Department of Cell Biology, Institute for Cancer Research, Montebello, 0310 Oslo, Norway
* Author for correspondence (e-mail: beata.grallert{at}labmed.uio.no)
Accepted 8 June 2002
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Checkpoint, S. pombe, orp1, DNA replication, Mitosis
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Much less is known about checkpoints in the G1 phase of the cell cycle.
This is mostly because G1 in fission yeast is very short under standard
laboratory growth conditions, rendering the investigation of the existence and
mechanism of such a checkpoint(s) difficult. However, several cell cycle
mutants arrest in the G1 phase of the cell cycle (i.e. before any DNA
replication has occurred and without proceeding to mitosis). This arrest might
be due to checkpoint activation, thereby providing a means to investigate G1
checkpoint(s) in fission yeast. We have used the orp1-4 replication
initiation mutant, which at the restrictive temperature either enters mitosis
in the absence of DNA replication (`cut' phenotype) or arrests with a 1C DNA
content without entering mitosis (`arrest' phenotype)
(Grallert and Nurse, 1996).
When the orp1-4 mutant is incubated at the restrictive temperature,
about 70% of the cells arrest, whereas 30% cut
(Grallert and Nurse, 1996
). In
the complete absence of orp1+ function, in the
orp1
mutant, all of the cells cut.
The orp1 gene encodes one of the components of the fission yeast
origin recognition complex (ORC). The ORC binds the replication origins
throughout the cell cycle and serves as a landing pad for other proteins
essential for the initiation of replication (reviewed by
Leatherwood, 1998). One of
these is the Cdc18 protein which, together with Cdt1
(Nishitani et al., 2000
), is
required for loading of the MCM proteins (minichromosome
maintenance) onto the origins, thus forming the pre-replication
complex (preRC).
In this work we have studied both the arrest and the cut phenotypes of the orp1-4 mutant. We show that orp1-4 cells arrest in G1 owing to a checkpoint that requires the checkpoint Rad proteins and Chk1, but not Cds1. Cdc2 is phosphorylated by Wee1 in G1 after activation of Chk1 and the arrest of orp1-4 cells depends on this phosphorylation. We also analyse the cutting and show that commitment to cutting occurs before S phase.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
2D gel analysis
Purified genomic DNA was digested with HindIII and KpnI
and analysed as described before (Muller
et al., 2000). After blotting, the membrane was probed with a
32P-labelled fragment within the 3kb HindIII-KpnI
fragment of the rDNA repeat of S. pombe
(Sanchez et al., 1998
).
Protein extracts and western blots
Protein extracts for western blotting were made by TCA extraction, as
described previously (Caspari et al.,
2000). For western blot analysis the following antibodies were
used: anti-phosphotyrosine Cdc2 (Sigma C0228) at a dilution of 1:400;
anti-pSTAIRE against Cdc2 (Santa Cruz sc-53) at a dilution of 1:2000; anti-HA
(Babco) at a dilution of 1:1000); anti-myc (PharMingen 9E10) at a dilution of
1:1000; and anti-
-tubulin (Sigma T5168) at a dilution of 1:20,000. The
secondary antibodies were either HRP or AP conjugates, used at a dilution of
1/5000. Detection was performed using the enhanced chemiluminescence procedure
(NEN ECL kit). Cdc2 and phosphorylated Cdc2 was measured using ECF detection
(Amersham) and quantified with a phosphoimager and the Image Quant software.
Chk1 and Wee1 phosphorylation was followed by Super Signal (Pierce)
detection.
Cds1 kinase activity
Cds1 was immunoprecipitated and Cds1 kinase activity was measured using MBP
as substrate as described (Lindsay et al.,
1998). Quantification of the kinase activity was performed using a
phosphoimager and the ImageQuant software.
Flow cytometry and microscopy
About 107 cells were spun down for each sample and fixed in 70%
ethanol before storing at 4°C. Samples were processed for flow cytometry
as described (Sazer and Sherwood,
1990), stained with Sytox Green (Molecular Probes S-7020,
http://pingu.salk.edu/fcm/protocols/ycc.html)
and analysed with a Becton-Dickinson FACSCalibur machine.
4',6-diamidino-2-phenylindole (DAPI) was used for nuclear staining, and
Calcofluor was used for cell wall and septum staining as described previously
(Moreno et al., 1991
).
Synchronous cells
Partially synchronous cells were selected from exponentially growing
cultures by lactose gradient centrifugation on 10-30% gradients, as described
(Carr et al., 1995).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
The G1 arrest of orp1-4 cells is checkpoint dependent
About 70% of the orp1-4 mutant cells arrest prior to DNA
replication at the restrictive temperature. We combined orp1-4 with a
number of checkpoint mutations. Mutation of any of several checkpoint
rad genes in orp1-4 abolished the arrest
(Fig. 2A), demonstrating that
it is checkpoint dependent. Consistently, phosphorylation of Rad9, a marker of
checkpoint activation (Caspari et al.,
2000), was clearly induced in orp1-4 cells
(Fig. 2B). As a positive
control, the extent of Rad9 phosphorylation was the same as that in a
cdc17ts mutant at the restrictive temperature
(Fig. 2B), conditions that are
known to require checkpoint activation for arrest. We conclude that arrest of
the orp1-4 mutant is checkpoint dependent.
|
The orp1-4 rad double mutants appear to enter mitosis (cut) earlier than the orp1-4 single mutant (Fig. 2A). To verify that the double mutants cut earlier and not only more extensively, we selected synchronous orp1-4, orp1-4 rad26 and orp1-4 cdc18-K46 cells after lactose gradient centrifugation and followed cutting at the restrictive temperature. Both double mutants were found to cut significantly earlier than the single mutant (Fig. 2C). In these experiments, the level of cutting of the orp1-4 mutant cells was less than shown above (Fig. 2A), because in the present experiment the cells were synchronized in early G2 phase and therefore reached the arrest point later than the asynchronous cells described in Fig. 2A.
Chk1, but not Cds1, is involved in the checkpoint response
In the DNA replication or the G2/M damage pathways the targets of the
checkpoint Rad proteins are the effector kinases Chk1 and Cds1
(Walworth et al., 1993;
Murakami and Okayama, 1995
).
Deletion of cds1 did not affect entry into aberrant mitosis (cutting)
in orp1-4 (Fig. 3A),
showing that Cds1 is not required for the checkpoint. Consistently, Cds1
kinase activity was not induced in orp1-4 upon temperature shift
(Fig. 3B). This finding further
supports the above conclusion that orp1-4 cells do not arrest in S
phase, since Cds1 is known to be required for checkpoints activated during S
phase (Murakami and Nurse,
2001
; Lindsay et al.,
1998
; Murakami and Okayama,
1995
). Surprisingly, deletion of chk1 abolished the
checkpoint that ensures arrest of 70% of the orp1-4 cells
(Fig. 3A). When orp1-4
cells were shifted to the restrictive temperature, Chk1 became phosphorylated
(Fig. 3C), further suggesting
that Chk1 is required to prevent cutting. In contrast, in orp1-4
cdc18-K46, where the vast majority of cells enter mitosis in the absence
of DNA replication, phosphorylation of Chk1 did not increase upon shift-up
(Fig. 3C), strongly suggesting
that the phosphorylated Chk1 is indeed responsible for arresting
orp1-4 cells.
|
These results show that there is a checkpoint prior to DNA replication that is responsible for the arrest of 70% of orp1-4 cells. This checkpoint involves the checkpoint Rad proteins and Chk1 but not Cds1.
The checkpoint is independent of Rum1 and Ste9
The major target of the S/M and the G2/M checkpoints is the Cdc2 kinase,
which also plays an important role in the initiation of DNA replication. Since
inhibiting Cdc2 is a likely target of the checkpoint activated in
orp1-4 cells, we investigated whether the known mechanisms of Cdc2
inhibition are required for the G1 arrest of 70% of orp1-4 cells.
Rum1 can inhibit the mitotic CDK, Cdc2-Cdc13, in vitro and is required for
efficient proteolysis of Cdc13 (Moreno and
Nurse, 1994; Correa-Bordes and
Nurse, 1995
; Davis and Smith,
2001
; Correa-Bordes et al.,
1997
). Ste9 is required for efficient degradation of the mitotic
cyclin Cdc13 during G1 arrest (Yamaguchi
et al., 2000
; Blanco et al.,
2000
; Guo and Dunphy,
2000
). We constructed double mutants of orp1-4 with
rum1
and ste9
and compared cutting in the
double mutants and the orp1-4 single mutant. Both double mutants cut
with the same timing and to the same extent as the orp1-4 single
mutant (data not shown). We conclude that Rum1 and Ste9 are not required for
the arrest of orp1-4 cells prior to DNA replication.
Cdc2 is phosphorylated by Wee1 in arrested orp1-4 cells
In cycling cells, phosphorylation of Cdc2 is thought to start in late G1,
close to the G1/S transition (Hayles and
Nurse, 1995). The previously characterised S/M and DNA damage
checkpoint pathways delay entry into mitosis by maintaining phosphorylation of
Cdc2. When orp1-4 cells are incubated at the restrictive temperature,
phosphorylated Cdc2 can be detected
(Grallert and Nurse, 1996
).
This could result from the activation of the Chk1-dependent checkpoint pathway
(above), or Cdc2 could have been phosphorylated before checkpoint activation.
We shifted synchronous orp1-4 cells to the restrictive temperature
and followed both the appearance of phosphorylated Cdc2 and the activation of
the checkpoint, as marked by Chk1 phosphorylation. Chk1 and Cdc2 became
phosphorylated 2.5 hours and 3.5 hours after shift-up, respectively
(Fig. 4A,B), opening the
possibility that Cdc2 phosphorylation in orp1-4 is a result of
activation of the checkpoint and of Chk1.
|
cdc2 mutations conferring a wee phenotype, cdc2-1w and cdc2-3w, make Cdc2 insensitive to phosphorylation by Wee1 and independent of dephosphorylation by Cdc25, respectively. When we combined orp1 with the cdc2-1w and cdc2-3w mutations, both double mutants were found to cut more than the orp1-4 single mutant (Fig. 4C). This shows that phosphorylation of Cdc2 is important to keep the orp1-4 cell from entering aberrant mitosis.
Phosphorylation of Cdc2 can be carried out by either of two kinases, Wee1
or Mik1 (reviewed by Berry and Gould,
1996). To investigate which kinase is responsible for
phosphorylation of Cdc2 in arrested orp1-4 cells, we constructed
orp1-4 wee1-50 and orp1-4 mik1
double mutants.
Mutation of wee1, but not of mik1 exaggerated the cutting
phenotype of orp1-4 (Fig.
4D). Expression of mik1 is cell cycle regulated and the
protein is expressed in S phase. Activation of the intra-S phase and S/M
checkpoints results in further induction of mik1 in a
cds1-dependent manner (Christensen
et al., 2000
; Baber-Furnari et
al., 2000
; Borgne and Nurse,
2000
). Therefore, a low level of expression of Mik1 in
orp1-4 cells at the restrictive temperature
(Fig. 4E) is consistent with a
lack of effect of a mik1
mutation and is further evidence that
the cells have not entered S phase.
When the G2/M damage checkpoint is activated, the Wee1 protein accumulates
and is phosphorylated in a Chk1-dependent manner
(Raleigh and O'Connell, 2001).
We investigated whether a similar process takes place in the pre-replication
checkpoint arresting orp1-4 cells. When asynchronous orp1-4
cells were arrested at the restrictive temperature an accumulation of Wee1
could be observed and at later times phosphorylated Wee1 was detected
(Fig. 4F). We conclude that
phosphorylation of Cdc2 in orp1-4 cells is due to Wee1 activity prior
to DNA replication and occurs after and possibly in response to checkpoint
activation.
Activation of a damage checkpoint pathway in G1 prevents cutting in
germinating orp1 spores
The above results suggest that there is a checkpoint prior to DNA
replication that requires the checkpoint Rad proteins and Chk1 and acts via
inducing and/or maintaining phosphorylation of Cdc2. In the orp1-4
mutant this checkpoint might be activated by aberrant origin structures
recognized as DNA damage (see Discussion). One prediction of this model is
that if we inflict DNA damage in the absence of Orp1 and thereby induce this
G1 checkpoint, cutting should be prevented. To test this hypothesis, we
irradiated germinating orp1 spores derived from an
orp1-4/orp1
diploid. These orp1
spores contain
the temperature-sensitive Orp1 from the parental diploid and, when germinated
at the restrictive temperature, all of them enter mitosis in the absence of
DNA replication (Grallert and Nurse,
1996
). The spores were irradiated with a UV dose (254 nm) that
gave a 50% survival of wild-type cells. Irradiation of the germinating
orp1
spores strongly reduced cutting
(Fig. 5), suggesting that
inflicting DNA damage in G1 in the absence of Orp1 can induce a checkpoint
that inhibits the mitotic machinery.
|
orp1-4 cells become committed to cutting early in G1
Entry into aberrant mitosis (cutting) in orp1-4 cells can be
observed already 4 hours after a shift to the restrictive temperature, but
only about 30% of the cells cut even after 6 hours
(Grallert and Nurse, 1996). We
argue that these cells enter mitosis from G1 (i.e. before the occurrence of
any DNA replication). First, if cells leaked into S phase before cutting, it
would be detectable by 2D gel analysis
(Fig. 1), although synthesis of
very short pieces of DNA would not be detected by this technique. Second, we
examined whether orp1-4 cells have to pass the cdc10 arrest
point before they become committed to cutting. Cdc10 is a transcription factor
that activates transcription of a number of genes required for initiation of
DNA replication (Aves et al.,
1985
; Tanaka et al.,
1992
; Lowndes et al.,
1992
). Therefore, temperature-sensitive cdc10 mutants
arrest at the restrictive temperature before any DNA replication has occurred
and with a 1C DNA content. If the orp1-4 cells have to pass the
cdc10 arrest point to become committed to cutting, we would expect an
orp1-4 cdc10ts double mutant not to cut at the restrictive
temperature. Even if the double mutant leaks through the cdc10 arrest
point, it would cut only after a delay compared with orp1-4. When
synchronous orp1-4 and orp1-4 cdc10-129 cells were selected
by lactose gradient centrifugation and shifted to the restrictive temperature,
the double mutant was not delayed at cutting compared with the single
orp1-4 mutant (Fig.
6). On the contrary, the double mutant cut earlier than the single
mutant. Asynchronous orp1-4 and orp1-4 cdc10-129 mutants cut
to the same extent and with similar timing (data not shown). We conclude that
commitment to cutting in orp1-4 occurs before entry into S phase and
not later than the cdc10 arrest point.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
It was shown earlier (Grallert and
Nurse, 1996) that chromosomes prepared from arrested
orp1-4 cells do not enter the gel upon PFGE. This result suggests the
presence of aberrant DNA structures but is also consistent with an early S
phase arrest. Two-dimensional gel analysis of ars3001 argues against
the latter interpretation and leaves us to conclude that aberrant DNA
structures are responsible for the arrest in orp1-4 cells.
In synchronous orp1-4 cells shifted to the restrictive temperature, Chk1 phosphorylation occurs before Cdc2 phosphorylation (Fig. 4A,B), leading us to speculate that activation of Chk1 brings about Cdc2 phosphorylation.
Of the two kinases that phosphorylate Cdc2, absence of Wee1 function
abolishes the G1 arrest of orp1-4 cells, strongly suggesting that
Wee1 phosphorylates Cdc2 during G1 in the present checkpoint
(Fig. 6). The following
observations suggest that Wee1 is activated by Chk1. Deleting
chk1+ in orp1-4 cells has the same consequences
as introducing a wee1-50 mutation (this work). Furthermore,
wee1- cells are insensitive to the overexpression of Chk1,
which can directly phosphorylate Wee1 in vitro
(O'Connell et al., 1997) and
activation of the G2/M damage checkpoint leads to accumulation and
phosphorylation of the Wee1 protein
(Raleigh and O'Connell, 2001
).
Accumulation and phosphorylation of Wee1 can also be observed in arrested
orp1-4 cells. Therefore, it is tempting to speculate that
phosphorylation and/or an increase of Wee1 activity are contributing
mechanisms to bring about and maintain a G1 arrest of orp1-4 cells
(Fig. 6). A direct way to
examine this suggestion would be to measure Cdc2 phosphorylation in orp1-4
chk1
and orp1-4 wee1 double mutants. However, these
mutants start cutting less than 2 hours after shift-up to the restrictive
temperature. Since Cdc2 becomes dephosphorylated as the cells go into mitosis
and cut (Fig. 4B), it is not
possible to determine whether the deletion of chk1 or wee1
results in lack of Cdc2 phosphorylation. In contrast to wee1,
mutation of mik1 has no effect on cutting in orp1-4 cells,
nor is mik1 expression induced at the restrictive temperature. Thus,
Mik1 is not a major target of the checkpoint induced in orp1-4
cells.
Most temperature-sensitive replication initiation mutants arrest in G1 or inside S phase, whereas several deletion mutants proceed to aberrant mitosis without completing S phase (cutting). In general, the tighter the mutants are, the more cutting occurs. These observations have led to the model that the preRC complex, in addition to initiating DNA replication, provides a signal that S phase is in preparation and mediates inhibition of mitosis through activation of a checkpoint pathway. An alternative explanation is that a preRC-independent checkpoint is activated in G1. A third alternative is that the cells attempt to replicate DNA in spite of compromised initiation, and that it is the S/M checkpoint pathway that prevents mitosis. The latter alternative has been excluded by the present experiments. We will discuss the two former alternatives below.
We suggest that the difference between the two populations of
orp1-4 cells (cutting versus arresting) is that the residual function
of the Orp1-4 protein allows changing the origin structure in 70% of the cells
to a structure that activates a checkpoint pathway
(Fig. 7). Although the preRC
does not assemble properly in orp1-4 cells
(Kearsey et al., 2000), some
steps of preRC formation might be performed, at least on some origins. This
incomplete preRC or the resulting origin structure might in turn activate the
checkpoint (Fig. 7). The
following observations support the idea that the arrested cells have proceeded
further than cutting cells have, and that formation of a certain structure,
possibly recognised as DNA damage, is responsible for the arrest. (1) The
cutting cells become committed to cutting at a point before the cdc10
arrest point (Fig. 6), while
the arresting cells arrest at or later than the cdc10 arrest point
(Grallert and Nurse, 1996
).
(2) orp1-4 cells rapidly lose viability upon shift to the restrictive
temperature, before cells undergoing aberrant mitoses can be observed (data
not shown). This argues for an irreversible formation of damage-like
structures. (3) Chromosomes prepared from arrested orp1-4 cells do
not enter the gel upon PFGE (Grallert and
Nurse, 1996
), consistent with the presence of aberrant DNA
structures. (4) Complete loss of Orp1 function or a combination of
orp1-4 with mutant alleles of genes encoding other preRC components
leads to increased entry into mitosis in the absence of DNA replication
relative to an orp1-4 single mutation
(Fig. 2C)
(Grallert and Nurse, 1996
),
strongly suggesting that the former mutants are unable to modify the origin
structure enough to activate a checkpoint. Consistently, no Chk1
phosphorylation was detected in orp1-4 cdc18-K46 double mutant cells
(Fig. 3C).
|
The checkpoint activated in orp1-4 cells is clearly different from
the intra-S checkpoint, since the former requires Chk1 but does not require
Cds1 (this work) while the latter has the opposite requirement
(Walworth et al., 1993;
Lindsay et al., 1998
).
We argue that complete lack of replication does not activate any checkpoint
and therefore results in entry into mitosis in the absence of DNA replication.
Induction of the checkpoint depends on an attempt to form a preRC. Formation
of an incomplete preRC results in checkpoint induction, but this does not
imply that the checkpoint is specifically activated by incomplete preRCs. Our
finding that irradiating germinating orp1 spores prevents
entry into mitosis points to the existence of a damage checkpoint activated in
G1 that can inhibit the mitotic machinery. We favour the interpretation that
this checkpoint is identical to the checkpoint activated in arresting
orp1-4 cells
Studying germinating spores carrying a deletion of the orp2 gene
resulted in the conclusion that there is no checkpoint for a lack of DNA
replication (Kiely et al.,
2000), consistent with our interpretation. However, the
orp2ts mutant was arrested in S phase and therefore those
experiments did not allow the detection of a checkpoint prior to DNA
replication.
Combination of the orp1-4 mutation with cdc10-129
(Fig. 1B), cdc18-K46
(Fig. 2C), checkpoint
rad genes (Fig. 2A),
or chk1 (Fig.
3A) makes the cells cut earlier and more extensively than the
single orp1-4 mutant. We suggest that the first two cut earlier and
more because the arrest in orp1 mutant cells is dependent on
attempting preRC formation. To follow our line of reasoning above, more preRC
formation will be achieved in the presence of Cdc18, which again will lead to
more checkpoint arrest and less cutting. Consistently, it has been shown that
Cdc18 overproduction can rescue the cutting phenotype of orp1-4
(Grallert and Nurse, 1996
). It
has been shown (Greenwood et al.,
1998
) that overexpressing the C-terminus of Cdc18 in wild-type
cells is sufficient for replication initiation and induction of a checkpoint
inhibiting mitosis, but since these cells were shown to be arrested in S phase
with between 1C and 2C DNA contents, the relevance for the present experiments
is uncertain. The early-cut phenotype observed when orp1-4 is
combined with checkpoint rad mutations suggests that the checkpoint
proteins might be physically present and active at the origin complex in
orp1-4 cells.
There are two contradictory reports on a G1 checkpoint in a
cdc10ts mutant. The Chk1 protein was found to be required
(Carr et al., 1995) and not
required (Hayles and Nurse,
1995
) for arrest of cdc10ts cells. In the two
papers different synchronisation methods were used, which might account for
the discrepancy. Since Cdc2 is not phosphorylated in arrested
cdc10ts cells (Hayles
and Nurse, 1995
), it is unlikely that the checkpoint pathways
arresting cdc10ts and orp1-4 cells are one and
the same. Consistently, Rum1, which is required only in pre-START G1, is
necessary for a cdc10ts arrest
(Moreno and Nurse, 1994
), but
not for arrest of orp1-4 cells (this work). However, we cannot
exclude the possibility that cdc10ts cells leak into later
parts of G1, although it was shown that they do not leak into S phase
(Carr et al., 1995
). If this is
indeed the case, the Chk1-dependent checkpoint reported earlier
(Carr et al., 1995
) might be
the same as the one arresting orp1-4 cells.
The checkpoint activated in orp1-4 cells appears to be similar to
the G2/M damage checkpoint in that it requires Chk1 and the maintenance of
Cdc2 phosphorylation. Also, failure of either checkpoint results in entry into
mitosis. We have provided several pieces of evidence that orp1-4
cells arrest in G1. Nevertheless, it is possible that the present G1/M
checkpoint mechanism is using the same molecular signals and targets as the
G2/M checkpoint. The phosphorylation state of Cdc2 and the levels and
activities of the mitotic regulators Cdc13 and Cdc25 may be the same during a
late G1 arrest as in a G2 arrest. In this context it is interesting to note
that orp1-4 cdc2-3w cells are checkpoint deficient
(Fig. 4C). This observation
raises the possibility that, in addition to Wee 1, Cdc25 might also be
targeted by Chk1 in orp1-4 cells, as in the G2/M checkpoint
(Raleigh and O'Connell, 2001).
In higher eukaryotes the biochemical differences during the G1 and G2 phases
are more extensive, because of the divergent evolution of CDKs and their
regulators. Therefore, failure of a G1 checkpoint cannot lead to premature
entry into mitosis, but only to premature entry into S phase.
Checkpoint activation in G1 in higher eukaryotes depends upon
phosphorylation and inhibition of CDKs, which is similar to the checkpoint
activated in orp1-4 cells. In human cells, CDK2 activity is rapidly
inhibited in response to activation of the G1/S damage pathway, by destruction
of CDC25A (Mailand et al.,
2000). It is noteworthy that hChk1 phosphorylates hCdc25A in vitro
(Sanchez et al., 1997
) and
destruction of Cdc25A is Chk1 dependent
(Mailand et al., 2000
). Less
is known about the possible involvement of Wee1 or Mik1 in the DNA damage
checkpoints in higher eukaryotes. Xenopus Chk1 phosphorylates Wee1
upon checkpoint activation, which leads to increased Wee1 kinase activity
(Lee et al., 2001
). In
Drosophila the rad3 homologue mei-41 and the
chk1 homologue GRP are involved in the slowing of the early embryonic
cycles, which indicates activation of a checkpoint once maternally provided
replication functions become limiting
(Fogarty et al., 1997
;
Sibon et al., 1999
;
Sibon et al., 1997
;
Zhou and Elledge, 2000
;
Rhind et al., 1997
).
Interestingly, mutation of Wee1 has similar phenotypic consequences
to mutation of Grp (Price et al.,
2000
) suggesting that this Grp-dependent checkpoint might target
Wee1. The relative contributions of inhibiting the phosphatases and inducing
the kinases might vary in different organisms, but the strategy of maintaining
CDK phosphorylation upon checkpoint activation in G1 appears to be
conserved.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aves, S. J., Durkacz, B. W., Carr, A. and Nurse, P. (1985). Cloning, sequencing and transcriptional control of the Schizosaccharomyces pombe cdc 10 `start' gene. EMBO J. 4, 457-463.[Abstract]
Baber-Furnari, B. A., Rhind, N., Boddy, M. N., Shanahan, P.,
Lopez-Girona, A. and Russell, P. (2000). Regulation of
mitotic inhibitor Mik1 helps to enforce the DNA damage checkpoint.
Mol. Biol. Cell 11,1
-11.
Berry, L. D. and Gould, K. L. (1996). Regulation of Cdc2 activity by phosphorylation at T14/Y15. Prog. Cell Cycle Res. 2,99 -105.[Medline]
Blanco, M. A., Sanchez-Diaz, A., de Prada, J. M. and Moreno,
S. (2000). APC(ste9/srw1) promotes degradation of mitotic
cyclins in G(1) and is inhibited by cdc2 phosphorylation. EMBO
J. 19,3945
-3955.
Borgne, A. and Nurse, P. (2000). The Spd1p S
phase inhibitor can activate the DNA replication checkpoint pathway in fission
yeast. J. Cell Sci. 113,4341
-4350.
Carr, A. M., Moudjou, M., Bentley, N. J. and Hagan, I. M. (1995). The Chk1 pathway is required to prevent mitosis following cell-cycle arrest at `start'. Curr. Biol. 5,1179 -1190.[Medline]
Caspari, T., Dahlen, M., Kanter-Smoler, G., Lindsay, H. D.,
Hofmann, K., Papadimitriou, K., Sunnerhagen, P. and Carr, A. M.
(2000). Characterization of Schizosaccharomyces pombe Hus1: a
PCNA-related protein that associates with Rad1 and Rad9. Mol. Cell
Biol. 20,1254
-1262.
Christensen, P. U., Bentley, N. J., Martinho, R. G., Nielsen, O.
and Carr, A. M. (2000). Mik1 levels accumulate in S phase and
may mediate an intrinsic link between S phase and mitosis. Proc.
Natl. Acad. Sci. USA 97,2579
-2584.
Correa-Bordes, J. and Nurse, P. (1995). p25rum1 orders S phase and mitosis by acting as an inhibitor of the p34cdc2 mitotic kinase. Cell 83,1001 -1009.[Medline]
Correa-Bordes, J., Gulli, M. P. and Nurse, P.
(1997). p25rum1 promotes proteolysis of the mitotic B-cyclin
p56cdc13 during G1 of the fission yeast cell cycle. EMBO
J. 16,4657
-4664.
Davis, L. and Smith, G. R. (2001). Meiotic
recombination and chromosome segregation in Schizosaccharomyces pombe.
Proc. Natl. Acad. Sci. USA
98,8395
-8402.
Edwards, R. J., Bentley, N. J. and Carr, A. M. (1999). A Rad3-Rad26 complex responds to DNA damage independently of other checkpoint proteins. Nat. Cell Biol. 1, 393-398.[CrossRef][Medline]
Fogarty, P., Campbell, S. D., Abu-Shumays, R., Phalle, B. S., Yu, K. R., Uy, G. L., Goldberg, M. L. and Sullivan, W. (1997). The Drosophila grapes gene is related to checkpoint gene chk1/rad27 and is required for late syncytial division fidelity. Curr. Biol. 7,418 -426.[Medline]
Grallert, B. and Nurse, P. (1996). The ORC1 homolog orp1 in fission yeast plays a key role in regulating onset of S phase. Genes Dev. 10,2644 -2654.[Abstract]
Greenwood, E., Nishitani, H. and Nurse, P.
(1998). Cdc18p can block mitosis by two independent mechanisms.
J. Cell Sci. 111,3101
-3108.
Guo, Z. and Dunphy, W. G. (2000). Response of
Xenopus Cds1 in cell-free extracts to DNA templates with double-stranded ends.
Mol. Biol. Cell 11,1535
-1546.
Hayles, J. and Nurse, P. (1995). A pre-start checkpoint preventing mitosis in fission yeast acts independently of p34cdc2 tyrosine phosphorylation. EMBO J. 14,2760 -2771.[Abstract]
Kearsey, S. E., Montgomery, S., Labib, K. and Lindner, K.
(2000). Chromatin binding of the fission yeast replication factor
mcm4 occurs during anaphase and requires ORC and cdc18. EMBO
J. 19,1681
-1690.
Kiely, J., Haase, S. B., Russell, P. and Leatherwood, J.
(2000). Functions of fission yeast orp2 in DNA replication and
checkpoint control. Genetics
154,599
-607.
Kim, S. M. and Huberman, J. A. (2001).
Regulation of replication timing in fission yeast. EMBO
J. 20,6115
-6126.
Kitamura, K., Maekawa, H. and Shimoda, C.
(1998). Fission yeast Ste9, a homolog of Hct1/Cdh1 and
Fizzy-related, is a novel negative regulator of cell cycle progression during
G1-phase. Mol. Biol. Cell
9,1065
-1080.
Leatherwood, J. (1998). Emerging mechanisms of eukaryotic DNA replication initiation. Curr. Opin. Cell Biol. 10,742 -748.[CrossRef][Medline]
Lee, J., Kumagai, A. and Dunphy, W. G. (2001).
Positive regulation of Wee1 by Chk1 and 14-3-3 proteins. Mol. Biol.
Cell 12,551
-563.
Lindsay, H. D., Griffiths, D. J., Edwards, R. J., Christensen,
P. U., Murray, J. M., Osman, F., Walworth, N. and Carr, A. M.
(1998). S-phase-specific activation of Cds1 kinase defines a
subpathway of the checkpoint response in Schizosaccharomyces pombe.
Genes Dev. 12,382
-395.
Liu, H. Y., Nefsky, B. S. and Walworth, N. C.
(2002). The Ded1 DEAD-box helicase interacts with Chk1 and Cdc2.
J. Biol. Chem. 277,2637
-2643.
Lowndes, N. F., McInerny, C. J., Johnson, A. L., Fantes, P. A. and Johnston, L. H. (1992). Control of DNA synthesis genes in fission yeast by the cell-cycle gene cdc 10+. Nature 355,449 -453.[CrossRef][Medline]
Mailand, N., Falck, J., Lukas, C., Syljuasen, R. G., Welcker,
M., Bartek, J. and Lukas, J. (2000). Rapid destruction of
human Cdc25A in response to DNA damage. Science
288,1425
-1429.
Martinho, R. G., Lindsay, H. D., Flaggs, G., DeMaggio, A. J.,
Hoekstra, M. F., Carr, A. M. and Bentley, N. J. (1998).
Analysis of Rad3 and Chk1 protein kinases defines different checkpoint
responses. EMBO J. 17,7239
-7249.
Moreno, S. and Nurse, P. (1994). Regulation of progression through the G1 phase of the cell cycle by the rum1+ gene. Nature 367,236 -242.[CrossRef][Medline]
Moreno, S., Klar, A. and Nurse, P. (1991). Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. Methods Enzymol. 194,795 -823.[Medline]
Moreno, S., Labib, K., Correa, J. and Nurse, P. (1994). Regulation of the cell cycle timing of Start in fission yeast by the rum1+ gene. J. Cell Sci. Suppl. 18, 63-68.[Medline]
Muller, M., Lucchini, R. and Sogo, J. M. (2000). Replication of yeast rDNA initiates downstream of transcriptionally active genes. Mol. Cell 5, 767-777.[CrossRef][Medline]
Murakami, H. and Nurse, P. (2001). Regulation of premeiotic S phase and recombination-related double-strand DNA breaks during meiosis in fission yeast. Nat. Genet. 28,290 -293.[CrossRef][Medline]
Murakami, H. and Okayama, H. (1995). A kinase from fission yeast responsible for blocking mitosis in S phase. Nature 374,817 -819.[CrossRef][Medline]
Nishitani, H., Lygerou, Z., Nishimoto, T. and Nurse, P. (2000). The Cdt1 protein is required to license DNA for replication in fission yeast. Nature 404,625 -628.[CrossRef][Medline]
O'Connell, M. J., Raleigh, J. M., Verkade, H. M. and Nurse,
P. (1997). Chk1 is a Wee 1 kinase in the G2 DNA damage
checkpoint inhibiting cdc2 by Y15 phosphorylation. EMBO
J. 16,545
-554.
Price, D., Rabinovitch, S., O'Farrell, P. H. and Campbell, S.
D. (2000). Drosophila wee1 has an essential role in the
nuclear divisions of early embryogenesis. Genetics
155,159
-166.
Raleigh, J. M. and O'Connell, M. J. (2001). The
G2 DNA damage checkpoint targets both Wee1 and Cdc25. J. Cell
Sci. 113,1727
-1736.
Rhind, N. and Russell, P. (1998). Tyrosine
phosphorylation of cdc2 is required for the replication checkpoint in
Schizosaccharomyces pombe. Mol. Cell. Biol.
18,3782
-3787.
Rhind, N., Furnari, B. and Russell, P. (1997). Cdc2 tyrosine phosphorylation is required for the DNA damage checkpoint in fission yeast. Genes Dev. 11,504 -511.[Abstract]
Sanchez, Y., Wong, C., Thoma, R. S., Richman, R., Wu, Z.,
Piwnica-Worms, H. and Elledge, S. J. (1997). Conservation of
the Chk1 checkpoint pathway in mammals: linkage of DNA damage to Cdk
regulation through Cdc25. Science
277,1497
-1501.
Sanchez, J. A., Kim, S. M. and Huberman, J. A. (1998). Ribosomal DNA replication in the fission yeast, Schizosaccharomyces pombe. Exp. Cell Res. 238,220 -230.[CrossRef][Medline]
Sazer, S. and Sherwood, S. W. (1990). Mitochondrial growth and DNA synthesis occur in the absence of nuclear DNA replication in fission yeast. J. Cell Sci. 97,509 -516.[Abstract]
Sibon, O. C., Stevenson, V. A. and Theurkauf, W. E. (1997). DNA-replication checkpoint control at the Drosophila midblastula transition. Nature 388, 93-97.[CrossRef][Medline]
Sibon, O. C., Laurencon, A., Hawley, R. and Theurkauf, W. E. (1999). The Drosophila ATM homologue Mei-41 has an essential checkpoint function at the midblastula transition. Curr. Biol. 9,302 -312.[CrossRef][Medline]
Sipiczki, M. and Ferenczy, L. (1977). Protoplast fusion of Schizosaccharomyces pombe Auxotrophic mutants of identical mating-type. Mol. Gen. Genet. 151, 77-81.[Medline]
Tanaka, K. and Russell, P. (2001). Mrc1 channels the DNA replication arrest signal to checkpoint kinase Cds1. Nat. Cell Biol. 3,966 -972.[CrossRef][Medline]
Tanaka, K., Okazaki, K., Okazaki, N., Ueda, T., Sugiyama, A., Nojima, H. and Okayama, H. (1992). A new cdc gene required for S phase entry of Schizosaccharomyces pombe encodes a protein similar to the cdc10+ and SWI4 gene products. EMBO J. 11,4923 -4932.[Abstract]
Walworth, N. C. and Bernards, R. (1996). rad-dependent response of the chk1-encoded protein kinase at the DNA damage checkpoint. Science 271,353 -356.[Abstract]
Walworth, N., Davey, S. and Beach, D. (1993). Fission yeast chk1 protein kinase links the rad checkpoint pathway to cdc2. Nature 363,368 -371.[CrossRef][Medline]
Wan, S. and Walworth, N. C. (2001). A novel genetic screen identifies checkpoint-defective alleles of Schizosaccharomyces pombe chk1. Curr. Genet. 38,299 -306.[CrossRef][Medline]
Wright, J. A., Keegan, K. S., Herendeen, D. R., Bentley, N. J.,
Carr, A. M., Hoekstra, M. F. and Concannon, P. (1998).
Protein kinase mutants of human ATR increase sensitivity to UV and ionizing
radiation and abrogate cell cycle checkpoint control. Proc. Natl.
Acad. Sci. USA 95,7445
-7450.
Yamaguchi, S., Murakami, H. and Okayama, H.
(1997). A WD repeat protein controls the cell cycle and
differentiation by negatively regulating Cdc2/B-type cyclin complexes.
Mol. Biol. Cell 8,2475
-2486.
Yamaguchi, S., Okayama, H. and Nurse, P.
(2000). Fission yeast Fizzy-related protein srw 1p is a
G(1)-specific promoter of mitotic cyclin B degradation. EMBO
J. 19,3968
-3977.
Zhou, B. B. and Elledge, S. J. (2000). The DNA damage response: putting checkpoints in perspective. Nature 408,433 -439.[CrossRef][Medline]