Centro de Investigación del Cáncer, CSIC/University of Salamanca, Campus Unamuno, 37007 Salamanca, Spain
* Author for correspondence (e-mail: pedross{at}usal.es)
Accepted 18 October 2002
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Summary |
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Key words: Meiosis, Checkpoint, Mek1, Meiotic recombination, Cell cycle, Fission yeast
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Introduction |
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Meiosis is a specialized type of cell division that generates haploid
gametes from diploid parental cells because a single round of DNA replication
is followed by two consecutive nuclear divisions. During meiotic prophase, a
complex series of interactions between homologous chromosomes (or homologs)
occur. First, chromosomes search for and associate with the homologous
partners (pairing). In most (but not all) organisms, these associations are
stabilized by synapsis, which is the formation of an elaborate proteinaceous
structure (the synaptonemal complex; SC) that holds homologs close together
along their entire length. Concomitantly, DNA recombination between homologous
chromosomes takes place. In addition to the exchange of genetic information,
the result of these interactions is the formation of physical connections
between homologs, called chiasmata, which promote correct chromosome
segregation during the first meiotic division (reviewed by
Roeder, 1997;
Smith and Nicolas, 1998
;
Zickler and Kleckner, 1999
;
Lee and Amon, 2001
).
Meiotic cells possess a surveillance mechanism referred to as the
`pachytene checkpoint' or the `meiotic recombination checkpoint' that monitors
these critical meiosis-specific events. Meiotic recombination is initiated by
DNA double-strand breaks (DSBs), which are repaired using nonsister chromatids
as templates. In response to defects in recombination that lead to
accumulation of unrepaired DSBs and/or other recombination intermediates, the
pachytene checkpoint triggers meiotic cell cycle arrest or delay to prevent
meiotic chromosome missegregation (Roeder
and Bailis, 2000).
A number of studies in the budding yeast Saccharomyces cerevisiae
have identified several components of the pachytene checkpoint
(Roeder and Bailis, 2000). DNA
damage checkpoint proteins that respond to DSBs in vegetative cells also
monitor these lesions during meiosis; however, there are differences between
the mitotic DNA damage checkpoint and the meiotic recombination checkpoint.
First, some DNA damage checkpoint proteins (e.g., Chk1, and Rad9) are not
required for the pachytene checkpoint
(Lydall et al., 1996
)
(P.A.S.-S. and G. S. Roeder, unpublished). Second, it has been proposed that
meiotic DSBs are monitored in a meiosis-specific chromosomal context and are
not recognized as `general' damage (Xu et
al., 1997
). Third, some crucial pachytene checkpoint proteins,
such as the nucleolar silencing factor Pch2 or the Mek1 kinase, are produced
only during meiosis (Rockmill and Roeder,
1991
; San-Segundo and Roeder,
1999
). Fourth, the DNA damage checkpoint and the pachytene
checkpoint act on different targets of the cell cycle machinery to block cell
cycle progression (Leu and Roeder,
1999
).
The pachytene checkpoint has been extensively studied only in S.
cerevisiae, but its operation in worms, flies and mammals has been also
reported (Edelmann et al.,
1996; Pittman et al.,
1998
; Yoshida et al.,
1998
; Ghabrial and Schupbach,
1999
; Gartner et al.,
2000
; MacQueen and Villeneuve,
2001
; Abdu et al.,
2002
). In fact, most (if not all) yeast pachytene checkpoint
proteins have homologs in other organisms
(Roeder and Bailis, 2000
).
However, although the fission yeast Schizosaccharomyces pombe is a
model organism widely used in checkpoint studies during the mitotic cell cycle
(Murakami and Nurse, 2000
),
little is known about surveillance mechanisms of meiosis-specific processes,
in particular meiotic recombination.
Here we show that the meiotic recombination checkpoint does indeed operate
in S. pombe, and we describe a role for a meiosis-specific kinase,
Mek1, in this control mechanism. Mek1 contains a forkhead-associated (FHA)
domain. FHA motifs are usually implicated in protein-protein interactions
regulated by phosphorylation (Durocher et
al., 1999). We present evidence indicating that fission yeast Mek1
prevents entry into the first meiotic division (MI) until recombination is
completed. The Mek1-dependent negative regulation of MI entry is achieved by
maintaining phosphorylation of Cdc2 at Tyr15, at least in part, through
inhibition of the Cdc25 phosphatase.
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Materials and Methods |
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To clone the mek1+ gene, the mek1+
cDNA was amplified by PCR using cDNA obtained from a 3-hour pat1
meiotic culture (see Fig. 1)
with primers mek1-N
5'-TTTTCTCGAGCATATGGACTTTTTATCACATGCCATG-3'
(XhoI site, underlined; NdeI site, italicized) and mek1-C
5'-TTTTCCCGGGCTAGCGGCCGCCTAGCCGGGAATGTTTAAGAGG-3'
(SmaI and NotI sites, underlined; an added stop codon,
italicized). The PCR product was digested with XhoI-SmaI and
cloned into the same sites of the pREP3X vector
(Forsburg, 1993), producing
plasmid pSS123, which contains mek1+ cDNA under the
nmt1(3x) promoter. The NdeI-SmaI fragment
from pSS123 containing mek1+ was cloned into the same
sites of pREP41-EGFP-N (Craven et al.,
1998
), generating plasmid pSS124, which expresses
mek1+, N-terminally tagged with GFP, from the
nmt1(41x) promoter. Plasmid pLV1 was constructed by cloning a
PstI-EcoRI fragment from pSS124 containing
nmt1-GFP-mek1+ into the same sites of the integrative
vector pJK148 (Keeney and Boeke,
1994
).
|
Genetic procedures
Spore viability was assayed by tetrad dissection. The frequency of meiotic
intergenic recombination was determined by random spore analysis. Crosses were
performed on MEA plates and, after 2 days, spores were isolated, grown on YES
plates and replica-plated to minimal medium. The number of recombinant spores
was counted and normalized to the total number of viable spores.
Northern and western blotting
RNA preparation and northern blot analysis were performed as described
previously (Blanco et al.,
2001) using a mek1+ PCR fragment amplified
with oligomers mek1-N and mek1-C as a probe. Total protein extracts were
prepared as described elsewhere (Blanco et
al., 2000
). For western blot analysis,
60 µg of total
extracts were run on 12% SDS-PAGE gels, transferred to nitrocellulose and
probed with the following antibodies: mouse monoclonal anti-HA (12CA5; 0.15
mg/ml), rabbit polyclonal anti-Cdc2 (C2; 1:200 dilution), rabbit polyclonal
anti-phospho-Cdc2(Tyr15) (Cell Signaling Technology; 1:1000 dilution) and
mouse monoclonal anti-tubulin (TAT1; 1:1000 dilution). Goat anti-rabbit or
goat anti-mouse antibodies conjugated to horseradish peroxidase (Amersham)
were used as secondary antibodies (1:3500 and 1:2000 dilution, respectively).
Immunoblots were developed using the Luminol Reagent (Santa Cruz
Biotechnology) or the SuperSignal kit (Pierce).
Flow cytometry
Flow cytometric analysis was performed on a Becton-Dickinson FACSscan using
propidium iodide staining of cells (Sazer
and Sherwood, 1990).
Microscopy
For the analysis of meiotic progression, cells were fixed in 70% ethanol
and processed for DAPI staining of nuclei as described previously
(Moreno et al., 1991). To
study the subcellular localization of Mek1-HA, immunofluorescence analysis was
performed essentially as described previously
(Santos and Snyder, 1997
),
except that PEMBAL buffer (100 mM PIPES pH 6.9, 1 mM EGTA, 1 mM MgSO4, 1% BSA,
0.1% azide, 0.1 M L-lysine HCl) was used instead of PBS. Mouse monoclonal
anti-HA antibody (HA.11, Convance) was used at 1:150 dilution. Goat anti-mouse
antibody conjugated to CY3 (Jackson ImmunoResearch Labs) was used as the
secondary antibody (1:200 dilution). Cells were visualized using a Zeiss
Axioplan2 fluorescence microscope equipped with a Hamamatsu CCD camera.
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Results |
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The S. pombe mek1+ gene was cloned by polymerase chain reaction (PCR) using specific primers and genomic DNA or cDNA as the template (see Materials and Methods; Fig. 1C). No PCR product corresponding to mek1+ was amplified when cDNA obtained from vegetative cells was used (Fig. 1C). However, a PCR fragment of the expected size was obtained when cDNA from S. pombe cells at the early stages of meiosis was used (Fig. 1C), indicating that the mek1+ gene is only expressed in meiotic cells. The difference in size of the mek1+ fragment amplified from genomic DNA or from cDNA is consistent with the presence of the two predicted introns in the gene.
The S. pombe Mek1 protein is produced during meiotic
prophase and localizes to the nucleus
Expression of the mek1+ gene was monitored by northern
blot analysis during a pat1-driven synchronous meiosis
(Fig. 2A-C). Consistent with
the results shown above, no expression of mek1+ is
detected in vegetatively growing cells; its expression is induced at the same
time as the onset of premeiotic S phase and reaches the maximum level during
the period corresponding to meiotic prophase; then, mek1+
mRNA levels decrease as cells enter into the first meiotic division
(Fig. 2A-C). The production of
the Mek1 protein was analyzed by western blot using anti-HA antibodies in a
meiotic time course of pat1-114 diploid cells expressing a functional
version of mek1+ tagged with three copies of the HA
epitope. The kinetics of Mek1-HA production is similar to the one described
above for mek1+ mRNA
(Fig. 2D).
|
To determine the subcellular location of Mek1, immunofluorescence analysis of pat1-114 mek1-HA diploid cells was carried out using anti-HA antibodies. The Mek1 protein localizes to the nucleus of meiotic cells during the horse-tail movement period (Fig. 2E). No staining is detected in control cells lacking the HA epitope (data not shown).
Spore viability and meiotic recombination are reduced in the
mek1 mutant
To study Mek1 function during meiosis in fission yeast, the
mek1+ gene was deleted. The mek1 mutant completes
meiotic divisions and sporulation, generating morphologically normal
four-spore asci. However, tetrad dissection revealed that spore viability is
reduced in the mek1 mutant compared to wildtype (64% versus 89%,
respectively). Although the overall decrease in spore viability of
mek1 is not dramatic, the fraction of tetrads containing four viable
spores is significantly reduced compared to wildtype (16% versus
78%; Fig. 3A). No excess
of tetrads containing 4, 2 and 0 viable spores is observed, suggesting that
spore death in the mek1 mutant is not due to chromosome
nondisjunction during meiosis I. Intergenic recombination was examined in the
leu1-his5 interval on chromosome II. The mek1 mutant
displays a
2.4-fold reduction in meiotic recombination in this region
(Fig. 3B).
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Mek1 regulates meiotic cell cycle progression in fission yeast
Using pat1 strains to induce synchronous meiosis, kinetics of
meiotic progression were examined in the mek1 mutant in comparison
with the wildtype (Fig. 4A). In
the mek1 mutant, the first meiotic division occurs reproducibly
30 minutes faster than in the otherwise isogenic wildtype
(Fig. 4A). Since
mek1+ expression is induced at the time of premeiotic S
phase (although its peak of expression is reached at prophase;
Fig. 2), DNA replication was
carefully monitored by FACS during synchronous meiosis in mek1 and
wild-type cells at 15 minute time points
(Fig. 4B). This analysis
revealed that premeiotic DNA replication takes place with the same kinetics in
both wild-type and mek1 cells. Therefore, since entry into meiosis I
occurs earlier in mek1, this implies that meiotic prophase is shorter
in the absence of Mek1.
|
To further characterize this observation, the effect of expressing high
levels of Mek1 was also studied. A green fluorescent protein (GFP)-tagged
version of mek1+ was placed under control of the
thiamine-regulated nmt1 promoter and integrated at the leu1
locus (see Materials and Methods). To induce overexpression of
mek1+, thiamine was removed 14 hours prior to transferring
the cells to medium lacking nitrogen (Fig.
4C). The production of Mek1-GFP was followed by microscopic
examination of the cells throughout the experiment. A control culture, in
which thiamine was always present and therefore mek1+
expression was repressed, was also examined. FACS analysis revealed that both
cultures were blocked in G1 to the same extent and underwent premeiotic S
phase with similar kinetics (Fig.
4D); however, nmt1-driven expression of
mek1+ resulted in a significant delay (1 hour) of the
first meiotic division (Fig.
4E). Interestingly, the Mek1-GFP signal disappeared as cells
entered meiosis I, and binucleate cells containing GFP signal were rarely
observed (data not show). Thus, these results suggest that the Mek1 kinase
negatively regulates entry into meiosis I.
Ectopic overexpression of mek1+ in vegetative
cells causes cell cycle arrest by inhibiting Cdc25 function
In order to understand how Mek1 regulates cell cycle progression, high
levels of the protein were produced in vegetative cells using the
nmt1 promoter. Interestingly, ectopic overproduction of Mek1 results
in inhibition of growth (Fig.
5A). Microscopic examination revealed that Mek1-overproducing
cells are highly elongated and contain a single undivided nucleus
(Fig. 5B), a phenotype that
resembles the G2/M arrest induced by activation of the DNA integrity
checkpoints or by overproduction of the Cds1 or Chk1 checkpoint kinases
(Furnari et al., 1997;
Boddy et al., 1998
).
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In principle, the G2/M arrest triggered by mek1+
ectopic overexpression may be caused by inhibition of the Cdc25 phosphatase or
activation of the Wee1 or Mik1 kinases. To identify which cell cycle
regulator(s) is the target of Mek1, the protein was overproduced in mutants
defective in either Cdc25 or Wee1 function. Since cdc25+
is an essential gene, to analyze whether Cdc25 is involved in the
Mek1-dependent arrest, a cdc2-3w strain, which bypasses the
requirement for Cdc25 was used (Russell
and Nurse, 1986). Like in the wild-type cells, overexpression of
mek1+ in cdc2-3w results in cell elongation, but
this phenotype is largely suppressed in a cdc2-3w cdc25
strain
(Fig. 5C). By contrast,
wee1-50 cells overexpressing mek1+ at the
restrictive temperature still manifest the elongation phenotype
(Fig. 5C). These observations
suggest that Cdc25, but not Wee1, is a target of Mek1.
Our results are consistent with the possibility that S. pombe Mek1
may be the meiosis-specific counterpart of the FHA family Cds1 checkpoint
kinase (Fig. 1). When DNA
integrity checkpoints are activated, Cds1 and Chk1 phosphorylate and inhibit
Cdc25; several residues of Cdc25 phosphorylated by Cds1 have been identified
(Zeng et al., 1998;
Furnari et al., 1999
;
Zeng and Piwnica-Worms, 1999
).
In order to investigate whether the Mek1-induced G2/M arrest in vegetative
cells is also mediated by phosphorylation of Cdc25, the effect of Mek1
overproduction was examined in the cdc25-9A mutant, which contains
nine Cds1 phosphorylation sites changed to alanine and is impaired in the
checkpoint response to DNA damage and replication blocks
(Zeng and Piwnica-Worms,
1999
). Importantly, the cell cycle arrest phenotype caused by Mek1
overproduction is significantly less severe in cdc25-9A cells
compared to wildtype (Fig. 5D).
Thus, our results suggest that Mek1 phosphorylates Cdc25 on at least some of
the same residues as Cds1, leading to inactivation of Cdc25 function and
resulting in cell cycle arrest. Nevertheless, since
nmt1-mek1+ cdc2-3w cdc25
and
nmt1-mek1+ cdc25-9A cells, in the absence of
thiamine, still show a partial arrest (Fig.
5; data not shown), targets of Mek1 other than Cdc25 may
exist.
A Mek1-dependent meiotic recombination checkpoint also operates in
fission yeast
The results reported above revealed that the consequences of high levels of
Mek1 in vegetative cells to a certain extent mimic the DNA integrity
checkpoint responses. However, mek1+ is normally expressed
only during meiotic prophase; therefore, we investigated whether Mek1 carries
out a checkpoint function during meiosis in fission yeast. The alterations
observed in meiotic progression when mek1+ is deleted or
when mek1+ is overexpressed
(Fig. 4) are also consistent
with such a regulatory role for Mek1.
In several organisms, including budding yeast, C. elegans and
mouse, defects at intermediate steps in the meiotic recombination process
trigger the so-called `pachytene checkpoint' or `meiotic recombination
checkpoint', which blocks meiotic cell cycle progression, thus preventing the
formation of defective gametes. To study whether a similar response also
occurs in fission yeast, meiotic progression in the S. pombe meu13
mutant, which is defective in chromosome pairing and meiotic recombination
(Nabeshima et al., 2001), was
carefully examined using pat1-driven synchronous meiosis. Meu13 is
the homolog of the S. cerevisiae Hop2 protein; the hop2
mutant triggers the pachytene checkpoint in budding yeast
(Leu et al., 1998
). The
meu13 mutant completes meiosis and sporulation, as described
previously (Nabeshima et al.,
2001
), but displays a
30 minute delay in entering meiosis I
compared to wildtype (Fig. 6A).
For example, at the 4 hour time point, only
15% of meu13 cells
had undergone meiosis I, compared with
50% in the wild-type strain (see
arrows in Fig. 6A; a
representative time course is presented, but the meu13 delay has been
observed in three independent experiments). Introduction of a rec12
mutation, which abolishes initiation of meiotic recombination suppresses this
delay (data not shown), suggesting that it is due to the presence of
recombination intermediates that trigger the meiotic checkpoint.
Interestingly, mutation of mek1+ also alleviates the
meiotic delay of meu13 (Fig.
6A); the meu13 mek1 double mutant proceeds to the first
meiotic division with similar kinetics to that of the mek1 single
mutant (i.e., even faster than wildtype;
Fig. 4). Moreover, whereas
spore viability in meu13 only shows a slight reduction in comparison
with wildtype (62% versus 77%, respectively), it is significantly reduced in
the meu13 mek1 double mutant (40%). Thus, during meiosis in fission
yeast, Mek1 participates in a surveillance mechanism that delays cell cycle
progression in response to defective recombination, which is important to
promote viability of the meiotic progeny.
|
The above results (Fig. 5) suggest that Cdc25 is a target of Mek1. Since Cdc25 dephosphorylates Tyr15 of Cdc2 promoting G2/M transition, the status of Tyr15 phosphorylation was examined during synchronized meiosis of wild-type, mek1, meu13 and meu13 mek1 strains (Fig. 6B). In agreement with the checkpoint-dependent meiotic delay of the meu13 mutant, phosphorylation of Tyr15 persists longer than in wildtype (compare the 4 hour time point in Fig. 6B). By contrast, in both mek1 and meu13 mek1 strains, dephosphorylation of the Cdc2 tyrosine 15 occurs earlier, consistent with higher levels of Cdc25 phosphatase activity in the absence of Mek1 and correlating with a faster meiotic progression in these strains.
The cdc25-9A mutant is defective in the meiotic
recombination checkpoint
Our overexpression studies in vegetative cells suggest that the
Mek1-dependent regulation of Cdc2 tyrosine 15 phosphorylation is exerted
through inhibition of Cdc25. To directly demonstrate that Cdc25 is required
for the fission yeast meiotic recombination checkpoint, meiotic progression
was examined in strains carrying the phosphorylation-deficient
cdc25-9A allele (Fig.
7). Like deletion of mek1+, the
cdc25-9A mutant alleviates the meiotic delay of meu13; for
example, at the 4 hour time point, 20% of the meu13 cells had
undergone meiosis I, compared with
60% in the cdc25-9A meu13
double mutant (see arrows in Fig.
7).
|
In summary, our results indicate that the meiotic recombination checkpoint in fission yeast inhibits entry into meiosis I by Mek1-dependent inhibitory phosphorylation of Cdc25, which contributes, at least in part, to maintaining Cdc2 phosphorylated on tyrosine 15 (Fig. 8).
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Discussion |
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The mek1 mutant of S. cerevisiae is proficient in
pairing, but displays reduced (although not abolished) meiotic recombination,
reduced spore viability, makes only short stretches of SC and is defective in
meiotic sister chromatid cohesion
(Rockmill and Roeder, 1991;
Bailis and Roeder, 1998
). By
contrast, inactivation of the chk-2 gene in C. elegans
results in a strong pairing defect and lack of crossover recombination, but SC
formation and chromosome morphogenesis are apparently normal
(Higashitani et al., 2000
;
MacQueen and Villeneuve, 2001
;
Oishi et al., 2001
). Our
results indicate that, like the budding yeast homolog, S. pombe Mek1
is also required for normal levels of meiotic interhomolog recombination and
spore viability. Because fission yeast lacks SC, no role for S. pombe
Mek1 in SC development can be proposed, but the characteristic horse-tail
morphology adopted by the prophase nucleus in S. pombe
(Chikashige et al., 1994
)
appears to be normal in the mek1 mutant (L. P.-H., S.M. and
P.A.S.-S., unpublished).
Despite the different meiotic phenotypes resulting from inactivation of
these meiotic FHA kinases during an unperturbed meiosis in these model
organisms, there is a common role for them; both budding and fission yeast
Mek1, as well as Chk2 in worms, are essential components of the meiotic cell
cycle control mechanism called pachytene checkpoint or meiotic recombination
checkpoint, which arrests or delays meiotic cell cycle progression when
recombination is incomplete (this work)
(Bailis and Roeder, 2000;
MacQueen and Villeneuve,
2001
). It is possible that checkpoint function, like that of the
mitotic counterparts of this protein family, could be the evolutionarily
conserved role for these proteins, and the diverse meiotic phenotypes observed
may reflect the peculiarities of meiosis in the different organisms as
revealed, for example, by the different relationship between synapsis and
initiation of recombination in S. cerevisiae and C. elegans
or the absence of SC in S. pombe
(Villeneuve and Hillers,
2001
).
Meiotic defects in the S. pombe mek1 mutant
We have shown that the mek1 mutant in S. pombe shows
reduced interhomolog meiotic recombination, decreased spore viability and a
shorter meiotic prophase. In principle, the defect in meiotic recombination
can be explained by a reduction in the number of initiating events (i.e.,
DSBs) and/or by increasing the number of DSBs repaired using a sister (instead
of a nonsister) chromatid. In S. cerevisiae, Mek1 appears to be
required both for the generation of wild-type levels of DSBs
(Xu et al., 1997) as well as
for the proper choice of recombination partner
(Thompson and Stahl, 1999
).
Alternatively, the accelerated progression through prophase in the fission
yeast mek1 mutant may cause entry into meiosis I with
unrepaired/unresolved recombination intermediates in a fraction of cells,
resulting in reduced recombination frequency and spore inviability. The
observed random pattern of spore death in mek1 is consistent with
this possibility and suggests that nondisjunction of homologs at meiosis I, as
a consequence of the failure to recombine, is not the only cause of spore
death in mek1 as this would result in an excess of asci with two or
zero viable spores (Molnar et al.,
1995
).
In contrast to the original checkpoint definition
(Hartwell and Weinert, 1989),
the direct participation of checkpoint proteins in the monitored cell cycle
event appears to be the rule rather than the exception. For example, in
addition to the mek1 phenotypes mentioned above, other pachytene
checkpoint mutants in budding yeast, such as rad24, rad17 and
mec1-1, show decreased crossing over, increased ectopic
recombination, increased unequal sister-chromatid exchange, defective
chromosome synapsis and reduced spore viability
(Lydall and Weinert, 1995
;
Lydall et al., 1996
;
Grushcow et al., 1999
;
Thompson and Stahl, 1999
). The
same observation applies for DNA damage and replication checkpoint proteins
such as Rad53 (another FHA Kinase member;
Fig. 1) and Mec1, which perform
essential functions during DNA replication in the mitotic cell cycle
(Desany et al., 1998
).
Mek1-dependent regulation of meiotic cell cycle
We have observed that kinetics of meiotic progression in fission yeast
depends on Mek1 dosage. Lack of Mek1 results in a more rapid entry into
meiosis I, whereas high levels of Mek1 lead to delayed meiotic progression.
Premeiotic S phase is not affected, suggesting that Mek1 function negatively
regulates the prophase to meiosis I transition. Supporting this notion,
western blot analysis of Mek1 throughout meiotic time courses revealed that
the protein rapidly disappears as cells enter the first meiotic division.
Since little is known about the molecular mechanisms controlling the
meiotic cell cycle in fission yeast in comparison with the regulation of the
mitotic cell cycle, we used ectopic overexpression of
mek1+ in vegetative cells as a tool for identifying
potential cell cycle targets of Mek1. We found that high levels of Mek1 in
vegetative cells lead to G2/M arrest, the same effect observed when the
homologous Cds1 checkpoint kinase is overproduced
(Boddy et al., 1998). In S.
pombe, the G2/M transition depends on the phosphorylation status of Cdc2
on Tyr15 (reviewed by Moser and Russell,
2000
). The Wee1 and Mik1 kinases inhibit Cdc2 activity by
phosphorylation of Tyr15, whereas the Cdc25 phosphatase activates Cdc2 by
removing the phosphate of Tyr15, thus promoting the G2/M transition. Our
results suggest that Mek1-induced cell cycle arrest partly results from
inhibition of Cdc25 rather than activation of Wee1, because the effect of Mek1
overproduction, as manifested by cell elongation, is diminished in
cdc25
and cdc25-9A strains, but is not significantly
altered in the absence of Wee1 function. The cdc25-9A mutant lacks
the relevant Cds1 phosphorylation sites and shows a much weaker cell cycle
arrest response to high Mek1 levels. Given the sequence similarity between
Mek1 and Cds1 (Fig. 1), these
results strongly suggest that Mek1 directly phosphorylates Cdc25, promoting
its inhibition. Nevertheless, our results indicate that Cdc25 is not the only
target of Mek1; the possible involvement of Mik1 in the Mek1-dependent
regulation of cell cycle remains to be investigated.
The meiotic recombination checkpoint in S. pombe: a role for
Mek1
Our results show that defects at intermediate steps in the recombination
pathway (induced by a meu13 mutation) trigger a meiotic cell cycle
delay in fission yeast mediated by inhibitory phosphorylation of the
cyclin-dependent kinase Cdc2 on Tyr15. Like its budding yeast homolog, S.
pombe Mek1 is an important component of this meiotic recombination
checkpoint; mutation of mek1+ alleviates the
meu13 delay. Although the checkpoint-induced delay is not very
prolonged (30 minutes), it appears to be important for the viability of
the meiotic products. The extent of the meiotic delay in meu13
roughly correlates with a delay in DSBs repair
(Shimada et al., 2002
);
therefore it is formally possible that S. pombe mutants with more
profound defects in recombinational repair of DSBs may exhibit stronger
meiotic delays. However, although detailed analysis of meiotic progression in
such mutants has not been reported, there is no evidence for a robust meiotic
block in fission yeast. For example, the rhp51 mutant is able to
complete meiosis and sporulation despite the presence of unrepaired DSBs
(Zenvirth and Simchen, 2000
;
Boddy et al., 2001
).
The pachytene checkpoint pathway has been extensively studied in S.
cerevisiae, and several components have been identified (see
introduction) (reviewed by Roeder and
Bailis, 2000). It has been proposed that Mek1-dependent
phosphorylation of Red1 is required for checkpoint-induced arrest in response
to unrepaired recombination intermediates. Once recombination has been
completed, dephosphorylation of Red1 by Glc7 allows pachytene exit and entry
into meiosis I (Bailis and Roeder,
1998
; Bailis and Roeder,
2000
). Pachytene arrest is achieved, at least in part, by
inhibition of Cdc28/Clb1 activity, both by Swe1-mediated inhibitory
phosphorylation of Cdc28 on Tyr19 (the equivalent of Tyr15 in S.
pombe Cdc2) and by limiting Ndt80-dependent transcription of
CLB1 (Chu and Herskowitz,
1998
; Hepworth et al.,
1998
; Leu and Roeder,
1999
; Tung et al.,
2000
). An additional branch of the checkpoint, which targets the
Sum1 transcriptional repressor has been recently reported
(Lindgren et al., 2000
). When
the pachytene checkpoint is activated, the Swe1 kinase accumulates in a
hyperphosphorylated (and presumably activated) form
(Leu and Roeder, 1999
);
however, the molecular mechanisms regulating Swe1 stability and
phosphorylation are unknown.
As mentioned above, in S. cerevisiae, phosphorylation of Red1 (a
component of the lateral elements of the SC) by Mek1 is important for
transducing the pachytene checkpoint signal. In S. pombe, however,
there is no SC and, although SC-like structures called linear elements have
been described (Bahler et al.,
1993), BLAST searches reveal that no obvious RED1 homolog
exist in the fission yeast genome
(http://www.sanger.ac.uk/Projects/S_pombe/).
Therefore, targets of Mek1 other than Red1 must exist in fission yeast. Our
experiments expressing Mek1 in different S. pombe mutant backgrounds
during vegetative growth suggest that Mek1 phosphorylates Cdc25. Moreover, we
have shown that the phosphorylation-deficient cdc25-9A mutant is
impaired in the meiotic checkpoint response to the presence of recombination
intermediates. We thus provide the first evidence of a direct connection
between an effector kinase of the meiotic recombination checkpoint (Mek1) and
a key component of the cell cycle machinery (Cdc25). Consistent with our
findings, an essential role for Cdc25 in promoting meiosis I has been reported
(Iino et al., 1995
).
As in budding yeast, here we show that the meiotic recombination checkpoint
in S. pombe regulates the phosphorylation of Cdc2 on Tyr15; however,
we propose that the checkpoint-induced meiotic delay would, in part, be
mediated by the Mek1-dependent inhibitory phosphorylation of Cdc25 and not by
the activation of Wee1. This contrasts with the situation in S.
cerevisiae, in which Mih1 (the Cdc25 homolog) seems to be dispensable for
pachytene checkpoint function (Leu and
Roeder, 1999). Thus, although the meiotic checkpoint arrests or
delays meiosis by maintaining inhibitory phosphorylation of the
cyclin-dependent kinase in both budding and fission yeast, different cell
cycle regulators are targeted in each organism. It has been recently described
that the Mrc1 protein is required for activation of Rad53 and Cds1 during the
replication checkpoint in both budding and fission yeast
(Alcasabas et al., 2001
;
Tanaka and Russell, 2001
).
Whether activation of Mek1 by the pachytene checkpoint also requires Mrc1 or
other adaptor proteins remains to be tested.
A model for the fission yeast meiotic recombination checkpoint
pathway
In agreement with our observations, during the preparation of this paper,
the existence of a meiotic recombination checkpoint in S. pombe has
also been reported (Shimada et al.,
2002). These authors show that the mitotic DNA integrity
checkpoint Rad proteins also respond to unrepaired DSBs during meiosis in
fission yeast, as they do in S. cerevisiae
(Lydall et al., 1996
). It has
been recently shown that Mec1 and the Rad24 group of budding yeast checkpoint
proteins do indeed localize to the sites of DSBs, acting as sensors of damage
(Kondo et al., 2001
;
Melo et al., 2001
;
Hong and Roeder, 2002
).
Combining our results and those of Shimada et al., we propose the following
model for the action of the meiotic recombination checkpoint in fission yeast
(Shimada et al., 2002
)
(Fig. 8). The presence of
ongoing recombination (presumably unrepaired DSBs) is sensed by the group of
Rad checkpoint proteins, generating a signal that results in activation of the
Mek1 kinase. Mek1, in turn, phosphorylates Cdc25, and possibly other as yet
unknown substrate(s), which contribute to the maintenance of Tyr15
phosphorylation of Cdc2, thus inhibiting meiosis I entry. When recombination
has been completed, the inhibitory signal disappears and dephosphorylation of
Cdc2 on Tyr15 promotes the first meiotic division.
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