Imperial Cancer Research Fund Molecular Oncology Laboratory, University of Oxford Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, UK
* Author for correspondence (e-mail: norbury{at}icrf.icnet.uk)
Accepted 26 October 2001
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Summary |
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Key words: Schizosaccharomyces pombe, Cohesin, Mitosis, Sister chromatid cohesion
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Introduction |
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S. cerevisiae Pds5 is essential for maintaining viability during
the mitotic cell cycle. Temperature sensitive S. cerevisiae pds5
mutants were identified in a screen for selective loss of viability during a
brief mitotic arrest in comparison with a G1 arrest
(Hartman et al., 2000). On
shifting such pds5 mutants to the restrictive temperature, most cells
fail to complete mitosis, being delayed in anaphase or telophase. This
terminal phenotype suggests that the cohesion-promoting activity of Pds5 is
essential for cell viability. This interpretation is strengthened by the
finding that the bimD6 mutation in A. nidulans can be
suppressed by mutation of sudA, which encodes an Smc3-like protein
(Holt and May, 1996
). The
bimD6 terminal phenotype also indicates an essential role for BIMD in
the successful completion of mitosis
(Denison et al., 1993
).
However, the bimD6 allele contains a nonsense mutation that would be
predicted to lead to the production of a short truncated peptide
(van Heemst et al., 2001
).
Thus, the BIMD protein may be non-essential for mitotic growth at low
temperatures. Spo76, the BIMD functional homologue in Sordaria
macrospora (van Heemst et al.,
1999
), is dispensable for vegetative growth, in contrast to the
essential nature of S. cerevisiae Pds5. Although spo76
mutants exhibit clear defects in meiotic and mitotic chromosome cohesion, in
the mitotic cycle they show only a comparatively subtle delay in the
transition from prometaphase to metaphase
(van Heemst et al., 1999
).
A. nidulans bimD mutants are hypersensitive to DNA damage
(Denison et al., 1993), and
this sensitivity could be attributable to a role of BIMD in chromatid
cohesion. Furthermore, mutation of Schizosaccharomyces pombe rad21
(the SCC1 orthologue) confers defects in the repair of double-strand
DNA breaks and mis4 mutants (which are defective in cohesin loading)
are sensitive to ultraviolet (UV) irradiation
(Birkenbihl and Subramani,
1992
; Furuya et al.,
1998
). Similarly, a requirement for cohesion in postreplicative
DNA repair has recently been demonstrated in S. cerevisiae
(Sjögren and
Nasmyth, 2001
). Cohesion therefore appears to promote DNA repair
from the undamaged sister chromatid and hence affords resistance to a variety
of forms of DNA damage. This interpretation is supported by the observation
that, during the selection of templates for recombinational DNA repair in
vivo, sister chromatids are preferred to homologous chromosomes
(Kadyk and Hartwell,
1992
).
Despite these studies on Pds5 and its orthologues in diverse species, there is no clear consensus regarding the role played by this protein in sister chromatid cohesion. The recent completion of the genome sequence of S. pombe, which is only distantly related to S. cerevisiae, A. nidulans and S. macrospora has allowed the identification of the only S. pombe gene significantly related to PDS5. Here, we describe the functional characterisation of this fission yeast gene, which we designate pds5+, and its involvement in the maintenance of genome stability.
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Materials and Methods |
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Fission yeast strains and methods
Conditions for growth, maintenance and genetic manipulation of fission
yeast were as described previously (Moreno
et al., 1991). A complete list of the strains used in this study
is given in Table 1. Except
where otherwise stated, strains were grown at 30°C in YE or EMM2 medium
with appropriate supplements. Where necessary, gene expression from the
nmt1 promoter was repressed by the addition of 5 µM thiamine to
the growth medium.
|
Minichromosome loss assays were performed using strains as shown in
Table 1 containing the
non-essential ade6-M216 marked Ch16 minichromosome derivative of
chromosome 3 (Niwa et al.,
1986). The ade6-M216 allele complements an unlinked
ade6-M210 marker in these strains such that they remain
ade+ as long as the minichromosome is maintained. Chromosome loss
was measured in the progeny from a single ade+ cell after a known
number of generations during which selection for adenine prototrophy had been
relaxed by growth on YE agar. Rates of chromosome loss per generation were
calculated exactly as described elsewhere
(Murakami et al., 1995
;
Stewart et al., 1997
),
according to the formula:
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Gene disruption and related techniques
The one-step disruption method was used, following PCR-mediated generation
of the entire ura4+ gene flanked by 80 bp segments from
the 5' and 3' regions of pds5+, using
oligonucleotides PDS5A and B (Table
2). Following transformation of a diploid strain 428h/429h,
ura+ progeny were screened for the desired integration pattern by
diagnostic PCR reactions using primer pairs spanning the presumptive
recombination sites (details of the additional primers used for this purpose
are available from the authors on request). Meiosis and sporulation were
induced by plating onto malt extract agar, and tetrad dissection was performed
with an MSM micromanipulator (Singer Instruments, UK) as described elsewhere
(Moreno et al., 1991).
Construction of the chromosomally HA- and GFP-tagged pds5 strains
(pds5-HA and pds5-GFP) was accomplished by an analogous
method using primers TAGA and B (Table
2). The pds5::LEU2 allele was generated by a secondary
one-step disruption of the pds5::ura4+ allele using
primers as described previously (Wang et
al., 2000a
).
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Antibodies and immunoblotting
Immunoblotting was performed essentially as described elsewhere
(Ausubel et al., 1995) using
Mini-Protean electrophoresis equipment (Bio-Rad, Hercules, CA) and a semi-dry
transfer apparatus (Hoefer) in conjunction with Hybond ECL membranes (Amersham
Pharmacia, UK). Proteins were detected using enhanced chemiluminescence (ECL,
Amersham Pharmacia, UK) following one hour incubations at room temperature
with the respective primary and horseradish peroxidase-conjugated anti-mouse
antibodies (Sigma, Poole, UK). The mouse anti-influenza hemagglutinin (HA)
monoclonal HA-11 (Covance Research Products, Berkeley, CA) was used at 1
µg/ml for detection of HA-tagged Pds5. Cdc2 was detected using the mouse
monoclonal antibody Y100 (generated by J. Gannon and kindly provided by H.
Yamano).
Gel filtration chromatography
Chromatographic separation of S. pombe lysates was carried out
using a superose-6TM column attached to an FPLC workstation (Amersham
Pharmacia, UK). Cells from a 100 ml culture of the pds5-HA strain in
mid-exponential growth were washed and resuspended in 300 µl FPLC buffer
(20% glycerol, 20 mM Tris-Cl pH 7.5, 100 mM NaCl, 0.1 mM EDTA, 1 mM
ß-mercaptoethanol, 60 mM ß-glycerophosphate, 0.1 mM sodium
orthovanadate, 1 mM PMSF) with additional protease inhibitors
(CompleteTM, Roche) and lysed by bead beating (3x30 seconds). After
clearing by centrifugation (16,000 g, 15 minutes) the lysate was
loaded onto the column that had been preequilibrated with 50 ml FPLC buffer.
The eluate was collected in 0.5 ml fractions and these were analysed by
SDS-PAGE and immunoblotting. The column was calibrated using the standards:
blue dextran (2000 kDa), thyroglobin (670 kDa), apoferritin (443 kDa),
ß-amylase (200 kDa), BSA (66 kDa) and carbonic anhydrase (29 kDa).
Microscopy
Cells fixed in 3.8% formaldehyde were washed in phosphate-buffered saline
and stained with 4',6-diamidino-2-phenylindole (DAPI) before examination
by fluorescence microscopy. Images were acquired using a Zeiss Axioskop
microscope equipped with a Planapochromat 100x objective, an Axiocam
cooled CCD camera and Axiovision software (Carl Zeiss Ltd, Welwyn Garden City,
UK), and were assembled using Adobe PhotoShop. In some experiments living
cells growing in EMM2 medium were stained by the addition of 5 µg/ml
bis-benzimide (Hoechst 33342, Sigma) before examination by fluorescence
microscopy. Visualisation of GFP-Swi6 in living cells embedded in 0.6% LMP
agarose was performed at room temperature (approximately 22°C) as
described elsewhere (Pidoux et al.,
2000).
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Results |
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The one-step gene disruption method was used in a
ura4-/ura4- diploid S. pombe strain to replace
one copy of the entire pds5 open reading frame with the
ura4+ selectable marker. After induction of meiosis and
sporulation, microdissection of tetrads showed that the
pds5::ura4+ alleles segregated 2:2, indicating that, in
contrast to its S. cerevisiae orthologue, S. pombe
pds5+ is not essential for mitotic growth. Microscopic
examination of exponentially growing cells with the
pds5::ura4+ genotype (pds5) showed that
there were no gross abnormalities in cell growth or division associated with
pds5 deletion (Fig.
1B).
Deletion of pds5 causes hypersensitivity to DNA damage
Since A. nidulans bimD6 mutant cells display increased sensitivity
to DNA damaging agents (Denison et al.,
1993), we were interested to know if the same might be true of
S. pombe pds5
cells. Exposure of pds5
cells and those of a pds5+ control strain to UV showed
that the pds5
strain is UV-hypersensitive, with a tenfold
reduction in cell viability after exposure to 200 J/m2
(Fig. 2A). Examination of the
cells 12 hours after UV irradiation at 150 J/m2 (a dose which only
marginally reduced viability of the pds5+ control) showed
that most of the pds5
cells were highly elongated and had
fragmented and/or misshapen nuclei. The sensitivity of pds5
cells was not restricted to UV-induced DNA damage, as they were also mildly
hypersensitive to the alkylating agent methyl methanesulphonate (MMS) and the
radiomimetic agent bleomycin (Fig.
2B). While this sensitivity was significant, it was not as extreme
as that seen in a strain lacking the checkpoint signalling kinase Rad3
(rad3
; Fig.
2B).
|
Mutants defective in the establishment of sister chromatid cohesion or in
elements of checkpoint signalling have been shown in many cases to lose
viability when DNA replication is inhibited. However, in contrast to their
sensitivity to DNA damaging agents, pds5 cells were not
sensitive to hydroxyurea (which inhibits ribonucleotide reductase and hence
DNA replication), in comparison with those of a rad3
strain
(Fig. 2C).
Pds5 is required for maintenance of viability when mitosis is
arrested
S. cerevisiae pds5 mutants show a characteristic loss of viability
during arrest in mitosis, and it was therefore of interest to determine if the
same is true of the S. pombe pds5 strain. In comparison with a
pds5+ strain, pds5
cells were
hypersensitive to the spindle poison thiabendazole (TBZ;
Fig. 3A) and deletion of
pds5 showed synthetic lethality with the nda3-KM311 ß
tubulin mutation (Hiraoka et al.,
1984
) at 23°C, at which temperature the cold-sensitive
nda3-KM311 single mutant is still able to grow
(Fig. 3B). These data suggest
that pds5 is involved in maintenance of viability during delayed
progression through mitotic metaphase. In order to define the
pds5
defect more precisely, release from an
nda3-KM311-induced metaphase block was monitored by microscopy of
pds5+ and pds5
cells. By 9 minutes after
release most of the pds5+ cells had entered anaphase with
two apparently equal daughter nuclei (Fig.
3C). Under the same conditions the pds5
cells
appeared to enter anaphase on schedule, but showed an unusually high
proportion of cells with lagging chromosomes
(Fig. 3C). At 2 hours after
release these cells showed a variety of apparently lethal abnormalities,
including total failure of chromosome segregation and the `cut' (cell untimely
torn) phenotype (data not shown). We conclude that failure to complete mitosis
probably accounts for the observed genetic interaction between pds5
deletion and nda3-KM311, and for the sensitivity of the
pds5
strain to TBZ.
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Sensitivity of S. cerevisiae pds5 mutants to anti-microtubule
agents has been ascribed to their failure to maintain sister chromatid
cohesion during mid-mitotic arrest
(Hartman et al., 2000;
Panizza et al., 2000
). Sister
centromere separation in living S. pombe cells can be monitored
conveniently by the use of a strain expressing a green fluorescent protein
(GFP)-tagged version of the kinetochore component Ndc80
(Wigge and Kilmartin, 2001
).
Ndc80-GFP appears as a single fluorescent spot in interphase cells, while in
anaphase the clustered kinetochores are seen as two spots, one in each
daughter nucleus. This pattern of Ndc80-GFP distribution was seen in
nda3-KM311 pds5
cells grown at the permissive temperature of
30°C (Fig. 3D). When this
strain was shifted to the restrictive temperature of 18°C to induce arrest
in a metaphase, most cells (95%) still showed a single Ndc80-GFP spot,
indicating that the sister centromeres were clustered. The remaining cells had
three well-defined masses of condensed chromatin, presumably corresponding to
the three S. pombe chromosomes, with an Ndc80-GFP spot associated
with each (Fig. 3D). Of a total
of 500 cells examined, none with more than three Ndc80-GFP spots was seen.
Thus, in contrast to the corresponding S. cerevisiae mutant, S.
pombe pds5
cells showed no indication of premature sister
centromere separation during metaphase arrest.
Elevated rate of chromosome loss on deletion of pds5
Given the chromosome segregation defects observed on release of
pds5 cells from metaphase arrest, we reasoned that Pds5 might
also be required to ensure the normal fidelity of chromosome segregation
during unperturbed mitosis. To address this point, pds5+
and pds5
strains containing a non-essential minichromosome
(Ch16) carrying an adenine biosynthetic marker were used to measure rates of
chromosome loss (Fig. 4A). This
assay showed that spontaneous loss of the minichromosome was 30-fold more
frequent in the pds5
strain in comparison with the
pds5+ control (mean loss rates were 0.00714 and 0.00024
per generation, respectively). We conclude that, while not essential,
pds5+ makes an important contribution to maintenance of
genome stability in S. pombe.
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Although the gross nuclear morphology of most pds5 cells
appeared normal (Fig. 1B), we
were interested to see if the elevated rate of mini-chromosome loss would be
reflected in chromosome segregation defects as scored by microscopy. To
address this point, pds5
and pds5+ strains
were constructed containing an integrated GFP-tagged swi6 gene. Like
the endogenous chromodomain protein Swi6, GFP-Swi6 localizes to the nucleus
and is concentrated at the heterochromatic centromeres and telomeres
(Pidoux et al., 2000
).
Anaphase chromosome separation, monitored in living cells by GFP-Swi6
fluorescence microscopy, appeared normal in the pds5+
background, as well as in the majority of pds5
cells. Abnormal
anaphase progression was nonetheless detectable in approximately 5% of the
pds5
cells, but not in the pds5+ control
(Fig. 4B,C). In some cells this
took the form of segregation, with apparently normal kinetics, of two unequal
masses of GFP-Swi6 (Fig. 4B).
In other pds5
cells an extended bridge of GFP-Swi6 persisted
for some time between the presumptive daughter nuclei
(Fig. 4C, upper panels), while
still others contained discrete centers of GFP-Swi6 fluorescence, presumably
corresponding to entire lagging chromosomes
(Fig. 4C, lower panels).
Chromosome segregation defects at the frequencies seen would be sufficient to
account for the elevated rate of minichromosome loss in pds5
cells.
One possible explanation for the observed mitotic defects in
pds5 cells would be that, in the absence of Pds5, some aspect
of centromere function or spindle microtubule attachment is defective in a
significant proportion of cells. To address this possibility, the effect of
combining the pds5 deletion with deletion of the bub1
spindle checkpoint gene (Bernard et al.,
1998
) was investigated. In the absence of bub1, cells
with centromeric defects fail to delay the onset of anaphase appropriately and
suffer catastrophic chromosome missegregation. Tetrad dissection following
sporulation of a pds5
/pds5+
bub1
/bub1+ diploid strain showed that
pds5
bub1
double mutants grew much more slowly than the
equivalent single mutants (Fig.
4D). Examination of DAPI stained samples from liquid cultures
showed that bub1
cells suffered chromosome mis-segregation
events (Fig. 4E), as reported
previously (Bernard et al.,
1998
). These were more frequent than those seen in
pds5
cells, but were not sufficient to cause a significant
growth disadvantage such as that seen in pds5
bub1
mutants (Fig. 4D). The growth
disadvantage in the latter was associated with a variety of chromosome
missegregation events (Fig.
4E). Interestingly, no such genetic interaction was seen between
pds5
and deletion of mad2, another spindle checkpoint
gene (data not shown).
Meiosis is frequently abnormal in the absence of pds5
In S. macrospora, Spo76 is required for sister chromatid cohesion
both in mitosis and in meiosis. Having established that Pds5, the only protein
in S. pombe significantly related to Spo76, has a role in the mitotic
cell cycle, we therefore made a preliminary investigation of the possibility
that Pds5 might also have a role in meiosis. Induction of meiosis and
sporulation in a homozygous pds5 diploid strain gave rise to
asci, 30% of which had fewer than four spores, which were in most cases
misshapen and abnormally sized and had substantially reduced viability
(Fig. 5). The DNA content of
these abnormal spores was frequently either greater or less than that of
normal haploid spores (Fig. 5A,
lower panels). By contrast, atypical asci of this sort represented <1% of
those formed in parallel by a
pds5+/pds5+ diploid strain. Completion
of meiosis and/or sporulation at the normal frequency thus depends at some
level on pds5+ function.
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Characterisation of Pds5 protein in S. pombe
Targeted recombination was used to add an HA epitope tag sequence to the
3' end of the pds5 open reading frame in its normal chromosomal
context, generating the pds5-HA strain (see Materials and Methods).
Immunoblotting of pds5-HA whole cell lysates with an anti-HA antibody
showed a single protein band of approximately 140 kDa, consistent with the
predicted relative molecular mass for Pds5 of 138874
(Fig. 6; and data not shown).
Immunoblotting of anti-PDS5 immunoprecipitates from human cells has been used
to demonstrate an interaction between PDS5 and cohesin subunits, although most
PDS5 was not found to be stably associated with cohesin as judged by sucrose
gradient fractionation of cell extracts
(Sumara et al., 2000). No size
fractionation data have been published to date for S. cerevisiae Pds5
or any of its fungal orthologues, however. We therefore used gel filtration
chromatography to estimate the apparent size of S. pombe Pds5 in a
cell lysate prepared from the pds5-HA strain
(Fig. 6). Under the conditions
used, the HA-tagged Pds5 migrated as a single peak with an apparent size
between 670 and 2000 kDa. Parallel fractionation of an extract from a
rad21-HA strain showed that the Rad21-HA protein (and, by extension,
other previously characterized cohesin components) was also present in a
single peak with a similar size distribution to that seen for Pds5-HA.
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The relationship between Pds5 and cohesin
To monitor the localisation of Pds5 in living cells, the one step gene
replacement method was used to generate a strain (pds5-GFP) encoding
a C-terminally GFP tagged version of Pds5. The GFP-tagged protein appeared to
be functional as judged by the lack of hypersensitivity to UV of the
pds5-GFP strain compared with pds5
(Fig. 2A,B). Examination of
living pds5-GFP cells by fluorescence microscopy showed that Pds5-GFP
(and, by inference Pds5) was predominantly localised to the nucleus, within
which it gave a diffusely speckled signal
(Fig. 7A). There were no
obvious differences in this pattern among cells at different cell cycle
stages, and in nda3-KM311 cells arrested in a metaphase-like state by
incubation at the restrictive temperature of 18°C for 12 hours the
Pds5-GFP signal co-localised with condensed chromosomes.
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Localisation of Pds5 to chromatin in S. cerevisiae is dependent
upon the integrity of the Scc1/Mcd1 cohesin component
(Hartman et al., 2000;
Panizza et al., 2000
). To see
if the same might be true of the orthologous S. pombe proteins, the
localisation of Pds5-GFP was monitored in a temperature-sensitive
rad21-K1 strain (Fig.
7B). At the permissive temperature of 25°C, Pds5-GFP was again
predominantly nuclear, but after inactivation of the Rad21 cohesin component
by shifting to the restrictive temperature of 36°C the Pds5-GFP signal was
diffusely localised throughout the cell. No such change in localisation was
seen on shifting a pds5-GFP (rad21+) strain from
25°C to 36°C (Fig. 7B).
Thus Pds5 localisation to the nucleus is cohesin-dependent in S.
pombe, as it is in S. cerevisiae. By contrast, Rad21-GFP
remained in the nuclear compartment on deletion of pds5, although in
pds5
rad21-GFP cells the Rad21-GFP appeared more punctate than
it did in pds5+ cells
(Fig. 7C).
To investigate further this apparent relationship between pds5 and cohesin,
the meiotic progeny of a diploid rad21-K1 pds5 strain
(Table 1) were characterised
following tetrad microdissection (Fig.
8A). From 102 tetrads, the numbers of pds5+
rad21+, pds5+ rad21-K1 and
pds5::LEU2 rad21+ segregant colonies visible after 5 days
growth at 25°C were 77, 45 and 78, respectively. These numbers are lower
than the 102 that would be expected for independently segregating markers, and
indicate that there was an overall loss of spore viability attributable to
heterozygosity at the pds5 and rad21 loci in the diploid.
Nonetheless, haploid segregants bearing either the rad21-K1 or the
pds5::LEU2 allele were able to grow and form colonies reasonably
efficiently. By contrast, no rad21-K1 pds5::LEU2 segregant colonies
were visible after 7 days growth. In many cases the positions occupied by
spores of this genotype could be deduced from the genotypes of the other
segregants. Microscopic examination showed that these rad21-K1
pds5::LEU2 cells were highly elongated, suggesting that partial loss of
rad21 function in the absence of pds5 leads to a failure of
cell cycle progression (Fig.
8A).
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Further genetic interactions between the pds5 deletion and genes
encoding components of the sister chromatid cohesion pathway were sought.
S. pombe Mis4 is a cohesin loading factor/adherin orthologous to
S. cerevisiae Scc2. Introduction of the temperature sensitive
mis4-242 mutation into a pds5 background revealed a
synthetic lethality phenotype at 28°C, at which temperature each of the
corresponding single mutants grew relatively normally
(Fig. 8B). Reducing the
temperature to 25°C allowed the pds5
mis4-242 cells to
grow, albeit slowly. On microscopic examination these cells showed a variety
of aberrant morphologies indicative of cell cycle defects, including extensive
elongation and chromosome missegregation
(Fig. 8B, lower panel). No such
defects were seen in the single mis4-242 mutant grown at the same
temperature. Thus pds5 can be linked genetically both to cohesin
itself and to a cohesin loading factor.
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Discussion |
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In some respects, S. pombe pds5+ more closely resembles
S. macrospora SPO76 than S. cerevisiae PDS5 or A.
nidulans bimD. Specifically, the S. pombe gene is dispensable
for mitotic growth but is required for the efficient completion of meiosis and
sporulation (Figs 1,
5). Pds5 was found previously
to be required for chromosome condensation as well as cohesion in S.
cerevisiae (Hartman et al.,
2000), but we find no indication that S. pombe Pds5 is
required for the chromosome condensation seen in metaphase arrested cells
(Fig. 3D). If the link between
cohesion and condensation that has been described in S. cerevisiae
(Guacci et al., 1997
) also
operates in the fission yeast, the competence of pds5
cells
for chromosome condensation would be consistent with their lack of overt
cohesion defects (Fig. 3).
However, Mis4, which is required for sister chromatid cohesion in S.
pombe, is not required for condensation
(Furuya et al., 1998
),
suggesting that any such link may not be straightforward. Indeed, it has been
suggested that, in Xenopus, cohesin may not be involved in chromosome
condensation at all (Losada et al.,
1998
). An involvement in chromosome condensation in S.
cerevisiae but not in S. pombe might explain why Pds5 is
essential for viability in the former but not the latter.
What then might be the mitotic role of S. pombe Pds5? Despite the
lack of precocious sister chromatid separation in pds5 cells
under the conditions tested here, a number of lines of evidence point towards
an intimate connection between Pds5 and the cohesion process. First, like its
budding yeast counterpart, S. pombe Pds5 localises to nuclear foci in
a manner that is dependent on cohesin function
(Fig. 7A-C). The constitutive
localisation of Pds5 to this nuclear compartment throughout the S.
pombe cell cycle is reminiscent of the behaviour of the Rad21 cohesin
(Tomonaga et al., 2000
) and
Mis4 adherin (Furuya et al.,
1998
). The majority of cohesin remains associated with chromatin
throughout the mitotic cell cycle in S. pombe, in contrast to the
situation in budding yeast, where Scc1/Mcd1 cleavage at the onset of anaphase
is associated with dissociation of cohesin and Pds5 from the chromosomes
(Guacci et al., 1997
;
Hartman et al., 2000
;
Michaelis et al., 1997
;
Panizza et al., 2000
;
Toth et al., 1999
). The
persistence of Pds5-GFP in cells at all stages of mitosis
(Fig. 7A) suggests that, unlike
Scc1/Rad21, Pds5 may not be regulated by proteolysis. Disappearance of
Pds5-GFP from the nucleus on inactivation of Rad21
(Fig. 7C) suggests that the
putative bipartite NLS in Pds5 (Fig.
1A) is insufficient to confer constitutive nuclear localisation.
In the absence of functional cohesin, Pds5 may be subject to nuclear export or
degradation; distinction between these possibilities awaits further
investigation.
A role for S. pombe Pds5 in chromatid cohesion would also be
consistent with the genetic interactions between pds5, rad21 and
mis4 (Fig. 8). The
observed lack of hypersensitivity of pds5 cells to HU
(Fig. 2C) suggests that
pds5 may not be required for the establishment of cohesion in S
phase, in contrast to mis4, which when mutated confers HU
hypersensitivity (Furuya et al.,
1998
). A role in cohesion could also be suggested by the
sensitivity of pds5
cells to DNA damage
(Fig. 2). Mutation of other
genes involved in the establishment or maintenance of cohesion has previously
been shown to lead to DNA damage sensitivity
(Birkenbihl and Subramani,
1992
; Denison et al.,
1993
; Furuya et al.,
1998
;
Sjögren and
Nasmyth, 2001
). The elongation of pds5
cells seen
after UV irradiation (Fig. 2A)
suggests that cell cycle checkpoint responses to UV-induced DNA damage are
established in the absence of pds5, but that some aspect of DNA
repair or cell cycle resumption is defective, accounting for the observed
increase in UV sensitivity. A general role for Pds5 in cell cycle resumption
seems unlikely, however, as G2 arrest for up to 6 hours in a cdc25-22
pds5::ura4+ strain was not accompanied by any significant loss
of viability (data not shown). Instead, a role for Pds5 in promoting repair of
a variety of DNA lesions seems more likely
(Fig. 2). This function could
be an indirect consequence of a primary involvement of Pds5 in cohesion.
In line with the proposed connection between Pds5 and cohesion in S.
pombe, soluble Pds5 was present in a high molecular weight complex
similar in size to that containing the Rad21 cohesin component
(Fig. 6). This is consistent
with the behaviour of human PDS5, which could be co-immunoprecipitated with
cohesin components from crude cell lysates, although it was exclusively
localised to size fractions smaller than those containing cohesin when lysates
were separated by sucrose density gradient centrifugation
(Sumara et al., 2000).
Interestingly, size fractionation of pds5-HA lysates in the absence
of phosphatase inhibitors yielded a Pds5-HA peak with a much smaller average
size, consistent with that expected for the monomeric protein (data not
shown). This suggests that retention of Pds5 in a high molecular weight
complex, perhaps including cohesin itself, depends on the maintenance of
phosphorylation of one or more proteins. Despite this apparent co-migration,
we were unable to co-immunoprecipitate known cohesin components reproducibly
with Pds5-HA, or to precipitate Pds5-HA with an anti-Rad21 antibody (data not
shown). Any interaction between Pds5 and cohesin therefore appears quite
labile in soluble extracts. However, our data do not rule out the possibility
that such an interaction might be more stable in the context of
chromatin-associated cohesin.
In the mitotic cycle, pds5 cells had an elevated rate of
chromosome loss and lagging chromosomes were readily detected
(Fig. 4). These cells were also
unusually vulnerable to arrest at metaphase, as judged by TBZ sensitivity and
synthetic lethality with nda3-KM311 at 23°C
(Fig. 3). Microscopic
examination of pds5
cells released from an nda3-KM311
arrest suggested that chromosome segregation was frequently grossly abnormal
under these circumstances. In the absence of any evidence for precocious
sister separation, it is still possible that these observations reflect
altered cohesion. For example, Pds5 may be required for the correct
localisation of cohesin to specific chromosomal sites, or for the inhibition
of excessive cohesin loading. These possibilities would be consistent with our
observation that Rad21-GFP remains associated with chromatin in the absence of
Pds5, although the precise pattern of its chromatin localisation appears
subtly different (Fig. 7C). In
this case the frequent failure of pds5
cells to complete
mitosis properly after metaphase delay
(Fig. 3C) could be the result
of anaphase progression without complete loss of sister chromatid cohesion.
Similar defects, occurring at a lower frequency, could explain the 30-fold
elevation in minichromosome loss and the presence of lagging chromosomes in
pds5
cells (Fig.
4). As this aspect of the pds5
phenotype is
particularly marked after a metaphase delay, it would appear that the putative
regulatory role of Pds5 is more important during mitosis than it is in
interphase. Analogous defects in meiosis I and/or II could explain the
observed defects in spore formation (Fig.
5).
The sensitivity of pds5 cells to metaphase arrest does not
appear to reflect a loss of spindle checkpoint integrity, since nda3-KM311
pds5
cells were able to arrest in a metaphase-like state on being
shifted to the restrictive temperature
(Fig. 3C,D). The potent genetic
interaction observed between pds5 and bub1
(Fig. 4D,E) could suggest that
the spindle checkpoint is at least partially activated in pds5
cells in the absence of any additional perturbation. If this is the case Pds5,
through its putative role in `fine tuning' sister chromatid cohesion, may be
required for the efficient establishment of bipolar attachments of chromosomes
to spindle microtubules. Interestingly, we found no equivalent genetic
interaction between pds5 and mad2, while the mitotic delay
in S. pombe cohesin mutants was reported to be
mad2-dependent (Tomonaga et al.,
2000
). A recent study demonstrated that, in addition to its
spindle checkpoint function, Bub1 is required for centromeric cohesion during
meiosis (Bernard et al., 2001
).
The genetic interaction between pds5 and bub1
(Fig. 4) could therefore
indicate that both are also involved in mitotic cohesion.
Investigation of cohesin-related components in S. pombe should
prove complementary to studies in S. cerevisiae with respect to
understanding chromatid cohesion and its regulation in other organisms, as the
overall organisation of mitosis in the two yeasts differs in several respects
(Russell and Nurse, 1986).
Indeed, significant differences between these species in terms of cohesin
composition and proteolysis have been described recently
(Tomonaga et al., 2000
).
Specifically, Psc3 (the S. pombe orthologue of Scc3) is not stably
associated with cohesin, and only a minor fraction of Rad21 is subject to
proteolysis at the onset of anaphase. Genetic approaches in distantly related
simple eukaryotes, combined with biochemical investigations in these and other
systems, should eventually provide a comprehensive understanding of sister
chromatid cohesion and its regulation.
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