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Molecular and Cellular Biology, March 2001, p. 1997-2007, Vol. 21, No. 6
Department of Molecular Biology, The Scripps
Research Institute, La Jolla, California 92037
Received 1 September 2000/Returned for modification 3 October
2000/Accepted 15 December 2000
In budding yeast, anaphase initiation is controlled by
ubiquitin-dependent degradation of Pds1p. Analysis of pds1
mutants implicated Pds1p in the DNA damage, spindle assembly, and
S-phase checkpoints. Though some components of these pathways are
known, others remain to be identified. Moreover, the essential function of Pds1p, independent of its role in checkpoint control, has not been
elucidated. To identify loci that genetically interact with PDS1, we screened for dosage suppressors of a
temperature-sensitive pds1 allele, pds1-128,
defective for checkpoint control at the permissive temperature and
essential for viability at 37°C. Genetic and functional interactions
of two suppressors are described. RAD23 and
DDI1 suppress the temperature and hydroxyurea, but not radiation or nocodazole, sensitivity of pds1-128.
rad23 and ddi1 mutants are partially defective
in S-phase checkpoint control but are proficient in DNA damage and
spindle assembly checkpoints. Therefore, Rad23p and Ddi1p participate
in a subset of Pds1p-dependent cell cycle controls. Both Rad23p and
Ddi1p contain ubiquitin-associated (UBA) domains which are required for
dosage suppression of pds1-128. UBA domains are found in
several proteins involved in ubiquitin-dependent proteolysis, though no
function has been assigned to them. Deletion of the UBA domains of
Rad23p and Ddi1p renders cells defective in S-phase checkpoint control,
implicating UBA domains in checkpoint signaling. Since Pds1p
destruction, and thus checkpoint regulation of mitosis, depends on
ubiquitin-dependent proteolysis, we propose that the UBA domains
functionally interact with the ubiquitin system to control Pds1p
degradation in response to checkpoint activation.
When DNA is damaged or chromosomes
are incompletely replicated, cells become checkpoint arrested. These
checkpoints avoid replication of damaged template DNA and prevent
aberrant segregation of damaged or partly replicated chromosomes. In
budding yeast, proteolysis of anaphase inhibitors is regulated by these
checkpoint systems. Progression from metaphase to anaphase is inhibited
by Pds1p in Saccharomyces cerevisiae (6, 7, 29,
30). Before anaphase, Pds1p binds to Esp1p, inhibiting its
anaphase-promoting activity (3). During an unperturbed
cell cycle, Pds1p becomes polyubiquitinated at the
metaphase-to-anaphase transition by multienzyme anaphase-promoting
complex (APC)-cyclosome complexes. The modified forms are then
recognized and degraded by 26S proteasomes (7). Once
released from Pds1p, Esp1p activity induces the onset of anaphase.
pds1 mutants fail to execute checkpoint control in response
to DNA damage, spindle poisons, or replication inhibition (4, 29,
30). Pds1p is required for replication checkpoint control only
late in S phase, not in the context of an early S-phase replication block enforced by hydroxyurea (HU) (4, 29, 30). In the presence of 0.1 M HU, replication proceeds more slowly. Under these
conditions, cells perform other aspects of cell cycle progression, budding, and spindle assembly as rapidly as in the absence of HU; cell
cycle arrest in G2 is then necessary to delay anaphase while replication is completed. Growth in the presence of a
non-replication-arresting concentration of HU therefore challenges the
ability of cells to accurately couple S phase with mitosis. Although
pds1 mutants can prevent mitotic progression when
replication is blocked, defective S-phase checkpoint control can be
observed when replication is partially inhibited. Such experiments
revealed a novel checkpoint pathway that is essential late in S phase.
Components of this pathway, other than Pds1p and Mec1p (4;
D. J. Clarke, M. Segal, S. Jensen, and S. I. Reed, submitted
for publication), have not been identified. Moreover, the essential
function of Pds1p at high temperature is not known. To gain insight
into this function, we screened for dosage suppressors of
pds1-128 and have characterized two, encoded by
RAD23 and DDI1. Ubiquitin-associated (UBA)
domains located at the C termini of both Rad23p and Ddi1p are required for suppression of pds1-128. Interestingly, these domains
are found in several proteins involved in ubiquitin-dependent
proteolysis, though no function has been assigned to UBA domains.
Further experiments implicate these UBA domains in checkpoint
signaling: strains with mutant versions of the proteins lacking the UBA
domains are S-phase checkpoint defective.
Yeast strain construction and growth conditions.
All strains
are derivatives of BF264-15DU MATa ade1
his2 leu2-3,112 trp1-1a ura3 Dns (20).
Cultures were grown at 30°C, unless otherwise stated, in yeast
extract-peptone (YEP) medium containing 2% dextrose, raffinose, or
galactose. Strains were constructed according to standard procedures
(22) except for gene disruptions (27). pds1 Yeast strain genotypes.
Genotypes used are shown in Table
1. Oligonucleotides used are shown in
Table 2.
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.6.1997-2007.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Dosage Suppressors of pds1 Implicate
Ubiquitin-Associated Domains in Checkpoint Control
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
cells were temperature sensitive at 28°C on
YEP-dextrose plates in the BF264-15DU genetic background.
Rad23p and Ddi1p were epitope tagged at the C-terminal extremities of
their genomic coding regions with either six Myc or six His epitopes.
The C-terminal region of each coding sequence was amplified by PCR
using oligonucleotides that included NotI or SalI
sites and then cloned into tagging vectors in frame with the epitope.
Such constructs were linearized by restriction enzyme digestion within
the RAD23 or DDI1 open reading frame (ORF) and
then integrated at the endogenous RAD23 and DDI1
loci. GAL1:RAD23 and
GAL1:DDI1 strains were constructed by amplifying
the RAD23 and DDI1 coding sequences, cloning into a yeast integration vector behind a GAL1 promoter and then
integrating at the LEU2 locus. Epitope tags could then be
integrated behind the GAL1:RAD23 and
GAL1:DDI1 sequences.
rad23
uba2 and
ddi1uba mutant alleles, expressed
endogenously or under GAL1 promoter control, were
constructed by integrating epitope tags directly 3' of the UBA domain
sequences, thus truncating the corresponding coding sequence.
Temperature-sensitive esp1 strains were generated by
PCR-based mutagenesis (13, 18).
TABLE 1.
Yeast strain genotypes
TABLE 2.
Oligonucleotide used in plasmid or strain construction
Isolation of pds1-128 dosage suppressors. pds1-128 suppressors were isolated by transformation with an S. cerevisiae genomic library contained in a vector which is maintained at high copy number in yeast cells (YEp24 library [2]). Transformants were replicated onto YEP-galactose plates, and suppressors were selected at 37°C. About 160,000 transformants were analyzed to identify 110 colonies rescued in a plasmid-dependent manner; 80 of these colonies appeared wild type (WT) at 37°C on YEP-galactose medium. Of the 13 clones examined further, all contained PDS1; 30 of the 110 colonies grew more poorly than WT at 37°C on YEP-galactose medium. Plasmids isolated from each of these clones contained either ESP1, RAD23, or DDI1.
Cell biology protocols.
Samples were prepared for FACScan
analysis as described elsewhere (14). Spindles were
visualized by fluorescence microscopy by using strains expressing a
TUB1-GFP construct (23). Cells were gamma
irradiated in liquid medium (3.5 Gy per min). For G1 arrest, strains were grown overnight to saturation and then diluted to
an optical density of 0.15 to 0.2; then 
-factor (200 ng/ml) was
added; 90 to 100% arrest was complete within 2 to 2.5 h at 26 to
30°C. For experiments in which spindle morphology was analyzed, it
was necessary to grow cells overnight in synthetic medium to allow good
visualization of the spindles. When induction of genes from the
GAL1 promoter was required, overnight cultures were grown in
YEP-raffinose. Synchrony was carried out in rich medium as described in
the text.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Dosage suppressors of pds1-128.
A genetic strategy
was used to find proteins that functionally interact with Pds1p. We
screened for genes whose increased dosage suppressed the temperature
sensitivity of pds1-128. From a genomic S. cerevisiae library (maintained at high copy number in a yeast
episomal vector), three dosage suppressors were identified: (i)
ESP1 (13 independent isolates, representing six plasmid
types), (ii) RAD23 (4 isolates, one plasmid type), and (iii)
DDI1 (8 isolates, two plasmid types). Since a genetic
interaction between ESP1 and pds1 was previously
reported (7), two novel suppressors of pds1-128,
RAD23 and DDI1, were identified (Fig.
1a).
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Specificity of pds1-128 dosage suppression.
Additional criteria were used to assess the specificity of the genetic
interactions with pds1-128: first, whether the suppressors could rescue a pds1 null mutant (pds1); and
second, whether genetic interactions with esp1 (encoding a
protein that physically interacts with Pds1p) could be detected (Fig.
1a and b).
cells at 28°C, the restrictive
temperature of pds1 (strains harboring 2 µm plasmids or
integrated versions of RAD23 and DDI1 induced
from the GAL1 promoter). Thus, high dosage of
DDI1 or RAD23 cannot bypass the
pds1 temperature sensitivity.
If Rad23p and Ddi1p are specific regulators of Pds1p, a genetic
interaction with esp1 mutants should be detectable. To test this, for this purpose, we constructed WT or esp1 mutant
strains which express RAD23 or DDI1 from the
inducible GAL1 promoter. Expression of either gene in WT
cells had little effect on cell growth (not shown), but the lethality
of strains with temperature-sensitive esp1 alleles
(esp1-N5 and esp1-B7 [13]) was
enhanced by overexpression of RAD23 or DDI1 (Fig.
1b). This result can be rationalized in the context of the model
described above; Pds1p inhibits the anaphase-promoting function of
Esp1p, and Rad 23p and Ddi1p are putative positive regulators of Pds1p
function. Therefore, high-dosage RAD23 and DDI1
would be expected to further compromise mutant esp1
function, resulting in increased temperature sensitivity.
The genetic interactions of RAD23 and DDI1 with
pds1-128 were also examined in the context of
Pds1p-dependent checkpoint pathways. The ability of RAD23 or
DDI1 to rescue pds1-128 temperature sensitivity in mec1-1, rad53-1 or chk1 mutant backgrounds
was examined (Fig. 1c). Experiments with the YEp24-based plasmids,
maintained at high copy number in yeast cells, or with RAD23
and DDI1 expressed from the GAL1 promoter gave
essentially the same results. pds1-128 rad53-1 and
pds1-128 chk1 cells were temperature sensitive at 37°C,
as are pds1-128 cells, and viability was restored by high dosage of the suppressors (not shown). Using the same analysis, pds1-128 mec1-1 cells were only partly rescued by
RAD23 and DDI1 overexpression (up to 35°C).
pds1-128 mec1-1 cells were temperature sensitive at 35°C,
though mec1-1 single mutants were not temperature sensitive.
The inviability of pds1-128 mec1-1 cells overexpressing RAD23 or DDI1 at 36°C suggests that rescue is
partly dependent on Mec1p. Rad23p, Ddi1p, and Mec1p may function in a
common pathway that targets Pds1p.
Dosage suppression of the pds1-128 S-phase checkpoint defect. The genetic interactions of RAD23 and DDI1 with pds1-128 were examined in the context of documented pds1 checkpoint defects. We tested whether induction of GAL1:RAD23 or GAL1:DDI1 was able to suppress the sensitivity of pds1-128 to nocodazole, gamma irradiation, or HU to examine whether Rad23p and Ddi1p could function in the spindle assembly, DNA damage, or S-phase checkpoint pathways. There was no suppression of the gamma irradiation sensitivity of pds1-128 (data not shown). In contrast, a high-copy-number plasmid containing the pds1-128 allele (YEp-pds1-128) did rescue the gamma irradiation sensitivity. Thus, increasing the amount of the mutant Pds1-128p corrected the DNA damage checkpoint defect of these cells. Similarly, YEp-pds1-128 rescued the temperature sensitivity of pds1-128 cells. By Western blot analysis, pds1-128 cells have substantially less Pds1 than do WT cells (13). Therefore, in the context of the DNA damage checkpoint, RAD23 or DDI1 overexpression presumably cannot increase the amount of Pds1-128p. Overexpression of RAD23 or DDI1 was also unable to rescue the nocodazole sensitivity of pds1-128 cells (data not shown). In this case, however, YEp-pds1-128 was similarly unable to rescue lethality caused by nocodazole treatment. Therefore, it was not possible to infer whether or not RAD23 and DDI1 overexpression affects Pds1-128p levels in the context of the spindle assembly checkpoint.
In contrast, induction of GAL1:RAD23 or GAL1:DDI1 did suppress the sensitivity of pds1-128 cells to HU (Fig. 2a). Growth on HU plates was also restored by YEp-pds1-128 (not shown). Crucially, GAL1:RAD23 and GAL1:DDI1 could not rescue the HU sensitivity of pds1 strains,
revealing that rescue was dependent on the presence of Pds1-128p.
High-copy-number RAD23 did increase Pds1-128p levels, and
this effect was enhanced in the context of the S-phase checkpoint response (Fig. 2b). Moreover, overexpression of RAD23 was
not able to rescue the HU sensitivity of a rad53-1 strain
(another S-phase checkpoint-defective mutant), indicating that not all HU-sensitive checkpoint mutants are suppressed. The above experiments revealed that RAD23 and DDI1 are not dosage
suppressors of the DNA damage checkpoint defect of pds1-128
mutant cells but can suppress the S-phase checkpoint defect. The data
suggest that Rad23p and Ddi1p function specifically in a
Pds1p-dependent S-phase checkpoint control.
|
) or galactose (+) medium to repress or induce
GAL1:RAD23. Cellular protein extracts were separated by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis and
Pds1-128-p-HA was detected after Western blotting. Cdc28p levels served
as a loading control. A light exposure and a darker exposure of the
same blot are shown.
Role of Rad23p and Ddi1p UBA domains in dosage suppression of
pds1-128.
One striking similarity between the C
termini of Rad23p and Ddi1p is that both contain a UBA domain (Rad23p
has a second, internally located UBA domain [Fig.
3a]). These are small domains (each
about 10% of the full-length protein) of unknown function. Six UBA
domain-containing proteins have been identified in budding yeast.
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uba2 and
GAL1:ddi1uba) were unable to rescue the
temperature sensitivity of pds1-128, though rescue was
achieved by full-length versions of either protein (Fig. 3b).
Suppression was also achieved by a Rad23p construct that lacked the
N-terminal ubiquitin-like (UBL) domain and also by a Rad23p mutant
lacking the internal UBA domain. Thus, the C-terminal UBA domains of
Rad23p and Ddi1p seem to be the critical domains for
pds1-128 suppression. In the case of Ddi1p, we also constructed point mutants of the UBA domain. One such mutant, L426A,
could be expected to disrupt UBA integrity, based on structure prediction. In yeast two-hybrid experiments, this mutant was able to
form Ddi1p-Ddi1p homodimers (B. L. Bertolaet and S. L. Reed, unpublished data), demonstrating that other than the UBA domain, the
protein must be structurally intact. L426A did not, however, have the
ability to suppress pds1-128 temperature sensitivity (Fig.
3b). Thus, L426A specifically abolished the ability of Ddi1p to
suppress pds1-128. The C-terminal UBA domains of Rad23p and Ddi1p were also required for enhancement of esp1 temperature
sensitivity (Fig. 3d and data not shown).
The ability of the mutant forms to rescue the HU sensitivity of
pds1-128 was also examined. Induction of
GAL1:ddi1uba was unable to rescue
pds1-128 HU sensitivity, whereas deletion of UBA2 from
Rad23p produced a mutant that could only partially rescue the HU
sensitivity (Fig. 3c). Therefore, the UBA domain of Ddi1p was essential
for suppression of the pds1-128 HU sensitivity, and deletion
of the C-terminal UBA domain of Rad23p reduced the ability of Rad23p to
rescue the HU sensitivity of pds1-128 cells.
rad23 ddi1 null mutants are sensitive to HU and
defective in cell cycle progression in the presence of HU.
The
genetic and functional interactions of RAD23 and
DDI1 with pds1-128 suggested that Rad23p and
Ddi1p may function in the S-phase checkpoint pathway and that the
putative checkpoint function of Rad23p and Ddi1p depends on conserved
UBA domains. It could therefore be expected that loss-of-function
alleles of RAD23 or DDI1 would result in loss of
S-phase checkpoint control. The common UBA domains of Rad23p and Ddi1p
suggested that the two proteins may have some functional
redundancy. We therefore constructed RAD23 and
DDI1 deletion strains and tested their HU sensitivity. (Fig.
4a). rad23 ddi1 cells
were as sensitive to HU as are pds1-128 cells, though
rad23 and ddi1 single mutants were either
much less sensitive (rad23) or not sensitive at all
(ddi1). rad23 ddi1 cells were not
sensitive to gamma irradiation (Fig. 4b) or nocodazole (not shown).
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ddi1 cells are S-phase
checkpoint defective, a rebudding assay was performed (Fig.
5a). Cells released from G1
following -factor arrest were grown on solid medium containing 100 mM HU, and cell bodies per microcolony were counted. For WT pds1-128 and rad23 ddi1 cells, budding
occurred with very similar timing (not shown). The premature appearance
of colonies containing three or more cell bodies (rebudding) would
indicate an uncoupling of the normal timing of cell cycle progression
from S phase. In these experiments, pds1-128 cells rebudded
before WT cells. However, if anything, rad23 ddi1
cells took longer to rebud than WT cells, suggesting a slight delay
before or during mitosis (Fig. 5a). In a similar experiment, in which
the strains were gamma irradiated before plating onto rich medium, WT
and rad23 ddi1 cells rebudded with indistinguishable
timing (Fig. 5b), whereas pds1-128 cells rebudded before WT
and rad23 ddi1 cells, due to the DNA damage checkpoint
defect of this strain (Fig. 5b and reference 29).
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ddi1 cells
in the presence of HU, we directly compared S-phase progression with the onset of anaphase following release from G1 (scoring
budding, mitotic spindle formation using a TUB1-GFP
construct, and DNA replication by FACscan analysis [Fig. 5c]).
Budding, spindle formation, and DNA replication (FACScan data not
shown) occurred with similar timing in each strain. Short
G2 spindles were defined as those between 1 and 2 µm long
in these experiments. Strikingly, spindle elongation began in the
rad23 ddi1 cells at the same time as in
pds1-128 cells, when about two-thirds of the DNA had been
replicated. At this time, while spindles of WT cells remained 1 to 2 µm in length, spindles of rad23 ddi1 cells elongated
to 4 to 6 µm. However, while spindles fully elongated (to 8 to 12 µm) and then disassembled in a timely manner in pds1-128
cells, rad23 ddi1 spindles remained about 4 to 6 µm
in length (midsized spindles), consistent with a midanaphase delay.
rad23 ddi1 cells eventually elongated their spindles
fully and exited mitosis (not shown), but later than WT cells did, in
agreement with the rebudding experiments. These data suggest that
rad23 ddi1 cells may be defective for restraining
anaphase onset in HU but are unable to complete spindle elongation and
exit mitosis rapidly.
Role of Rad23p and Ddi1p UBA domains in S-phase checkpoint
control.
The potential dual roles of Rad23p in nucleotide excision
repair (NER) and checkpoint control might result in a somewhat
ambiguous checkpoint-defective phenotype in the rad23
ddi1 mutant. Although these cells appeared to initiate anaphase
before S phase was complete, they also appeared to be delayed for
progression through the latter parts of mitosis. To resolve this issue,
we attempted to create strains with mutant versions of Rad23p and Ddi1p
that would be defective for checkpoint control but not compromised in
NER or in the ability to progress through mitosis. We constructed
strains in which endogenous RAD23 and DDI1 were
replaced with mutant versions lacking the C-terminal UBA domains
(rad23uba2 and
ddi1uba), based on the genetic evidence that
these mutant versions cannot suppress pds1-128. The UBA
domain deletion alleles were epitope tagged to confirm that expression
levels of the resulting products were similar to those of the WT
proteins (not shown). We reasoned that since these domains are at least
partly required for suppression of pds1-128 HU sensitivity,
it may be possible to detect a checkpoint defect in strains with UBA
domain deletions. Either a rad23uba2 or a
ddi1uba mutation showed only a marginal
sensitivity to HU (not shown). In contrast, strains with both mutated
alleles were HU sensitive (Fig. 6a),
though not as sensitive as pds1-128
strains. This suggested that rad23uba2
ddi1uba cells might be partially defective in
S-phase checkpoint regulation. rad23uba2
ddi1uba cells were not sensitive to nocodazole,
gamma irradiation, or UV irradiation (not shown), correlating with the
failure of high dosage RAD23 and DDI1 to rescue
the sensitivity of pds1-128 to these agents and confirming
that the rad23uba2 ddi1uba
strain is NER competent.
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uba2
ddi1uba cells are S-phase checkpoint defective, the
rebudding assay was performed on medium containing 100 mM HU (Fig. 6b).
WT, pds1-128, and rad23uba2
ddi1uba cells budded with similar timing, but
pds1-128 and rad23uba2
ddi1uba cells clearly rebudded prematurely, well
before WT cells did. As a control, the rebudding assay was performed
following gamma irradiation of G1 cells (as described
for Fig. 5). In this case, the behavior of
rad23uba2 ddi1uba cells
was indistinguishable from that of WT cells, demonstrating that the DNA
damage checkpoint is intact in this mutant strain.
Next we compared S-phase progression with the onset of anaphase
(measuring spindle elongation or loss of sister chromatid cohesion)
following release from G1 (Fig. 6c and d). Spindle
formation and progression through S-phase occurred with similar timing
in WT and rad23uba2 ddi1uba
cells. WT cells reached G2 about 5 h following release
from G1 and then initiated anaphase ~30 to 40 min later.
In contrast, spindle elongation in the rad23uba2
ddi1uba mutant began earlier than in WT cells and
before the cells reached a 2C DNA content. The
rad23uba2 ddi1uba mutant cells
did not become delayed partway through spindle elongation, with 4- to
6-µm spindles, as was the case for the rad23 ddi1 mutant. Compared directly to pds1-128 cells (not shown),
anaphase began at an intermediate time in
rad23uba2 ddi1uba cells, later
than in pds1-128 cells but consistently earlier than in WT
cells. When loss of sister chromatid cohesion was used as an indicator
of anaphase onset, similar results were consistently obtained (Fig.
6d). These kinetics correlate well with the relative sensitivities of
rad23uba2 ddi1uba and
pds1-128 cells to HU and suggest that
rad23uba2 ddi1uba cells are
partially defective in S-phase checkpoint control. The advancement of
anaphase onset and of rebudding in the rad23uba2
ddi1uba mutant clearly implicates the UBA domains
of Rad23p and Ddi1p in cell cycle control.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Genetic interactions of pds1-128 with RAD23
and DDI1.
The key role of Pds1p in M-phase cell cycle
control has been well documented, but less is known about factors
regulating Pds1p in response to checkpoint signals. We have used a
genetic screen to identify proteins that interact with Pds1p. High
dosage of RAD23 or DDI1 suppressed the
temperature sensitivity of pds1-128 and rescued the HU
sensitivity of pds1-128 at the permissive temperature. Neither the temperature sensitivity nor the HU sensitivity was rescued
by RAD23 or DDI1 overexpression in
pds1 strains; there was no bypass of the cellular
requirement for Pds1p. In agreement, high-dosage RAD23 and
DDI1 enhanced the temperature sensitivity of esp1
mutants. This would be expected of proteins that positively regulate
Pds1p, since Esp1p separin activity, required for progression through
mitosis, is negatively regulated by Pds1p. These interactions implicate
Rad23p and Ddi1p in promoting Pds1p-dependent functions. Moreover,
RAD23 and DDI1 specifically corrected the S-phase
checkpoint defect of pds1-128 cells. Neither the gamma
irradiation sensitivity nor the nocodazole sensitivity of
pds1-128 was rescued by overexpressing RAD23 and
DDI1. Thus, these data implicate Rad23p and Ddi1p in subset
of the Pds1p-dependent checkpoint pathways.
UBA domains of Rad23p and Ddi1p are required for S-phase checkpoint
control.
Rad23p is a highly conserved protein with a NER function.
The role of Rad23 in NER depends on binding to the NER complex via its
XPC-binding domain and on an interaction with the proteasome cap via
its UBL domain (9-11, 16, 17, 24, 28). Almost nothing is
known about the function of Ddi1p. It has an upstream activation domain
identical to that of RAD23 and is induced by DNA damage and
HU, but unlike rad23 mutants, ddi1 strains
are not sensitive to UV light (data not shown) and are therefore not required for NER.
uba2
ddi1uba cells were not sensitive to gamma
irradiation or nocodazole but were HU sensitive and underwent an
advanced loss of sister chromatid cohesion and spindle elongation
compared to WT cells when grown in the presence of 100 mM HU.
rad23uba2 ddi1uba mutant cells
also exited mitosis and rebudded prematurely in the presence of 100 mM
HU. It is not clear whether this rapid exit from mitosis could
contribute to the HU sensitivity of these cells. Still, rapid exit from
mitosis could be accounted for by deregulation of Pds1p proteolysis
during anaphase in the rad23uba2
ddi1uba mutant. As well as regulating anaphase
onset, Pds1p plays a role in controlling exit from mitosis (1, 8,
15, 25). Although some Pds1p is degraded at the onset of
anaphase (7), a subfraction of Pds1p remains and is
localized on anaphase spindles (13). After anaphase onset,
Pds1p inhibits exit from mitosis (5, 26). Thus,
rad23uba2 ddi1uba cells may be
unable to efficiently regulate Pds1p proteolysis to control the onset
of anaphase and to ensure a timely disassembly of anaphase spindles and
subsequent exit from mitosis.
Regulation of Pds1p by Rad23p and Ddi1p?
DNA damage, spindle
perturbations, and replication inhibition increase Pds1p stability,
which in turn inhibits the onset of anaphase. We have shown that
RAD23 overexpression increases the level of Pds1-128p in a
manner that is enhanced by S-phase checkpoint activation. The proposal
that Rad23p and Ddi1p regulate Pds1p stability is an attractive one
given the dependence of rescue on UBA domains. The function of UBA
domains is not known, though they are present in different classes of
enzyme involved in ubiquitin-dependent proteolysis (12),
an intriguing coincidence given the dependence of Pds1p proteolysis on
the ubiquitin system. Rad23p also has a UBL domain at its N terminus;
this domain was recently shown to be required for a physical
interaction with 26S proteasomes, the particles that degrade
ubiquitinated Pds1p to initiate the onset of anaphase. However,
deletion of this domain did not abrogate the ability of Rad23p to
rescue pds1-128 when overproduced, and other data implicate
the UBL domain in the NER function of Rad23p (21, 28). An
alternative model for suppression of pds1-128 by
RAD23 and DDI1 was recently suggested by the
finding that both Rad23p and Ddi1p could bind to ubiquitin and that
this interaction was dependent on the UBA domains (Bertolaet and Reed,
unpublished). These data make it tempting to propose an intriguing
mechanism for Rad23p- and Ddi1p-dependent checkpoint regulation. Rad23p and Ddi1p could regulate Pds1p stability by binding to ubiquitinated Pds1p or to the ubiquitin ligase complex that ubiquitinates Pds1p via
the UBA domains (Fig. 7). Indeed, a
recent report described a novel activity of Rad23p as an inhibitor of
ubiquitin chain elongation (19). Rad23p was shown to bind
to a mono- or a diubiquitinated substrate and prevent extension of the
ubiquitin chain. A similar interaction with ubiquitinated Pds1p, or the
ubiquitin ligase complex, stimulated by ongoing DNA replication, may
provide one mechanism through which the S-phase checkpoint is enforced.
How UBA domains participate in such control systems is currently being addressed.
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ACKNOWLEDGMENTS |
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We thank T. Weinert for strains, A. Straight for the TUB1-GFP construct, and L. Prakash for anti-Rad23p antibodies.
M.S. was supported by EMBO and HFSP fellowships, D.J.C. was supported by an EMBO fellowship and a U.S. Army Medical Research Materiel Command Breast Cancer Research Fellowship, B.L.B. was supported by fellowships from the NIH and University of California Office of the President, and S.J. was supported by a fellowship from the Danish Medical Research Council. This work was supported by NIH grant GM38328 (S.I.R.).
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Rd., La Jolla, CA 92037. Phone: (858) 784-9836. Fax: (858) 784-2781. E-mail: sreed{at}scripps.edu.
Present address: Sanofi-Synthelabo, Centre de Recherche de Labege,
31676 Labege, France.
Present address: Triad Therapeutics, San Diego, CA 92121.
§ Present address: National Institute for Medical Research, Division of Yeast Genetics, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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| 1. | Alexandru, G., W. Zachariae, A. Schleiffer, and K. Nasmyth. 1999. Sister chromatid separation and chromosome re-duplication are regulated by different mechanisms in response to spindle damage. EMBO J. 18:2707-2721[CrossRef][Medline]. |
| 2. | Carlson, M., and D. Botstein. 1982. Two differentially regulated mRNAs with different 5' ends encode secreted with intracellular forms of yeast invertase. Cell 28:145-154[CrossRef][Medline]. |
| 3. | Ciosk, R., W. Zachariae, C. Michaelis, A. Shevchenko, M. Mann, and K. Nasmyth. 1998. An ESP1/PDS1 complex regulates loss of sister chromatid cohesion at the metaphase to anaphase transition in yeast. Cell 93:1067-1076[CrossRef][Medline]. |
| 4. | Clarke, D. J., M. Segal, G. Mondesert, and S. I. Reed. 1999. The Pds1 anaphase inhibitor and Mec1 kinase define distinct checkpoints coupling S phase with mitosis in budding yeast. Curr. Biol. 9:365-368[CrossRef][Medline]. |
| 5. |
Cohen, F. O., and D. Koshland.
1999.
Pds1p of budding yeast has dual roles: inhibition of anaphase initiation and regulation of mitotic exit.
Genes Dev.
13:1950-1959 |
| 6. |
Cohen-Fix, O., and D. Koshland.
1997.
The anaphase inhibitor of Saccharomyces cerevisiae Pds1p is a target of the DNA damage checkpoint pathway.
Proc. Natl. Acad. Sci. USA
94:14361-14366 |
| 7. |
Cohen-Fix, O.,
J. M. Peters,
M. W. Kirschner, and D. Koshland.
1996.
Anaphase initiation in Saccharomyces cerevisiae is controlled by the APC-dependent degradation of the anaphase inhibitor Pds1p.
Genes Dev.
10:3081-3093 |
| 8. |
Fraschini, R.,
E. Formenti,
G. Lucchini, and S. Piatti.
1999.
Budding yeast Bub2 is localized at spindle pole bodies and activates the mitotic checkpoint via a different pathway from Mad2.
J. Cell Biol.
145:979-991 |
| 9. |
Guzder, S. N.,
Y. Habraken,
P. Sung,
L. Prakash, and S. Prakash.
1995.
Reconstitution of yeast nucleotide excision repair with purified Rad proteins, replication protein A, and transcription factor TFIIH.
J. Biol. Chem.
270:12973-12976 |
| 10. |
Guzder, S. N.,
P. Sung,
L. Prakash, and S. Prakash.
1998.
Affinity of yeast nucleotide excision repair factor 2, consisting of the Rad4 and Rad23 proteins, for ultraviolet damaged DNA.
J. Biol. Chem.
273:31541-31546 |
| 11. |
He, Z.,
J. Wong,
H. S. Maniar,
S. J. Brill, and C. J. Ingles.
1996.
Assessing the requirements for nucleotide excision repair proteins of Saccharomyces cerevisiae in an in vitro system.
J. Biol. Chem.
271:28243-28249 |
| 12. | Hofmann, K., and P. Bucher. 1996. The UBA domain: a sequence motif present in multiple enzyme classes of the ubiquitination pathway. Trends Biochem. Sci 21:172-173[CrossRef][Medline]. |
| 13. |
Jensen, S.,
M. Segal,
D. J. Clarke, and S. I. Reed.
2001.
A novel role of the budding yeast separin Esp1 in anaphase spindle elongation: evidence that proper spindle association of Esp1 is regulated by Pds1.
J. Cell Biol.
152:27-40 |
| 14. | Lew, D. J., N. J. Marini, and S. I. Reed. 1992. Different G1 cyclins control the timing of cell cycle commitment in mother and daughter cells in the budding yeast Saccharomyces cerevisiae. Cell 69:317-327[CrossRef][Medline]. |
| 15. |
Li, R.
1999.
Bifurcation of the mitotic checkpoint pathway in budding yeast.
Proc. Natl. Acad. Sci. USA
96:4989-4994 |
| 16. | Masutani, C., M. Araki, K. Sugasawa, P. J. van der Spek, A. Yamada, A. Uchida, T. Maekawa, D. Bootsma, J. H. Hoeijmakers, and F. Hanaoka. 1997. Identification and characterization of XPC-binding domain of hHR23B. Mol. Cell. Biol. 17:6915-6923[Abstract]. |
| 17. | Mueller, J. P., and M. J. Smerdon. 1996. Rad23 is required for transcription-coupled repair and efficient overrall repair in Saccharomyces cerevisiae. Mol. Cell. Biol. 16:2361-2368[Abstract]. |
| 18. | Muhlrad, D., R. Hunter, and R. Parker. 1992. A rapid method for localized mutagenesis of yeast genes. Yeast 8:79-82[CrossRef][Medline]. |
| 19. | Ortolan, T. G., P. Tongaonkar, D. Lambertson, L. Chen, C. Schauber, and K. Madura. 2000. The DNA repair protein Rad23 is a negative regulator of multi-ubiquitin chain assembly. Nat. Cell Biol. 2:601-608[CrossRef][Medline]. |
| 20. | Richardson, H. E., C. Wittenberg, F. R. Cross, and S. I. Reed. 1989. An essential G1 function for cyclin-like proteins in yeast. Cell 59:1127-1133[CrossRef][Medline]. |
| 21. | Russell, S. J., S. H. Reed, W. Huang, E. C. Friedberg, and S. A. Johnston. 1999. The 19S regulatory complex of the proteasome functions independently of proteolysis in nucleotide excision repair. Mol. Cell 3:687-695[CrossRef][Medline]. |
| 22. | Sherman, F., G. Fink, and J. B. Hicks. 1982. Methods in yeast genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. |
| 23. |
Straight, A. F.,
W. F. Marshall,
J. W. Sedat, and A. W. Murray.
1997.
Mitosis in living budding yeast: anaphase A but no metaphase plate.
Science
277:574-578 |
| 24. | Sugasawa, K., C. Masutani, A. Uchida, T. Maekawa, P. J. van der Spek, D. Bootsma, J. H. Hoeijmakers, and F. Hanaoka. 1996. HHR23B, a human Rad23 homolog, stimulates XPC protein in nucleotide excision repair in vitro. Mol. Cell. Biol. 16:4852-4861[Abstract]. |
| 25. |
Tavormina, P. A., and D. J. Burke.
1998.
Cell cycle arrest in cdc20 mutants of Saccharomyces cerevisiae is independent of Ndc10p and kinetochore function but requires a subset of spindle checkpoint genes.
Genetics
148:1701-1713 |
| 26. |
Tinker, K. R., and D. O. Morgan.
1999.
Pds1 and Esp1 control both anaphase and mitotic exit in normal cells and after DNA damage.
Genes Dev.
13:1936-1949 |
| 27. | Wach, A., A. Brachat, R. Pohlmann, and P. Philippsen. 1994. New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 10:1793-1808[CrossRef][Medline]. |
| 28. |
Watkins, J. F.,
P. Sung,
L. Prakash, and S. Prakash.
1993.
The Saccharomyces cerevisiae DNA repair gene RAD23 encodes a nuclear protein containing a ubiquitin-like domain required for biological function.
Mol. Cell. Biol.
13:7757-7765 |
| 29. |
Yamamoto, A.,
V. Guacci, and D. Koshland.
1996.
Pds1p is required for faithful execution of anaphase in the yeast, Saccharomyces cerevisiae.
J. Cell Biol.
133:85-97 |
| 30. |
Yamamoto, A.,
V. Guacci, and D. Koshland.
1996.
Pds1p, an inhibitor of anaphase in budding yeast, plays a critical role in the APC and checkpoint pathways(s).
J. Cell Biol.
133:99-110 |
Molecular and Cellular Biology, March 2001, p. 1997-2007, Vol. 21, No. 6
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.6.1997-2007.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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