Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California 94143-0448
Minichromosome maintenance (MCM) proteins are essential DNA replication factors conserved among eukaryotes. MCMs cycle between chromatin bound and dissociated states during each cell cycle. Their absence on chromatin is thought to contribute to the inability of a G2 nucleus to replicate DNA. Passage through mitosis restores the ability of MCMs to bind chromatin and the ability to replicate DNA. In Drosophila early embryonic cell cycles, which lack a G1 phase, MCMs reassociate with condensed chromosomes toward the end of mitosis. To explore the coupling between mitosis and MCM-chromatin interaction, we tested whether this reassociation requires mitotic degradation of cyclins. Arrest of mitosis by induced expression of nondegradable forms of cyclins A and/or B showed that reassociation of MCMs to chromatin requires cyclin A destruction but not cyclin B destruction. In contrast to the earlier mitoses, mitosis 16 (M16) is followed by G1, and MCMs do not reassociate with chromatin at the end of M16. dacapo mutant embryos lack an inhibitor of cyclin E, do not enter G1 quiescence after M16, and show mitotic reassociation of MCM proteins. We propose that cyclin E, inhibited by Dacapo in M16, promotes chromosome binding of MCMs. We suggest that cyclins have both positive and negative roles in controlling MCM-chromatin association.
THE competence of a nucleus to replicate its DNA
oscillates during the cell cycle. For example, a G1
nucleus is able to replicate DNA given the appropriate signals, while a G2 nucleus is unable to do so given
the same signals (Rao and Johnson, 1970 Studies in a variety of experimental systems suggest that
the key mitotic event that restores the ability to replicate
DNA is the loss of cyclins or associated activities. In Saccharomyces cerevisiae, Schizosaccaromyces pombe, Drosophila, and vertebrate cultured cells, experimental removal
of mitotic cyclins or cdks allow DNA replication in a G2
nucleus (Broek et al., 1991 These data led us to ask if the mitotic loss of cyclin:cdk
activity couples passage through mitosis to chromosome
association of MCMs. Mitotic inactivation of cyclin:cdks
occurs by proteolytic degradation of the cyclin subunits. In
the Drosophila embryo cyclins A and B provide essential,
nonredundant, mitotic functions (Knoblich and Lehner,
1993 Fixation and Antibody Staining Procedures
For antibody staining, embryos were fixed and stained with purified primary antibodies, and diluted 1:50-1:500 using standard procedures. Specificity of rabbit polyclonal antibodies against each MCM has been described previously (Su et al., 1996 Heat Induction
Stable versions of mitotic cyclins were constructed essentially as described
previously (Sigrist et al., 1995 dacapo Mutants
Embryos were collected from heterozygous dap3x41 parents for 1 h and
aged at 25° for 5.5 h to reach cycle 16 before fixing. This is a mutant resulting from imprecise excision of the transposon from the dap3 line (de Nooij
et al., 1997). A Cyo wg-LacZ balancer was used to identify the genotype
to embryos by immunostaining for MCMs Associate with Chromosomes
as Mitosis Is Completed
Identification and characterization of three Drosophila
MCMs, MCM2, MCM4, and MCM5, have been described
(Treisman et al., 1995
During the embryonic cycles that lack a G1 phase, relocalization of MCMs to the nuclear region occurred as nuclei exit mitosis (Fig. 1). Association was first detected on
the mitotic chromosomes at late anaphase/telophase (Fig.
1, C-H, white arrows). Relocalization of MCMs precedes
and overlaps that of nuclear pore complexes (NPCs), as visualized by staining with WGA (Fig. 1, D and G; Finlay et al., 1987 Cyclin Degradation and
MCM-Chromosome Association
Next, we asked what mitotic event brings about chromosome association of MCMs. Chromosome association of
MCMs correlates temporally with progression from metaphase to telophase. Progression beyond metaphase in many
organisms is driven by proteolysis, the substrates of which
include mitotic cyclins. Possibly, it is the degradation of
one or more of these proteins that allows MCMs to associate with chromosomes. To ask if mitotic cyclins are involved, we analyzed chromatin association of MCMs when
cyclin degradation is prevented.
Deletion of the "destruction box" at the amino termini
of mitotic cyclins results in their stabilization in many systems, including Drosophila (Ghiara et al., 1991 To test if MCM-chromosome association requires cyclin
degradation, we analyzed three Drosophila MCMs after
induction of stable cyclins in cell cycles 14 and 15. All
three MCMs dispersed from the nucleus upon entry to mitosis in the presence of stable cyclins, as they do in normal
mitoses (not shown). During arrests in M14 and M15, reassociation with chromosomes occurred in the presence of
cyclin Bs but not cyclin As (Fig. 2; data not shown). In the
presence of cyclin As, DmMCM2 staining was uniform
throughout the cell, except for exclusion from the region
occupied by chromosomes (Fig. 2, panel 2, arrowheads).
This is similar to what is seen in metaphases of wild-type
embryos and heat-shocked w67 controls lacking the transgenes (not shown). In contrast, in the presence of cyclin
Bs, no such clearing of the DmMCM2 stain from the chromosomal region was seen. In most cells, colocalization of
DmMCM2 stain and DNA stain is observed, suggesting
concentration of DmMCM2 on chromosomes; this is similar to chromosomes in late anaphase of wild-type mitoses (e.g., Fig. 1, F-H, white arrows). The intensity of DmMCM2
stain on the chromosomes varied from cell to cell; this may
be because cells of a Drosophila embryo undergo M14 and
M15 asynchronously (Foe, 1989
While we cannot rule out that the lack of DmMCM2
stain on chromosomes that we report here and in Fig. 1 is
caused by epitope masking at certain stages of mitosis, this
notion is not consistent with the strong cytoplasmic staining of DmMCM2 seen at the same stages (e.g., in As arrest
in Fig. 2, panel 2).
These results indicate that cyclin As is able to prevent
DmMCM2 association with chromosomes, while cyclin Bs
lacks the ability to do so under our experimental conditions. Consistent with this idea, when cyclins As and Bs
were coexpressed, DmMCM2 was not associated with
chromosomes (Fig. 2, panel 3, arrowheads). Staining for
DmMCM4 and DmMCM5 showed that binding of these
MCMs paralleled that of DmMCM2 (data not shown).
MCM-Chromosome Association Is Delayed When
Mitosis Is Followed by a Prolonged Gap Phase
Our data show that all requirements for MCM-chromosome association are met during a mitotic arrest with cyclin Bs during embryonic mitoses M14 and M15. These mitoses are followed immediately by S phase and no gap
phase. Thus, many activities required for MCM-chromosome association and DNA replication are likely to be
present at these times. Later, upon the completion of M16,
many epidermal cells withdraw from the cell cycle for the
remainder of embryogenesis and enter a prolonged gap
phase (G117; Foe et al., 1993
The difference between M16 and earlier mitoses is also
evident in careful analysis of unperturbed embryos. We
found that chromosomes in late anaphase/telophase of
M16 did not accumulate MCMs, whereas chromosomes at
the same stage in earlier mitoses had acquired the MCM
stain (compare bracketed nuclei in Fig. 4 B to those in Fig.
4 A). We conclude that chromosome association of MCMs
does not occur or is greatly delayed in M16. One possible
basis for the difference we have observed between M16
and earlier mitoses is the presence of an inhibitor of
MCM-chromosome association at this terminal mitosis.
The data described below are consistent with this idea.
Note, however, that once cells finish M16 and enter the
next interphase, nuclear MCM staining is evident, presumably as a result of nuclear import (Fig. 4 C; see also Fig. 1,
F-H, and text).
We also examined embryos arrested in M16 because of
production of cyclin As or coproduction of cyclins As and
Bs, or because of a mutation in fizzy, which is required for
mitotic degradation of cyclins (Sigrist et al., 1995 Dap Inhibits MCM-chromosome association
Analysis of dacapo (dap) mutants suggests that this gene
has a role in MCM-chromosome association in M16. dap
encodes a p21/p27 type cdk inhibitor (Dap) whose developmentally regulated expression just before M16 contributes to the withdrawal of epidermal cells from the cell cycle (de Nooij et al., 1997; Lane et al., 1997). In homozygous
dap mutants, many epidermal cells enter an additional S
phase (S17) immediately after M16. Analysis of DmMCM2
in dap homozygotes showed that many late anaphase/telophase chromosomes acquire DmMCM2 in M16, unlike
those in M16 of wild-type or heterozygous sibling embryos
(Fig. 5). This is illustrated most clearly in embryos in
which the DNA has been stained with the fluorescent dye
Hoechst 33258 and DmMCM2 has been stained immunohistochemically. When the MCMs colocalize with DNA,
the histochemical stain efficiently quenches the fluorescent staining (e.g., see Fig. 4). Focusing on the bright cells,
one can see that the later stages of mitosis are not
quenched in cell cycle 16 of wild-type or heterozygous dap
embryos, but they are quenched in homozygous dap mutant embryos (Fig. 5). For example, late anaphase/telophase nuclei in wild type/heterozygotes are devoid of histochemical DmMCM2 stain and are clearly visible when
visualized for DNA (Fig. 5 A, boxes, and panels 1-3). In
contrast, late anaphase/telophase nuclei in homozygous
dap mutants have acquired the DmMCM2 stain and are barely visible when visualized for DNA (Fig. 5 B and panels 4-6). Not all anaphase/telophase figures acquire MCM
stain in dap mutants (e.g., Fig. 5 B, arrowhead). Likewise,
not all cells of the epidermis undergo S17 in a homozygous
dap mutant (de Nooij et al., 1997; Lane et al., 1997). Direct
analysis of DmMCM2 immunofluorescent staining also revealed association of MCMs with late anaphase/telophase nuclei of homozygous dap mutants (not shown). These
data suggest that expression of Dap in cycle 16 normally
inhibits chromosome association of DmMCM2.
A summary of data on MCM-chromosome interactions
is shown schematically in Fig. 6 and described below.
The normal cycle of association and dissociation of MCM
proteins from chromatin is thought to play a role in the
cell cycle oscillations in the competence of DNA for replication. To investigate whether the mitotic association of
Drosophila MCMs to chromatin is controlled by the destruction of cyclins, we have analyzed the ability of stabilized cyclins to block this association. Our results show
that stable cyclin A, but not stable cyclin B, can block this
mitotic association. Additionally, a difference in the behavior of MCMs during mitoses that are followed immediately by S phase (M14 and M15) and a mitosis that is followed by a prolonged G1 quiescence (M16) indicates that
MCM association may require other factors in addition to
the destruction of mitotic cyclins.
The association of MCM proteins to condensed chromosomes in M14 and M15 arrested with stable cyclin B demonstrates that this association requires neither decondensation of the chromosomes nor assembly of a nuclear
membrane. This finding is consistent with the observed kinetics of association of MCMs with chromosomes upon
exit from mitosis in unperturbed cycles of vertebrates
(Todorov et al., 1994 How might cyclins or cyclin-dependent kinases control
MCM behavior? Some members of the MCM family contain putative cdk phosphorylation sites. In Xenopus extracts, the phosphorylation state of a MCM homologue,
XMCM4, has been shown to change in concert with mitotic kinase activity (Coue et al., 1996 A differential ability of Drosophila cyclins A and B to
block MCM-chromosome association might be relevant to
previous findings that cyclin A, but not cyclin B, prevents
rereplication of DNA in G2 in Drosophila. That is, rereplication in cyclin A mutants occurs in the presence of cyclin
B (Lehner and O'Farrell, 1990 In many ways, our results parallel recent findings in Xenopus extracts (Mahbubani et al., 1997 Before cycle 16 of the embryo, each mitosis is followed
immediately by S phase. We suggest that any necessary
preparation for DNA synthesis must occur during mitosis
in these cycles to allow immediate progression into S
phase subsequently. Accordingly, we found that binding of
MCMs to chromosomes occurs during mitosis in precycle
16 mitoses we have examined. In contrast, M16 is followed by a long gap phase in epidermal cells. Thus, at this stage
in embryogenesis, there is an extended postmitotic period
during which cells might prepare for DNA synthesis and
binding of MCMs to chromosomes might occur at any
time before S phase. Indeed, at this stage, we find that
MCMs do not bind chromosomes in M16.
The change in MCM-chromosome association seen at
M16 is unlikely to be a direct outcome of the transition
from maternal to zygotic control of MCM transcription.
This is because in Drosophila embryos, maternal-zygotic
transition occurs earlier, during cycle 14 (Foe et al., 1993 While the result in M16 appears to contradict reports of
MCM-chromosome association during mitosis in mammalian cell cycles that contain a G1, we wish to stress that
G117 in Drosophila epidermal cells lasts for the rest of embryogenesis and terminates only upon hatching of the larvae. Thus, G117 may be different from G1 of cycling cells
and resemble a quiescent state. Indeed, although MCMs
appear to be chromatin-bound in G1 of cycling mammalian cells, they are downregulated in G0 cells (Schulte et
al., 1996 We conclude that, unlike in earlier mitoses, all requirements for chromosome association of MCMs are not met
in M16. Similarly, in an arrest with cyclin Bs during M16,
DmMCM2 was not present on chromosomes, unlike in cyclin Bs arrests at earlier mitoses. The inability of M16 chromosomes to bind MCMs appears to depend on dap, which
is expressed for the first time in G216 in epidermal cells. In
dap mutants, DmMCM2 binds chromosomes in M16 (this
report) and cells enter S phase instead of G117 (de Nooij
et al., 1997; Lane et al., 1997). Our data, therefore, implicate Dap as an inhibitor of chromosome association of
MCMs.
Dap is a specific inhibitor of cyclin E:cdk2 complexes,
and its role in promoting the arrest of cycle 17 cells in G1 is
thought to result from its contribution to the downregulation of cyclin E (Lane et al., 1997). Indeed, downregulation of cyclin E is known to be essential for G1 quiescence
in cycle 17, and expression of cyclin E is sufficient to drive
ectopic S phase (Knoblich et al., 1994 In contrast to our proposal that cyclin E:cdk2 stimulates
MCM-chromosome association, a recent report argued
that addition of high levels of cyclin E to Xenopus extracts
can inhibit MCM binding to sperm chromatin (Hua et al.,
1997 In conclusion, our data suggest a role for mitotic cyclin
degradation in chromosome binding of replication factors,
MCMs. Degradation of mitotic cyclins is essential for the
completion of mitosis. The completion of mitosis thus appears to be intimately linked to preparation for DNA replication by a common event, degradation of mitotic cyclins. As a result, the two major phases of the cell cycle,
mitosis and DNA replication, may be viewed, not as discrete phases that must be ordered, but rather, as events
that are coupled such that completion of one may occur
concurrently with preparation for the other.
; reviewed in Heichman and Roberts, 1994
; Coverley and Laskey, 1994
). Because a G1 state arises from a G2 state upon passage through
mitosis, mitosis restores the competence to replicate DNA.
Restoration of replication competence at mitosis is thought
to be marked and perhaps caused by association of minichromosome maintenance (MCM)1 proteins with chromatin (Todorov et al., 1994
; Kimura et al., 1994
; Chong et
al., 1995
; Madine et al., 1995b
; Todorov et al., 1995
; Coue
et al., 1996
; Schulte et al., 1996
). MCMs are evolutionarily conserved replication factors that function in an early step
of DNA replication (reviewed in Tye, 1994
, and Chong et
al., 1996
; see also Hennessy et al., 1991
; Thommes et al.,
1992
; Yan et al., 1993
; Miyake et al., 1993
). In a premitotic
nucleus, MCMs are not associated with chromatin; as vertebrate nuclei progress through mitosis, association of
MCMs with chromosomes occurs in parallel with mitotic
restoration of replication competence (Todorov et al.,
1994
; Schulte et al., 1996
; Coue et al., 1996
). To understand how passage through mitosis is coordinated with DNA
replication, we have asked what triggers chromosome association of MCMs during mitosis.
; Hayles et al., 1994
; Dahmann
et al., 1995
; Sauer et al., 1995
; Hayashi, 1996
; Coverley et
al., 1996
). These data demonstrate that mitotic cyclin:cdks
prevent DNA replication in G2, and they suggest that loss
of cyclin:cdks during mitosis restores the competence to replicate DNA. The mechanisms by which cyclin:cdks prevent rereplication in G2 may include preventing MCMs
from binding to chromatin for the following reasons: Cyclin:cdks can prevent an assembly of proteins called the
"prereplicative complex" on origins of DNA replication in
S. cerevisiae (Dahmann et al., 1995
; Piatti et al., 1996); prereplicative complexes are thought to contain MCMs (reviewed in Nasmyth, 1996
; Diffley, 1997). Additionally, an
inhibitor of MCM function is present in Xenopus mitotic
extracts and can be removed with Suc1 beads, a procedure
that depletes cdks (Mahbubani et al., 1997
).
). We therefore analyzed the consequence of preventing degradation of cyclins A and B on Drosophila MCMs.
We demonstrate here that mitotic association of MCMs
with chromosomes occurs when degradation of cyclin B
was prevented but not when degradation of cyclin A was
prevented. We infer from this data that mitotic degradation of cyclin A permits MCMs to associate with chromosomes. After the 16th mitosis (M16), epidermal cells of the
embryo withdraw from the cell cycle and enter a prolonged gap phase (G117). During M16, MCMs failed to associate with chromosomes despite the absence of cyclin A. The withdrawal of epidermal cells from the cell cycle depends on the turn-on of Dacapo, a cdk inhibitor (cki) specific for cyclin E:cdk2 (de Nooij et al., 1997; Lane et al.,
1997). In dacapo mutants, chromosome association of
MCMs is detected in M16, suggesting that Dap inhibits
this association normally. We infer that in addition to destruction of cyclin A, MCM-chromosome association requires an activity that can be inhibited by Dacapo.
MATERIALS AND METHODS
). Anti-
-tubulin antibody (Amersham,
Arlington Heights, IL) was used at 1:100 dilution. Primary antibodies
were detected either with fluorescent secondary antibodies (Jackson ImmunoResearch Laboratories; West Grove, PA) or by histochemical staining using the peroxidase reaction. DNA was stained with 10 mg/ml bisbenzamid (Hoechst 33258). WGA (Molecular Probes, Inc., Eugene, OR) was
used at 500 ng/ml.
). Briefly, stable cyclin A lacks amino acids
(aa) 2-170. Stable cyclin B lacks aa 3-47 and has a glycine as the second aa
residue, before aa 48 from the wild-type sequence (Sprenger et al., 1997
).
Both cyclins were produced from hsp70 promoter and carried as homozygous lines. w67, the isogenic stock used for carrying the transgenes, was
used as a control in all heat shock experiments. Drosophila embryos were
collected for 2 h and aged for either 2 h at room temperature (rt; to reach
cycle 14) or for 5 h at rt (to reach cycle 16) before heat shock. Embryos
were heat shocked by floating grape juice-agar plates on water at 37° for
30 min. After recovery at rt for 2 h, embryos were fixed for 20 min in a
two-phase mixture of heptane and fix (PBS + 10% formaldehyde) according to standard procedures, and were processed for antibody staining as
described above. The level of induced stable cyclins is about two- to three
fold over that of endogenous cyclins. Cell cycle and developmental stages of embryos were confirmed by the morphology of embryos and the density of cells.
-galactosidase (Amersham antibodies). Homozygotes were identified by the lack of
-galactosidase staining.
RESULTS
; Feger et al., 1995
; Su et al., 1996
,
1997
). Here, we first describe their localization during embryonic divisions as detected by immunostaining of fixed
Drosophila embryos. Fig. 1, A and B, shows nuclear staining for DmMCM2 during G2 of cycle 14, dispersal of staining during mitosis 14, and reaccummulation in telophase 14 (DmMCM4 and DmMCM5 staining was indistinguishable from DmMCM2; not shown). Although all interphase
cells exhibited nuclear MCM staining (see below), these
early cell cycles lack a G1 phase. Fig. 4 documents that
DmMCM5 is nuclear during G1 of cell cycle 17 (similar observations were made for DmMCM2 and DMCM4; not
shown). We conclude that these proteins are nuclear in G1,
S, and G2, and that they are dispersed from chromatin
upon entry into mitosis. This cell cycle pattern of localization is similar to the behavior of vertebrate MCMs (reviewed in Chong et al., 1996
).
Fig. 1.
Subcellular localization of
DmMCMs during embryonic divisions. Drosophila embryos were
fixed and stained with DmMCM2
antibody (A, E, and H), WGA (to
detect NPCs; D and G) and Hoechst
33258 (to visualize DNA; B, C, and
F). (A and B) The head region of an
embryo in transition from postblastoderm division cycle 14 to cycle 15 is shown. DmMCM2 antigen is
present in the nuclei of G2 cells in
cycle 14 (white, open arrows). As
cells enter mitosis, the chromatin
condenses, and DmMCM2 antigen
disperses from the nuclei into the
cytoplasm (white arrowheads).
DmMCM2 staining is again nuclear
in anaphase/telophase cells (black
arrows, see also C-H). (C-H) Reaccumulation of DmMCM2 to the nucleus at exit from mitosis during
precellular nuclear division cycles
(C-E) and postblastoderm cellular
divisions (cycles 15; F-H). The embryo in C-E was undergoing a wave
of mitoses such that nuclei toward the bottom of the figure are at a more
advanced state of mitosis. A progression from anaphase (top) to telophase
(bottom) is shown. In F-H, asynchronously dividing cells of a mitotic
domain in M15 are shown. Chromosomes early in anaphase do not accumulate detectable levels of DmMCM2
(C-E, top, F-H, arrowheads). Reaccumulation of DmMCM2 (E and
H) to the nuclear region begins in
late anaphase (white, open arrows)
and precedes the reappearance of
WGA staining (D and G). Nuclear
MCM stain increased as cells progressed into interphase (S phase of
cycle 16) and completely reformed
a nuclear envelope (F-H, black arrows). Bar, 20 µm.
[View Larger Versions of these Images (91 + 97K GIF file)]
Fig. 4.
Chromosomes do not associate with DmMCM5 in M16
of wild-type embryos. Embryos were fixed and stained with purified antibodies against DmMCM5 and Hoechst 33258 to visualize
DNA. The primary antibody was detected immunohistochemically for increased sensitivity (see Materials and Methods). Embryos were visualized simultaneously for DmMCM5 (in bright
field) and DNA (fluorescence). DmMCM5 signal appears as a
dark stain and, when it colocalizes with DNA, quenches the
DNA fluorescence. All interphase nuclei are dark reflecting nuclear localization of MCMs in interphase (white arrows). (A and
B) During cycle 15, DmMCM5 disperses from the nucleus in mitosis and reaccumulates on pairs of late anaphase/telophase chromosomes (A, bracket). Further reaccumulation is seen as nuclei
progress into interphase (A, arrow). This pattern of DmMCM5
relocalization is identical to the pattern detected by immunofluoresence in Fig. 1, F-H. In contrast, cells of the dorsal epidermis
that are going through M16 in a stage 11 embryo fail to acquire
DmMCM5 stain at a similar stage in mitosis (B, bracket). Note,
however, that when these cells have completed M16 and are in
G117, DmMCM5 stain is present in the nuclei (B and C, arrows),
presumably because of nuclear import (see Fig. 1 legend). A-C
are magnified from areas indicated by brackets in A, B
, and C
.
Embryos are shown anterior to the right and dorsal facing forward (A
) or down (B
and C
). Bar, 10 µm in A-C.
[View Larger Version of this Image (180K GIF file)]
; see also Stafstrom and Staehelin, 1984
). Therefore, this relocalization, at least initially, most likely results
from binding of MCMs to chromatin and not to nuclear
import. Similarly, localization of MCMs to chromatin precedes complete nuclear reformation in Xenopus (Coue et
al., 1996
). In both syncytial and postblastoderm cycles, nuclear MCM immunofluoresence increased further as cells progressed into interphase (e.g., Fig. 1, F-H, black arrows). This increase is possibly caused by import through
NPCs: While only some MCMs contain sequences with obvious similarity to nuclear localization signals, they form
complexes with each other, and analyses in Xenopus have
documented import into intact nuclei (Madine et al.,
1995a
).
; Holloway
et al., 1993
; Surana et al., 1993
; Sigrist et al., 1995
). Expression of stable cyclins from heat-inducible promoters and
the consequences during postblastoderm embryonic divisions of Drosophila have been described (Sigrist et al.,
1995
). Similar experiments using our stable cyclin constructs reproduced these observations (Sprenger et al., 1997
; see Materials and Methods). To restate briefly, in the presence of stable cyclins, cells enter mitosis but they arrest
with condensed chromosomes. In a mitotic arrest by stable
cyclin A (cyclin As), the chromosomes and spindle have
metaphase appearance and the sister chromatids remain
together. In contrast, in an arrest caused by stable cyclin B
(cyclin Bs) or coproduction of cyclins As and Bs, sister
chromatids are separated as they would be at the meta- phase-anaphase transition. Importantly, endogenous cyclins are degraded normally in an arrest caused by stable
cyclins; i.e., cyclin B is degraded in an arrest caused by cyclin As, and cyclin A is degraded in an arrest caused by cyclin
Bs (Sigrist et al., 1995
).
) and had been arrested in
mitosis for various lengths of time at fixation.
Fig. 2.
The effect of stable cyclins on association of DmMCM2
with chromosomes in mitosis. Embryos carrying heat-inducible
transgenes for cyclin As (As), cyclin Bs (Bs), or both (As+Bs) were
fixed and stained for DmMCM2 and DNA after heat induction. Cells indicated by arrowheads in the top two panels are magnified two times and shown in the bottom two panels. Note that regions occupied by chromosomes in panels 2 and 3 are devoid of
DmMCM2 stain. The local exclusion of the stain is more obvious
in a cyclin As arrest than in an arrest caused by coexpression of
cyclins As and Bs, perhaps because the chromosomes are more
tightly clustered in the former case. In contrast, cells arrested
with cyclin Bs (panel 1) showed no exclusion of the stain from
chromosomes, and in many cells, MCM stain on the chromosomes substantially exceeds the cytoplasmic signal (panel 1, arrowheads). Bar, 20 µm.
[View Larger Version of this Image (75K GIF file)]
). When cells were arrested in
M16 with cyclin Bs, DmMCM2 (Fig. 3 D) stain was excluded from the region occupied by chromosomes (Fig. 3
C). These data indicate that in a cyclin Bs arrest in M16, all
requirements for MCM-chromosome association are not
met, whereas they were met in cyclin Bs arrest in M14 or M15.
Fig. 3.
Chromosomes do not associate with DmMCM2 in a
stable cyclin B arrest during M16. Embryos carrying the heat-
inducible transgene for cyclin Bs were heat shocked, fixed after a
rest period at room temperature, and stained for -tubulin (B),
DNA (C), and DmMCM2 (D). At this stage in embryogenesis
(stage 14/15 in Campos-Ortega and Hartenstein, 1985
), epidermal cells had ceased to divide in wild-type embryos. Therefore,
cells blocked in M16 were identified by
-tubulin staining of mitotic spindles. A mitotic cell from the area indicated by the arrowhead in A (third thoracic segment) is shown at a higher magnification in B-D. Note the exclusion of DmMCM2 stain by
chromosomes (C and D, arrowheads). Bar, 5 µm in B-D.
[View Larger Version of this Image (143K GIF file)]
). We did
not see chromosome association of DmMCM2 in any of
these cases (data not shown).
Fig. 5.
MCM-chromosome
association in M16 in dacapo
mutants is detected by histochemistry. Homozygous dap
mutant embryos and their wild-type or heterozygous siblings were fixed and stained
for DmMCM2 and DNA.
Histochemical staining used
for detection of DmMCM2 results in a dark-colored deposit that quenches the DNA
stain when DmMCM2 and
DNA are colocalized. Lateral views of the posterior third of stage 11 embryos are
shown in A and B. Many of
the epidermal cells in this
view are completing M16
(dorsolateral epidermis). (A)
In wild type or heterozygotes, identified by -galactosidase stain in the wingless
pattern (arrowheads), many pairs of anaphase/telophase chromosomes lack the DmMCM2 stain and are seen as brightly fluorescent (boxes show examples magnified in panels 1-3). This is similar to what is seen for DmMCM5 in wild-type embryos undergoing M16 (Fig. 4 B). (B) In contrast, in dap homozygous mutants, identified by the absence of
-galactosidase stain, many anaphase/telophase chromosomes acquire DmMCM2 stain, which quenches the DNA signal. Consequently, chromosome pairs (boxes) are not readily visible, except when magnified as in panels 4-6. Bar, 20 µm in A and B; 7 µm in panels 1-6.
[View Larger Version of this Image (108K GIF file)]
Fig. 6.
A summary of cyclin profiles and
MCM-chromosome association. (A) In wild-type embryonic cycles in which S phase follows
M, cyclin E (dashed line) is present continuously,
and association of MCMs (dots) with chromosomes (bars) occurs as mitotic cyclins (solid
lines) are degraded during mitosis. (B) In M16,
cyclin E-associated activity is down regulated as
dacapo is expressed (arrow), and MCMs fail to
associate with chromosomes, even when mitotic
cyclins are degraded (Lehner and O'Farrell,
1989). (C) In dap mutants, cyclin E-associated
activity persists, MCMs associate with chromosomes as described in A, and M16 is followed by
S phase. We propose that cyclin:cdk activity is coupled to MCM-chromosome association in the following manner: a cyclin A-associated activity inhibits chromosome association of MCMs. After the loss of cyclin A in mitosis, chromosome association of MCMs requires a second cyclin-dependent activity that is provided by cyclin E:cdk2. The diagrams are not drawn to scale.
[View Larger Version of this Image (14K GIF file)]
DISCUSSION
; Coue et al., 1996
; Schulte et al.,
1996
) and Drosophila (this report). The ability of stable
cyclin A to block MCM association to mitotic chromosomes suggests either that the normal cyclin A can inhibit
this association or that the deletion of the destruction box
and flanking sequences produced a novel activity. While
we can not presently eliminate the latter alternative, we
suggest that inhibition of MCM association to chromatin
by cyclin A:cdc2 kinase might contribute to the failure of
MCMs to associate with chromosomes during metaphase.
The activation of the proteolytic machinery and destruction of cyclin A, and possibly other inhibitors of MCM-
chromosome association, would then time the beginning of the assembly of replication proteins onto DNA.
), and cdks can direct
phosphorylation of XMCM4 and promote its dispersal
from the nucleus in vitro (Hendrickson et al., 1996
). Direct
inhibition of MCM-chromosome association by mitotic
cyclin/cdk phosphorylation is suggested by these analyses. However, our efforts to detect differential modification of
MCMs on the basis of mobility on denaturing and native
gels have been negative (Su, T.T., and P.H. O'Farrell, unpublished data). In addition, we cannot exclude more indirect paths of regulation. For example, association of Xenopus MCMs to chromatin requires CDC6 and ORC2 (Coleman
et al., 1996
). Thus, cdks could influence the activity of
these proteins and thereby affect MCM-chromosome association indirectly.
; Sauer et al., 1995
). Perhaps
inhibition of MCM-chromosome association by cyclin A
contributes to the block to rereplication.
) in which MCMs
have been shown to contribute to replication licensing factor (RLF) activity (Blow and Laskey, 1988
; Chong et al.,
1995
; Madine et al., 1995a
). (a) RLF becomes active at
metaphase-anaphase transition; Drosophila MCMs binding to chromosomes follows metaphase-anaphase transition. (b) A cdk-dependent activity renders RLF inactive in
metaphase; cyclin A appears to prevent chromosome association of Drosophila MCMs, presumably until its degradation. (c) 6-Dimethylaminopurine, which prevents RLF
activation, has been shown to stabilize a mitotic cyclin; we
show here that stabilization of a mitotic cyclin prevents MCM-chromosome association. Thus, our results complement the biochemical data in Xenopus and, additionally,
provide evidence for the role of mitotic kinases in regulation of MCM behavior in vivo.
).
The switch from maternal to zygotic transcription of
DmMCM2, DmMCM4, and DmMCM5 also occurs at this
time (Treisman et al., 1995
; Su, T.T., and P.H. O'Farrell,
unpublished observations). Furthermore, mutants in MCM
genes show no defect until later stages, indicating that the
maternal supply of MCM gene products is sufficient for
these early stages we have examined (Feger et al., 1995
;
Treisman et al., 1995
).
).
). This raises the possibility that Dap inhibits MCM-chromosome association
by inhibiting cyclin E:cdk2. Perhaps cyclin E:cdk2 promotes chromosome association of MCMs. The behavior of
MCMs in mitoses up to and including M16 is consistent
with this idea (Fig. 6) First, cyclin E is present continuously in embryonic cycles in which S follows M (Knoblich
et al., 1994
); MCMs associate with mitotic chromosomes
after metaphase in these cycles (Fig. 6 A). Second, cyclin E
activity is absent in M16, which is followed not by S but by
G117 (Knoblich et al., 1994
); likewise, MCMs do not associate with M16 chromosomes (Fig. 6 B). In dap mutants, M16 is followed by S17, and cyclin E activity is thought to
persist (Fig. 6 C). On the basis of these data, we propose
that mitotic cyclin degradation creates a permissive state
for MCMs to associate with chromosomes, but that cyclin
E:cdk2 also contributes positively to this association. Observations in mammalian cultured cells suggests that mitotic association of MCMs and chromosomes can occur in
cell cycles that include a G1 and lack cyclin E during mitosis (Schulte et al., 1996
; Sherr, 1994
). We do not know the basis for these different behaviors, but we suggest that the
activity provided by cyclin E in Drosophila may be supplied otherwise, perhaps by a redundant function, in mammalian cells.
). However, this report emphasized the role of cyclin
E in the inhibition of DNA rereplication. We concur with
this emphasis and argue that cyclins make both positive
and negative inputs to DNA replication and the assembly of proteins (such as MCMs) at origins. In Drosophila, both
cyclins A and E are capable of driving G1 cells into S
phase, yet both cyclins A and E are able to inhibit postreplicative cells from rereplicating their DNA (Sauer et al.,
1995
; Sprenger et al., 1997
; Follette, P.J., R.J. Duronio and
P.H. O'Farrell, manuscript in preparation; reviewed in Su
et al., 1995, and Wuarin and Nurse, 1996
). In yeast, cyclins
have both stimulatory and inhibitory inputs into DNA
replication (reviewed in Diffley, 1996
; Nasmyth, 1996
). In
Xenopus, depletion of the endogenous cyclin E demonstrates that replication in Xenopus extracts requires cyclin
E (Fang and Newport, 1991
; Jackson et al., 1995
), yet high
levels of exogenous cyclin E block the binding of MCM
protein to newly added chromatin (Hua et al., 1997
).
These observations are consistent with the idea that cdk
activity has at least two inputs into cycles of replication,
one positive (perhaps leading to binding of MCMs to chromatin) and one negative (possibly inhibiting rebinding of
MCMs to chromatin).
Received for publication 30 July 1997 and in revised form 7 August 1997.
Address all correspondence to Patrick H. O'Farrell, Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143-0448. Tel.: (415) 476-4707; Fax: (415) 502-5143/5145; E-mail: ofarrell{at}cgl.ucsf.eduWe thank Anita Sil, Peter Follette, and Smruti Vidwans for critical reading of the manuscript; Wallace Marshall for WGA; Drs. de Nooij and Hariharan for dacapo stocks; and Frank Sprenger for sharing stable cyclin stocks before publication.
This work was supported by a National Institutes of Health (NIH) postdoctoral fellowship (GM15032) to T.T. Su and NIH grant (2-RO1-GM37193) to P.H. O'Farrell.
aa, amino acid; M16, mitosis 16; MCM, minichromosome maintenance (protein); NPC, nuclear pore complex; RLF, replication licensing factor; rt, room temperature.
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