapamycin
inhibition of TOR or by overexpression of a dominant ATG13
mutation. The inhibition of permanent DNA damage-induced
cell cycle arrest can be overcome by inhibiting autophagy, by
inhibiting vacuolar proteolysis, or by driving Esp1 into the nu-
cleus with a heterologous nuclear localization sequence (NLS).
In a screen for new checkpoint recovery-defective mutations
(Materials and Methods), we identified a transposon insertion in
the VPS51 gene, a component of the GARP complex. Subse-
quently we found that complete deletions of VPS51 or other
GARP genes (VPS52, VPS53, VPS54, and YPT6) prevent adap-
tation (Fig. 1), similar to the phenotype of a previously identified
adaptation-defective mutation tid1Δ (hereafter referred to as
rdh54Δ) (9). Adaptation was assayed by inducing a HO endonu-
clease-mediated unrepairable DSB at the MAT locus and ob-
serving cells under the microscope for arrest at G2/M (dumbbell-
shaped cells) and subsequent budding (6). GARP mutants all
maintain arrest with the characteristic large-budded phenotype
with a single DAPI-staining nucleus, usually at, or stretched
across, the bud neck. This phenotype eliminates the possibility
that mutants have completed mitosis but fail to bud in the next
Author contributions: F.D., V.V.E., J.C.H., A.A.-E., S.Y., and J.E.H. designed research; F.D.,
V.V.E., J.C.H., A.A.-E., V.R., and S.Y. performed research; F.D., J.C.H., A.A.-E., and S.Y.
contributed new reagents/analytic tools; F.D., V.V.E., J.C.H., A.A.-E., V.R., S.Y., and J.E.H.
analyzed data; and F.D., V.V.E., J.C.H., S.Y., and J.E.H. wrote the paper.
The authors declare no conflict of interest.
1Present address: Harvard Medical School, Boston, MA 02115.
2F.D., V.V.E., and J.C.H. contributed equally to this work.
3Present address: Samsung Advanced Institute of Technology, Cambridge, MA 02142.
4Present address: Hadassah Academic College, Jerusalem 91010, Israel.
5Present address: Department of Genetics and Development, Columbia University, New
York, NY 10032.
6To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
See Author Summary on page 15 (volume 110, number 1).
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| Published online November 19, 2012
cell cycle. We note that not all vacuolar protein-sorting (vps)
mutants, all of which cause carboxypeptidase Y to be secreted
rather than retained in the vacuole (26), exhibit an adaptation de-
fect; for example, vps25Δ and vps35Δ all display normal adap-
tation (Fig. 1).
To establish whether the defects in GARP mutants were
specific to cell cycle arrest provoked by DNA damage, we asked
if blocking cells with nocodazole before anaphase would also
lead to a recovery defect. Log-phase cells grown in YEPD liquid
medium were arrested for 12 h with 30 μg/mL nocodazole such
that >95% were arrested as large dumbbells. These cells were
then spread on YEPD without nocodazole to assess their ability
to resume cell cycle progression and form colonies. vps51Δ cells
exhibit a modest delay in cell cycle progression 8 h after release
from nocodazole, but by 24 h 100% of both WT and vps51Δ cells
progressed beyond the dumbbell stage (Fig. S1). Hence, noco-
dazole arrest, which triggers the spindle checkpoint activation,
does not cause the same defect, as does a single unrepaired DSB.
GARP Mutants Are Suppressed by Inactivating Vacuolar Protease Prb1.
It has been observed that GARP mutants, like other vps mutants,
cause mislocalization of vacuolar proteases (26); hence the ad-
aptation defect of GARP mutants could be due to the aberrant
proteolytic degradation of a key mitotic activator. We therefore
asked whether vps51Δ mutants are suppressed by deleting Pep4,
which activates several vacuolar proteases (27). We found a
striking suppression of vps51Δ by pep4Δ (Fig. 2A). In contrast,
there was no significant suppression of three adaptation-defective
mutations involved in DNA metabolism or dephosphorylation of
the checkpoint kinase cascade (rdh54Δ, srs2Δ, and ptc2Δ ptc3Δ).
Pep4 activates a number of vacuolar proteases. We established
that deleting the gene encoding one of these, proteinase B (PRB1),
showed the same suppression of the adaptation defects of vps51Δ
as pep4Δ, again without affecting the other adaptation-defective
mutations (Fig. 2B). These results lead to the suggestion that cells
with defects in retrograde vesicle trafficking fail to adapt because
of the Prb1-dependent degradation of a necessary mitotic activator.
Nuclear Accumulation of Pds1 and Esp1 Is Defective in GARP Mutants.
The checkpoint-mediated G2/M arrest is enforced by preventing
% cells adapted
(JKM179) and vesicular transport mutants were tested in the checkpoint ad-
aptation assay (Materials and Methods). Of these, WT cells adapt whereas
vps51Δ, vps53Δ, and ypt6Δ were found to be adaptation defective. vps52Δ has
a similar effect but was measured only at 24 h and is not shown for clarity.
Retromer mutant (vps35Δ) and ESCRT II mutant (vps25Δ) were not adaptation
defective. The rdh54Δ cells serve as positive control for the adaptation defect.
Cells were pregrown in YEP-Lactate (YEPL) media overnight and then ma-
nipulated onto minimal complete media supplemented with 1% yeast extract
and containing 2% galactose (CYG). The percentage of cells progressing be-
yond the “dumbbell” arrest stage (“percentage of adaptation”) is shown.
Vps51 is required for adaptation to the DNA damage checkpoint. WT
% cells adapted
% cells adapted
Fig. 2.(A and B) pep4Δ and prb1Δ can rescue the adaptation defect in vps51Δ cells but not in rdh54Δ, srs2Δ, and ptc2Δ ptc3Δ cells.
| www.pnas.org/cgi/doi/10.1073/pnas.1218065109Dotiwala et al.
separase, Esp1, from releasing sister-chromatid cohesion (28).
There are several constraints on Esp1. First, it is inhibited by
securin, Pds1, which is stabilized against APC/C-mediated deg-
radation by Chk1- and Rad53-dependent phosphorylation (29).
Second, Esp1 appears to be sequestered in the cytoplasm until
the proper time in the cell cycle (30). Nuclear import of Esp1 is
at least in part mediated by Pds1 acting as a chaperone (31)
although other chaperones may also play important roles (32).
To examine localization of Pds1 and Esp1, we integrated a GFP
tag in the genomic copy of PDS1 or ESP1. Both PDS1-GFP and
ESP1-GFP are functional, as the cells expressing these GFP fu-
sion proteins in the absence of wild-type PDS1 or ESP1 did not
show any detectable mitotic defects even at 37 °C, whereas esp1Δ
is lethal at all temperatures and pds1Δ is inviable at 37 °C. The
localization of Pds1-GFP and Esp1-GFP in rapidly growing cell
cultures is consistent with previous reports (28, 30, 33). Both
tagged proteins accumulated in the nucleus in medium-to-large
budded cells (Fig. S2).
When the cells were checkpoint arrested in G2/M phase by
induction of a DSB and examined 6 h later, Pds1-GFP accu-
mulated in the nucleus both in wild-type cells and in the adap-
tation-defective rdh54Δ strain (Fig. 3A). However, in vps51Δ,
Pds1-GFP is not concentrated in the nucleus and is apparently
much less abundant, as measured by total fluorescence intensity
of the cells (Fig. 3A). This delocalization of Pds1-GFP in vps51Δ
is specific to DNA damage-induced G2/M arrest; in large-bud-
ded growing cells and in nocodazole-arrested cells Pds1-GFP is
mostly found in the nucleus (Figs. S2 and S3). Like Pds1-GFP,
Esp1-GFP is strongly concentrated in the nucleus of DSB-arrested
wild-type and rdh54Δ cells 6 h after DSB induction; however, in
vps51Δ cells, nuclear accumulation of Esp1-GFP was not seen
(Fig. 3A). Remarkably, this defect in Esp1-GFP and Pds1-GFP
localization is rescued by deleting PEP4 (Fig.3A). These data
support the idea that—in the GARP mutants—the DNA damage
checkpoint response prevents nuclear accumulation of both Pds1
and Esp1, resulting in prolonged checkpoint-mediated arrest.
Pds1-GFP and Esp1-GFP were often found in endosome-like
structures in the cytoplasm in vps51Δ pep4Δ cells (Fig. 3A). Our
attempts to visualize both Pds1-GFP and endosome/vacuole
membranes were unsuccessful because vps51Δ has a fragmented
vacuole and is defective in the endosomal marker FM4-64 up-
take (34). However, the altered localization and degradation of
WT rdh54 vps51 vps51pep4
% nuclear GFP signal
arrested in G2/M phase for 6 h after galactose induction of HO endonuclease. Images were taken without fixation. (B) Quantitation of the images shown in A.
The percentage of cells displaying nuclear GFP signal was calculated in each case by counting 100 G2/M-arrested cells per sample. (C) Western blotting of Pds1-
GFP and Esp1-myc in the cells arrested by DNA damage. Yeast lysates were prepared 12 h after galactose induction of HO endonuclease. Western blotting of
Rho1 is shown as a loading control. Esp1 protein level was not affected 6 h after the DNA DSB. The anti-PSTAIR antibody was used as a reference control.
Pds1 and Esp1 are mislocalized in the cells arrested by DNA damage. (A) Localization of Esp1 and Pds1 during DSB-induced G2/M arrest. The cells were
Dotiwala et al.PNAS
| Published online November 19, 2012
Pds1 are not only seen in these vps51Δ mutant cells, but also can
be seen in pep4Δ cells after induction of an unrepaired DSB (Fig.
4). To further examine whether Pds1 is targeted to the vacuole
for degradation during DNA damage-induced G2 arrest, we
carefully quantified Pds1-GFP localization in the cell, as described
in Materials and Methods. In WT cells, 6 h after DSB induction,
Pds1-GFP accumulates in the nucleus and is absent from the
vacuole (less signal in the vacuole than in the cytoplasm, as seen
in Fig. 4). In contrast, in pep4Δ cells, Pds1-GFP signal in the
vacuolar area was much higher than in the cytoplasm. Vacuolar
accumulation of Pds1-GFP in pep4Δ was further confirmed by
staining the vacuole with FM4-64 (Fig. S4).
Degradation of Pds1 and Nuclear Localization Defects of Pds1 and
Esp1 in GARP Mutants Are Suppressed by PEP4 Deletion. Reduced
nuclear Pds1 and Esp1 could be due to their mislocalization,
degradation, or both.
By Western blot analysis we found that Pds1-GFP levels were
and that the level of Pds1 in vps51Δ strains was restored by deletion
of PEP4 (Fig. 3C). Importantly, defective nuclear accumulation
of Pds1-GFP in the vps51Δ strain was also restored by deletion of
PEP4 (Fig. 3A). The importance of Pds1 in nuclear localization of
Esp1 has already been established (33). These results are in
caused at least in part by the exclusion of Esp1 from the nucleus
when Pds1 is partially degraded and apparently mislocalized in re-
sponse to the DNA damage checkpoint in these mutant cells. Thus,
Consistent with the Western blotting data, Pds1-GFP is stabilized
in pep4Δ and the GFP signal in the nucleus is much stronger,
possibly explaining how pep4Δ could rescue the Pds1 localization
defect in vps51Δ. These results lead to the surprising conclusion
that cells with defects in retrograde vesicle trafficking fail to adapt
because of the Pep4- and Prb1-dependent degradation of one or
more necessary mitotic activators, including Pds1.
GARP Mutants Are Suppressed by Driving Separase into the Nucleus.
Because Esp1 seems to be excluded from the nucleus after DNA
damage in vps51Δ cells, we hypothesized that we could suppress
the mislocalization of Esp1 by fusing it to the NLS of SV40 (30).
In our experiments the fusion protein gene was transcribed un-
der the control of the normal chromosomal ESP1 promoter at its
normal chromosomal location (Materials and Methods). Indeed,
Esp1-NLSSV40efficiently suppressed the adaptation defects of
vps51Δ (Fig. 5). Esp1-NLSSV40does not affect the permanent
arrest of rdh54Δ or ptc2Δ ptc3Δ, which presumably affects events
entirely within the arrested nucleus. Esp1-NLSSV40did partially
suppress srs2Δ; in addition, wild-type cells adapt more completely
when Esp1-NLS SV40 (superscripted as in other instances) is
expressed. As we do not know the mechanism by which either
srs2Δ or rdh54Δ blocks the process of adaptation, we cannot
account for the difference. These data argue strongly that the
disruption of normal vesicle traffic results in the retention or
inactivation of Esp1 in a cytoplasmic compartment and this can
be overcome when Esp1 is driven into the nucleus
Inhibition of Adaptation in vps51Δ Requires Autophagy via the CVT
Pathway.If Pds1 and/or other factors that are needed to facilitate
Esp1 entry into the nucleus are degraded in vps51Δ strains, then
there should be a pathway to direct these proteins to the vacuole
or some other vesicular compartment harboring Prb1. Two ma-
jor ways in which this might occur should involve either the
targeted (CVT) or general autophagy pathways (35, 36).
We first examined Atg1 and Atg5 mutations that affect both
autophagy pathways (37). In otherwise WT cells, a defect in
autophagy did not affect adaptation, as seen by examining atg1Δ
and atg5Δ mutations (Fig. 6 A and B). However, when we con-
structed atg5Δ vps51Δ and atg1Δ vps51Δ double mutants, we
found that these mutations suppressed the adaptation defect of
vps51Δ (Fig. 6 A and B). Furthermore, deletion of atg5Δ did not
suppress adaptation-defective mutants rdh54Δ, srs2Δ, and ptc2Δ
ptc3Δ. We then examined atg17Δ and atg11Δ that are specifically
required for the bulk and CVT pathways, respectively (37). As
seen in Fig. 6B, atg11Δ suppressed the permanent arrest of
vps51Δ cells whereas atg17Δ did not. These results support the
idea that the DNA damage-induced response involves the tar-
geted degradation of mitotic regulators via the CVT pathway and
that this phenotype is exacerbated in GARP mutants. We note
that vps51Δ cells with knockouts in ESCRT2 (vps25Δ) or the
retromer complex (vps35Δ) are still adaptation defective (Fig. S5).
Deletion of VPS64 is synthetically lethal with pds1Δ at normally
permissive temperatures (31), which could suggest that it plays
an alternative role in transport of Esp1. However, a deletion of
vps64Δ did not have an adaptation defect, nor did it suppress the
vps51Δ adaptation defect. (Fig. S5).
Induction of Increased Autophagy Causes an Adaptation Defect in
Wild-Type Cells. Increased autophagy, in response to nutrient
deprivation or after alkylation-mediated DNA damage, can be
triggered by inactivating the TOR complex with rapamycin
(20, 38). We asked whether the normal adaptation response of
wild-type cells could be blocked by inhibiting TORC1 with rapa-
mycin (Materials and Methods). Because rapamycin-inhibited
can be targeted to vacuole/endosomes. The cells were arrested in G2/M
phase for 6 h after galactose induction of HO endonuclease. Pds1-GFP signal
intensity was pseudocolored to reflect their signal intensity. These samples
were prepared, taken at the same time in the same condition, and the
images were equally processed. Arrows point to the position of vacuoles.
Prevention of vacuolar degradation by pep4Δ reveals that Pds1-GFP
% cells adapted
vps51Δ. Adding the nuclear localization signal (NLS) from SV40 to the C
terminus of Esp1 suppresses the adaptation defect of vps51Δ and partially of
srs2Δ but has no effect on two other adaptation-defective mutations.
Artificial nuclear targeting of Esp1 rescues the adaptation defect of
| www.pnas.org/cgi/doi/10.1073/pnas.1218065109 Dotiwala et al.
cells complete mitosis but fail to complete cytokinesis (39), we
could not use our standard adaptation assay. Instead we moni-
tored the proportion of cells suffering a single DSB that arrested
with a single nucleus (thus adaptation defective) or could complete
mitosis and become binucleate. As shown in Fig. S6, rapamycin
provoked a failure of adaptation. When this assay was repeated
in an atg5Δ strain, we found that nuclear division could proceed,
supporting the idea that hyperactivation of autophagy by rapa-
mycin results in permanent G2/M arrest following induction of a
single unrepaired DSB (Fig. S6).
Because rapamycin has a number of pleiotropic effects (39),
we searched for another method to specifically induce autophagy
following DNA damage. We took advantage of the discovery
that overexpression of a nonphosphorylatable form of ATG13
(ATG13-8SA) induces autophagy without affecting normal cell
cycle progression (40). Here, too, we found that WT cells suffering
a DSB were unable to adapt when ATG13-8SA was overexpressed;
moreover, deleting either ATG1 or PEP4 suppressed the action
of ATG13-8SA overexpression and allowed adaptation (Fig. 7A).
This adaptation defect is dependent on a functional DNA damage
response as a deletion of the MEC1 checkpoint kinase completely
suppressed the effect of ATG13-8SA overexpression, as did a
strain lacking the HO endonuclease cut site (Fig. 7A). Further-
more, we confirmed that the failure to adapt was correlated with
an inability to complete nuclear division by assessing the number
of mononucleate (hence arrested) cells. As seen in Fig. S7, ∼80%
of ATG13-8SA overexpressing cells remain arrested with a single
nucleus at 24 h.
We then examined the localization of both Pds1-GFP and
Esp1-GFP following DNA damage while overexpressing ATG13-
8SA. In these cells both Pds1-GFP and Esp1-GFP are unable to
localize properly following DNA damage, mirroring what we
observe in the GARP mutants (Fig. 8 and Fig. S8). Finally,
we found that atg11Δ was also able to suppress the effect of
ATG13-8SA overexpression whereas atg17Δ did not (Fig. 7A).
These results mimic what was seen in the GARP mutations and
suggest that increasing autophagy in wild-type cells is sufficient
to block adaptation via the CVT pathway.
Rad53 Checkpoint Kinase Is Dephosphorylated in GARP Mutants After
DNA Damage. We had previously shown that during the process of
adaptation the checkpoint kinase RAD53 is robustly phosphor-
ylated in response to DNA damage and subsequently is dephos-
phorylated at the time of adaptation. In some adaptation-defective
mutants, such as rdh54Δ, ptc2Δ, ptc3Δ, or cdc5-ad, Rad53 remains
hyperphosphorylated for at least 24 h (7). In contrast to other
adaptation-defective mutations, vps51Δ cells also dephosphory-
lated Rad53 with kinetics similar to those of WT cells (Fig. 7B),
suggesting that hyperphosphorylation of Rad53 is not responsible
for the permanent arrest defect of GARP mutants. Moreover,
Rad53 is dephosphorylated after 12 h in ATG13-8SA overex-
pressed cells (Fig. 7B), suggesting that these cells closely mimic
the enhanced autophagy conditions seen in vps51Δ cells. These
results suggest that, whereas the DNA damage response is re-
quired to establish cell cycle arrest and designate Pds1’s degra-
dation by the CVT pathway, when autophagy is hyperactivated,
WT vps51 atg11 vps51
% adapted at 24h
Deletion of ATG5 suppresses vps51Δ but has little effect on other adapta-
tion-defective mutants. (B) Deletion of ATG1 and ATG11 but not ATG17
suppresses the adaptation defect of vps51Δ. ** denotes statistically signifi-
cant difference compared with vps51Δ cells. Error bars reflect SEM of three
Inhibition of autophagy rescues the adaptation defect of vps51Δ. (A)
% adapted at 24h
WTWTatg1 pep4 atg11atg17WT(-cs) mec1
+ pGAL ATG13-8SA
0 3 6 9 12 24 27
0 3 6 9 12 15 18 24
WT+ pGAL ATG13-8SA
tation assay was performed as described in Materials and Methods except
that strains harboring plasmids were grown in selective media containing
raffinose overnight to maintain the plasmid and then unbudded cells were
spread onto YEP-Galactose plates and monitored for adaptation at 24 h.
** denotes a statistically significant difference compared with the WT strain
overexpressing ATG13-8SA. Error bars reflect SEM of three independent
experiments. (B) The Rad53 checkpoint kinase is dephosphorylated in GARP
mutants with WT kinetics. The indicated strains were grown in YEP-Lactate
and DNA damage was induced by adding galactose to the cultures. Samples
were taken at various time points denoted and Western blot was performed
as described in Materials and Methods.
(A) Induction of autophagy prevents adaptation in WT cells. Adap-
Dotiwala et al. PNAS
| Published online November 19, 2012
this sequence of events makes it impossible for Esp1 to accu-
mulate in the nucleus and execute the release of anaphase.
Recovery Defects of vps51Δ Involve Prb1-Mediated Degradation and
Autophagy. Recovery is defined as the process by which cells turn
off the DNA damage checkpoint and resume the cell cycle after
repairing the DNA DSB. To assess recovery we used strain
YMV80, which repairs a HO-induced DSB through single-strand
annealing, in which the flanking homologous sequences are
separated by 25 kb and repair requires 6 h (12). To assay recovery,
strains in the YMV80 background were grown in preinduction
media (YEP-Raffinose or YEP-Lactate) and the cells were then
plated on YEPD and YEP-Gal. For each strain viability was
measured as a ratio of the number of colonies on YEP-Gal to
those on YEPD, as well as the percentage of cells arrested as
dumbbells at 24 h. Among previously studied adaptation-defective
mutants only srs2Δ, ptc2Δ ptc3Δ, and sae2Δ are recovery defective
(12). Approximately 35% of vps51Δ cells fail to resume cell cycle
progression (Fig. 9). We also found that overexpression of ATG13-
8SA, which prevented adaptation in WT cells, also prevents re-
covery as measured by colony formation on galactose plates (Fig. 9).
As in adaptation assays, we found that atg11Δ and atg1Δ suppressed
the recovery defect of overexpressing ATG13-8SA (Fig. 9B).
After DNA damage provokes G2/M arrest, there is a transient
activation of the CVT-mediated degradation of Pds1 and the
failure of Esp1 to enter the nucleus to allow resumption of mitosis.
This process is exacerbated when the CVT pathway of autophagy
is hyperactivated, in GARP mutants, by overexpression of ATG13-
8SA or by rapamycin treatment; under these conditions cells
cannot adapt and show reduced recovery even when damage is
repaired. This arrest can be bypassed in at least four ways: (i) by
inactivating the DNA damage checkpoint; (ii) by driving Esp1
into the nucleus without its normal chaperone; (iii) by blocking
autophagy, specifically with mutations that appear to block CVT
pathway; or (iv) by preventing vacuolar proteolysis.
It is striking that deletion of PRB1 or PEP4 can suppress
vps51Δ, not only where the vacuole is fragmented, but also when
ATG13-8SA is overexpressed and vacuolar morphology is ap-
parently normal. One might have imagined that once cargo such
as Pds1 was delivered to the vacuole, it would remain sequestered
and could not escape, undegraded, and thus could not enable
Esp1 to enter the nucleus and initiate anaphase; but preventing
proteolysis does not trap sufficient Pds1 (or some other compo-
nent) to block mitosis. We suggest that there is some sort of
“clogging” of the CVT pathway so that enough Pds1 remains at
in some way reversible in the absence of proteolysis. We note that
theabundance of Pds1is increasedin a pep4Δstrain, back towild-
type levels (Fig. 3); moreover, there is strong localization not only
in the vacuole but also in the nucleus (Fig. 4).
We note that previous studies have suggested that Vps51,
apart from its role in endosome-to-Golgi retrograde transport, is
essential for Cvt targeting (34), whereas our results suggest that
vps51Δ and other GARP mutants actually exacerbate autophagy
via this pathway. One possible explanation for this apparent
% nuclear localization of GFP
and Esp1-GFP. The images were taken as described in Fig. 3A. Representative
images are shown in Fig. S6. The graph denotes the percentage of G2/M-
arrested cells that showed nuclear localization of GFP 6 h after galactose
induction. At least 100 G2/M-arrested cells were counted per sample.
Overexpression of ATG13-8SA causes mislocalization of Pds1-GFP
cells at 24 h
% G2/M arrested cells
vps51Δ prevents recovery from DNA damage. WT (YMV80), vps51Δ, cdc5-ad,
and YMV80+pGAL ATG13-8SA were pregrown in YEP-Lactate and equal
numbers of cells were spread onto YEPD plates or YEP-Gal plates. The ratio
of the number of viable colonies on YEP-Gal vs. YEPD is presented as per-
centage of recovery (solid bars). Cells were pregrown in YEP-Lactate liquid
media and HO expression was induced with galactose for 6 h. Cells were
then spread onto YEP-Gal plates and the cell cycle progression of individual
cells was monitored at 24 h after induction. The percentage of cells that
have not yet recovered and are still arrested at the two-cell body “dumb-
bell” stage is presented as percentage of dumbbells (shaded bars). (B) The
block in adaptation imposed by overexpression of ATG13* is suppressed by
deleting ATG1 or ATG11.
Overexpression of ATG13-8SA (here designated ATG13*) and
| www.pnas.org/cgi/doi/10.1073/pnas.1218065109 Dotiwala et al.
difference is the effects we see are coupled to the induction of
the DNA damage checkpoint, which may alter several cytoplas-
mic targets in addition to the well-documented effects on septins
(19) and dynein-dependent nuclear positioning (41). Thus, the
idea that DNA damage should manifest its response by an al-
teration of cytoplasmic function is not without precedent.
We interpret the defects of GARP mutants in resuming cell
cycle progression after DNA damage as a result of an alteration
in the localization and stability of a key mitotic activator. Most
likely this activator is Esp1 and/or an associated chaperone or
cytoplasmic receptor, because we can suppress the adaptation
and recovery defects of vesicle transport mutants by driving Esp1
into the nucleus with an SV40 NLS. Previous studies have sug-
gested that the transport of Esp1 into the nucleus is highly reg-
ulated, so that even when securin is absent, mitosis is not
executed prematurely (32). After DNA damage there appear to
be additional constraints that prevent Esp1 entry or activity. For
example, even in a pds1Δ strain, there is a substantial cell cycle
delay in response to a single DSB, a delay that is not seen in a
strain lacking the ATR homolog, Mec1 (41). The basis of the
inhibition of Esp1 activity is not known; we suggest that separase
could be restrained in the cytoplasm by association with a trans-
membrane-bound receptor, e.g., in the way that the cyclin Cln3 is
bound at the endoplasmic reticulum (ER) surface and released
by the chaperone Ydj1 (42). Here the release and/or transport of
Esp1 appear to depend on Pds1. Recently, Sarin et al. (31)
identified several possible chaperones whose deletions are syn-
thetically lethal with the absence of Pds1. These proteins are
candidates to be cochaperones with Pds1 in coordinating the
localization of Esp1, but deleting VPS64 did not mimic vps51Δ.
Still, there are important questions remaining. For example, is
adaptation or recovery accompanied by a change in APC-Cdc20
activity? We do know that APC activity is not required for ad-
aptation as it is reflected in the loss of Rad53 hyperphosphorylation
(7, 8) but it should continue to be important for the nuclear deg-
radation of Pds1. It is also intriguing that maintenance of check-
point-mediated arrest of yeast cells to DNA damage (i.e., a partial
loss of telomere protection) depends on APC-Cdh1 (43).
Defects in GARP mutants have revealed that the DNA dam-
age checkpoint prevents nuclear accumulation of Esp1. Our data
suggest that Pds1 is the key factor that is targeted for degradation
in the vacuole. It is likely that transporting excess Pds1 to the
vacuole is ensuring stable cell cycle arrest in G2 phase and that
this transport is likely mediated via the CVT pathway. Consistent
with this observation we find that atg1Δ cells, which are defective
for both autophagy pathways, show a reduced duration of check-
point arrest and adapt more extensively. Thus, the sequestration
and/or degradation of key mitogenic factors by autophagy could
be one way by which cells can maintain a robust checkpoint arrest
in response to DNA damage. We note that other factors may also
be sequestered or degraded, so that the degradation of Pds1
might not be the only, or even most important, alteration. It is
important to note that we also see evidence of vacuolar locali-
zation of Pds1-GFP in normal cells after the induction of an
unrepaired DSB when vacuolar degradation is blocked by PEP4
deletion. The GARP mutants exacerbate trafficking of Pds1 to
the vacuole, but these mutants are exacerbating a normal, under-
Rab6, the homolog of Ypt6, results in mitotic defects that suggest
a second role for this G protein, much as we suggest here (44).
Recently there have been other reports of the autophagic
degradation of proteins critical for DNA repair. The Sae2 pro-
tein is lost from the nucleus and degraded via autophagy in cells
suffering DNA damage when treated with an inhibitor of histone
deacetylase (25). Similarly, the Rnr1 protein of the ribonucleotide
reductase complex is degraded after alkylation damage; more-
over, as we report here, inhibition of the TOR complex exacer-
bates this response (20). The degradation of Sae2 upon valproic
acid treatment is partially rescued by deletion of ATG19, a gene
specifically required for the CVT pathway (25). However, these
experiments did not rule out the possibility that some amount of
the degradation could be mediated via the bulk autophagic
pathway as well. In this paper, we show that deletion of either
ATG1 or ATG11 but not ATG17 suppresses the permanent check-
point arrest of both vps51Δ and ATG13-8SA overexpressing cells.
We suggest, on the basis of our results with the fate of Pds1, that
these nuclear proteins are likely targeted for degradation via the
Another recent report suggests that DNA damage induced by
sublethal doses of camptothecin or etoposide results in autoph-
agy-induced cell death in HeLa cells (45), and treatment of
MCF7 breast cancer cell lines with rapamycin appears to inhibit
both homologous recombination and nonhomologous end-join-
ing pathways (46). However, in the latter study it is not clear
whether the defect is due to autophagic induction or some other
effect of rapamycin treatment. We note that the specific induc-
tion of autophagy in strains that robustly activate the DNA dam-
age checkpoint, but that eventually manage to repair the lesion,
resulted in a dramatic drop in viability. This could be due to an
inability to resume mitosis after DNA damage, defects in homol-
ogous recombination, or a combination of both factors. Further
work will address the contribution of autophagy in these diverse
pathways of homology-driven repair.
As we noted before, the GARP proteins may play roles in
several steps in vesicle transport, but are characterized as being
defective in Golgi-to-ER retrograde transport because that is the
step most clearly affected. However, GARP mutants are just one
of many subsets of vesicle transport gene mutations that com-
monly affect vacuolar protein sorting; but other vps mutations
such as those affecting the Retromer complex or the GET com-
plex have no effect in adaptation after DNA damage. We have,
however, found deletion mutations in other vesicle transport
functions that exhibit similar adaptation defects, including the
ARF-GAP Gcs1 and the TRAPP complex protein Gsg1.
Materials and Methods
Yeast Media, GAL-HO Induction, Adaptation, and Recovery Assays. Yeast cells
were grown in YEPD [1% yeast extract, 2% peptone, 2% dextrose (wt/vol)]
media for normal growth and before spot assays. YEP plus 3% lactic acid
(YEP-Lactate) or YEP plus 2% raffinose (YEP-Raffinose) was used as a pre-
induction medium for most adaptation and recovery assays. Strains carrying
plasmids were either pregrown in synthetic media with raffinose or grown
in synthetic media with dextrose and then inoculated into YEP-Lactate for
preinduction growth of 12–16 h. We have not observed any difference
resulting from these preinduction regimes. For adaptation assays unbudded
G1 cells in the JKM179 background were micromanipulated into grids on
plates of synthetic complete media with 2% galactose supplemented with
1% yeast extract (CYG) or on YEP-Galactose (YEP-Gal) plates and cell cycle
progression was monitored at 8, 24, and 48 h (6). The percentage of cells
with three or more cells or buds is plotted as a measurement of adaptation.
To assay recovery, YMV80 cells (12) were grown overnight in YEP-Lactate
and equal numbers of cells were spread onto YEPD (HO off) and YEP-Gal
plates (HO on). The percentage of recovery is the ratio of the number of
viable colonies (YEP-Gal/YEPD) after 3 d growth at 30 °C. To assess a delay in
recovery we induced HO expression by adding galactose to YEP-Lactate
liquid for 6 h and then plated the cells on YEP-Gal plates. Plates were in-
cubated overnight at 30 °C and the number of cells in individual micro-
colonies was determined 24 h after the initial HO induction.
Strains, Plasmids, and PCR Manipulations. All yeast strains are described in
Table S1. Strain JKM179 and its MATa derivative, JKM139, used for adap-
tation assays, and strain YMV80, used for recovery assays, have been de-
scribed in refs. 6 and 12. All other strains were created by transformation of
either PCR-generated or plasmid-derived deletion fragments or by standard
genetic crosses. Standard PCR conditions and Taq polymerase were used in all
cases. Oligo sequences are available on request. The double-deletion strains
were created by crossing the appropriate single mutants and dissecting
tetrads after sporulating the diploid. We also used rdh54Δ::URA3 (YSL301),
srs2Δ::LEU2 (YSL302), and ptc2Δ::URA3 ptc3Δ::KAN (MCM298). Strains
Dotiwala et al.PNAS
| Published online November 19, 2012
without an HO cut site were created by picking rare colonies that grew on
galactose plates. Such colonies have cut and then religated MAT by impre-
cise end joining, generating a sequence that is no longer recognized by the
HO endonuclease. The JKM179 strain expressing Esp1-NLSSV40, under the
endogenous Esp1 promoter, was created by transforming with the URA3-
marked plasmid pFD026 after cutting with BstAPI.
pFD026 was constructed by amplifying 1.5 kb of 3′ Esp1 ORF, using Esp1
forward and Esp1 reverse primers (NLS sequence included in the primers),
and inserting it into pRS306 using BamHI and EcoRI. Single-copy Esp1 NLSSV40can
be introduced into yeast by transforming after cutting pFD026 with BstAPI.
PDS1-GFP and ESP1-GFP strains were constructed using a single-step PCR
method (47). A 6-aa flexible linker (Gly-Gly-Ser-Gly-Gly-Ser) was inserted
between the ORFs and GFP to secure functionality of the fusion proteins.
Screen for Novel Recovery Mutants. To identify novel checkpoint recovery
mutants, the wild-type recovery strain YMV80 (12) was mutagenized with
the mTn-LacZ-LEU2 library (48), and colonies were assayed for recovery in
a replica-plating regime. A total of 17,500 independent colonies were ana-
lyzed, and the two strongest secondary positive mutants were analyzed
further. One mutant with delayed colony formation (T11) was found to have
the transposon inserted at nucleotide 132 of the 495-nt VPS51 ORF. The
second mutant (T20) was found to have an insertion at nucleotide 1,435 of
the 2,880-nt MSH1 ORF. Cells lacking MSH1 grow poorly on plates contain-
ing ethanol and glycerol as the carbon source, which is the second step in
our screening regime; hence the apparent recovery defect in this strain is
most likely a false positive. This screen was not extended to identify all
Microscopy. Cells were imaged using a Zeiss 200M inverted or a Zeiss AX10
microsope (Carl Zeiss) equipped with an MS-2000 stage (Applied Scientific
Instrumentation), a Lambda LS 175-W Xenon light source (Sutter Instru-
ments), and 63× Plan-Fluar objectives. Image acquisition was performed
with a CoolSnap HQ camera (Roper Scientific). The microscope and camera
were controlled by SlideBook software (Intelligent Imaging Innovations).
All image manipulations and fluorescence intensity measurements were
carried out using SlideBook software and figures were prepared with
Western Blots. Western blotting of Rad53 was performed as described in ref. 7
with anti-RAD53 antibodies (provided by M. Foaini). Briefly, indicated strains
were grown in YEP-Lacate, galactose was added to induce the DSBs, and
they were collected at various time points indicated. Cells were collected by
centrifugation and washed with 20% TCA. All purification steps were per-
formed on ice with prechilled solutions. Cell pellets were resuspended in
500 μL 20% TCA and subjected to glass bead lysis. The suspension was col-
lected, 1 mL of 5% TCA was added to wash the beads, and wash minus the
glass beads was added to the suspension. The precipitated proteins were
collected by centrifugation. Pellets were washed with 1 mL 100% ethanol
and proteins solubilized in 200 μL 1 M Tris (pH8.0)/300 μL 3× SDS/PAGE
loading buffer [60 mM Tris (pH 6.8), 2% SDS, 10% glycerol, 100 mM DTT,
0.2% bromophenol blue]. After 20 min at 95 °C, insoluble material was re-
moved by centrifugation and the supernatant analyzed further (7). Western
blotting of Pds1-GFP and Esp1-GFP was performed as described in ref. 49,
using mouse monoclonal anti-GFP (Roche).
ACKNOWLEDGMENTS. We thank Scott Emr, Richard Kahn, Anita Corbett,
Gerald Johnston, Alan Tartakoff, Vytas Bankaitis, Ilia Ouspenski, Marie-Claude
Marsolier-Kergoat, Nevan Krogan, John McCusker, David Schild, Mike Gustin,
Mike Snyder, Yoshiaki Kamada, and David Stillman for plasmids, advice, and
discussion. Scott Emr suggested the possibility that vacuolar proteolysis could
be involved, and Jonathan Weissman, Sean Collins, Maya Schuldiner, Vlad
Dinec, and other members of the Weissman laboratory provided an invalu-
able sounding board when J.E.H. spent part of a sabbatical at University of
California, San Francisco (UCSF). We thank David Pellman for the use of his
microscope. We thank Kathryn Patterson for help in early stages of this
project. This work was supported by National Institutes of Health Grant
GM61766 (to J.E.H.). J.C.H. was a Fellow of the Leukemia and Lymphoma
Society. J.E.H. benefited from a sabbatical visit to UCSF and support from
UCSF. S.Y. is supported by a Massachusetts Life Sciences Consortium grant.
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Dotiwala et al. 10.1073/pnas.1218065109
% cells beyond G2/M arrest
30 μg/mL nocodazole, such that >95% were arrested as large dumbbells. Plating of individual cells on YEPD plates without nocodazole showed that vps51Δ did
not prevent cells from resuming budding and growth.
Resumption of growth after nocodazole arrest is not affected by vps51Δ. Cells grown in log phase in YEPD liquid medium were arrested for 12 h with
taken and processed equally.
Pds1-GFP and Esp1-GFP localization in cycling cells. GFP signals of living cells from log-phase culture in SC medium were imaged. Both pictures were
Dotiwala et al. www.pnas.org/cgi/content/short/12180651091 of 6
Fig. S3.Localization of Pds1-GFP in nocodazole-arrested cells. The cells were arrested for 4 h with 15 μg/mL nocodazole and the GFP signal was then imaged.
FM4-64 Pds1-GFP merged
Fig. S4. Pds1-GFP accumulates in the vacuole in pep4 cells after DNA damage. The vacuolar membrane of the pep4 cells was visualized with FM4-64 stain.
% cells adapted
the Retromer complex (vps35Δ) or the ESCRT complex (vps25Δ) or by deleting the Vps64 cochaperone. The adaptation defect is, however, rescued but per-
turbing autophagy (atg5Δ).
Adaptation defect in vps51Δ cells is not affected by deleting the Vps64 chaperone. The adaptation defect in vps51Δ cells is not rescued by perturbing
Dotiwala et al. www.pnas.org/cgi/content/short/1218065109 2 of 6
- +Rap - +Rap
% bi-nucleate cells
α-factor, and then induced with galactose for 3 h so that almost all cells in the population suffer DNA damage. At this point rapamycin was added at 2 μg/uL
final concentration to induce autophagy. The cells were collected at the indicated time points after galactose induction, fixed with ethanol, and DAPI stained .
The percentage of large-budded binucleate cells (hence adapted) was assessed at each time point by counting 200 cells per sample.
Addition of rapamycin prevents adaptation in WT cells. JKM179 and atg5Δ were synchronized in G1 by α-factor treatment, filtered to remove
ATG13-8SA overexpressing conditions. The indicated strains were induced with galactose for 6 h and then imaged.
ATG13-8SA (ATG13*) overexpression promotes mislocalization of Pds1 and Esp1. Live cell images are shown of Pds1-GFP and Esp1-GFP in either WT or
Dotiwala et al. www.pnas.org/cgi/content/short/12180651093 of 6
% Binucleate cells at 24h
for 24 h and then DAPI stained to monitor nuclear division. The percentage of binucleate cells is shown for each sample and was calculated by counting at least
150 cells per sample. (Right) Representative images are shown.
ATG13-8SA overexpression in WT cells prevents nuclear division after DNA damage. WT and ATG13-8SA overexpressing cells were galactose induced
Dotiwala et al. www.pnas.org/cgi/content/short/1218065109 4 of 6
Table S1. Yeast strains used in this study
Strain Relevant genotypeReference
JKM179 MATa yku70Δ::URA3
hoΔ hmlΔ::ADE1 MATα hmrΔ::ADE1 GAL-HO::ade3
JKM179 sml1Δ::KAN mec1Δ::NAT
JKM179 MATaΔ leu2-HOcs HIS4::NAT::U2
JKM179 Esp1- NLSSV40:URA3
JKM179 Esp1- NLSSV40:URA3 vps51Δ::KAN
JKM179 Esp1- NLSSV40:URA3 rdh54Δ::URA3
JKM179 Esp1- NLSSV40:URA3 srs2Δ::LEU2
JKM179 Esp1- NLSSV40:URA3 ptc2Δ::URA3 ptc3Δ::KAN
JKM179 pep4Δ::KAN vps51Δ::KAN
JKM179 pep4Δ::KAN rdh54Δ::URA3
JKM179 pep4Δ::KAN srs2Δ::LEU2
JKM179 pep4Δ::KAN ptc2Δ::URA3 ptc3Δ::KAN
JKM139 prb1Δ::KAN vps51Δ::KAN
JKM179 prb1Δ::KAN rdh54Δ::URA3
JKM179 prb1Δ::KAN srs2Δ::LEU2
JKM179 prb1Δ::KAN ptc2Δ::URA3 ptc3Δ::KAN
JKM179 atg5Δ::KAN vps51Δ::KAN
JKM179 atg5Δ::KAN rdh54Δ::URA3
JKM179 atg5Δ::KAN srs2Δ::LEU2
JKM179 atg5Δ::NAT ptc2Δ::URA3 ptc3Δ::KAN
JKM139 vps35Δ::KAN vps51Δ::KAN
JKM179 vps25Δ::HPH vps51Δ::KAN
JKM179 vps64Δ::NAT vps51Δ::KAN
JKM179 pep4Δ::KAN esp1::ESP1-GFP-TRP1
JKM179 pep4Δ::KAN pds1::PDS1-GFP-TRP1
JKM179, vps51Δ::URA3, atg11Δ::KAN
JKM179, vps51Δ::URA3, atg1Δ::KAN
JKM179, vps51Δ::URA3, atg17Δ::KAN
JKM179, mec1Δ::NAT, sml1Δ::KAN,pGAL-ATG13-8SA::URA3
JKM179, atg1Δ:: KAN,pGAL-ATG13-8SA::URA3
JKM179 (HOcs deleted), pGAL-ATG13-8SA::URA3
Lee et al. (1)
Lee et al. (1)
Lee et al. (2)
Shroff et al. (3)
Vaze et al. (4)
Vaze et al. (4)
Dotiwala et al. www.pnas.org/cgi/content/short/1218065109 5 of 6
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