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J. Biol. Chem., Vol. 275, Issue 50, 39773-39778, December 15, 2000
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From the Department of Biological Chemistry and Molecular
Pharmacology, Harvard Medical School, Boston, Massachusetts 02115
Received for publication, September 5, 2000
To investigate how the protein kinase cdc7
stimulates DNA replication in metazoans, a soluble cell-free
replication system derived from Xenopus eggs was used. DNA
was incubated in egg cytosol to form prereplication complexes
and then in nucleoplasmic extract to initiate DNA synthesis. We find
that cdc7 is greatly enriched in nucleoplasmic extract and that this
high concentration is essential for efficient DNA replication,
supporting previous models that the nucleus activates replication
indirectly by sequestering essential components. cdc7 binds to
chromatin at the G1/S transition before initiation occurs,
and it dissociates from chromatin as S phase progresses. The chromatin
association of cdc7 requires chromatin-bound MCM. In turn, cdc7 is
required to load the initiation factor cdc45 onto the DNA. Finally,
efficient replication is observed when chromatin is exposed first to
cdc7 and then to cdk2 but not when it is exposed to cdk2 before cdc7.
Therefore, the cdc7- and cdk2-dependent initiation steps
can be separated, indicating the existence of a novel, stable
initiation intermediate. Moreover, the data suggest that cdk2 can only
act after cdc7 has executed its function.
Eukaryotic cells regulate the initiation of DNA replication via
the ordered assembly and disassembly of replication complexes at
origins of replication (1). During the G1 phase of the cell cycle, prereplication complexes
(pre-RCs)1 are assembled
through the sequential binding of cdc6 and the MCM complex with a
chromatin-bound origin recognition complex (ORC). At the
G1/S transition, pre-RCs are activated for replication by
an S phase-specific cyclin-dependent kinase (cdk2) and a
second cdk-like kinase, cdc7. Activation of pre-RCs causes their
disassembly through the loss of MCM from the origin. De novo
binding of MCM to chromatin is inhibited by S and M phase-specific
cdks, such that new pre-RCs cannot be formed until cdk activity is
destroyed in mitosis. As a result, replication can initiate only once
from pre-RCs that were assembled during the G1 phase of the
cell cycle.
At the G1/S transition, the initiation factor cdc45
associates with the pre-RC to generate a preinitiation complex. In
yeast, this event appears to require both the action of cdk and
cdc7/dbf4 (2, 3), and temperature shift experiments suggest that cdk exerts its function before cdc7 (4). In Xenopus egg
extracts, cdc45 loading has been shown to require cdk2 (5, 6). After cdc45 binding, the next detectable event in initiation is the unwinding
of the origin, a process that requires the eukaryotic single-stranded
DNA-binding protein, RPA (6, 7, 43). Although the helicase that
unwinds the DNA is not known, there is mounting evidence it may be MCM
(8-11). Once the origin has been unwound sufficiently, DNA polymerase
cdc7 was first isolated in budding yeast, and its activity was shown to
depend on a regulatory subunit, dbf4, which is expressed in
mid-G1 and S phase (reviewed in Ref. 12). The requirements for cdc7 and dbf4 in replication initiation can be completely bypassed
by a point mutation in MCM5 called bob1 (13), and MCM2 mutations are suppressed by mutations in dbf4 (14). Moreover, several
MCM subunits are phosphorylated by cdc7 in vitro and
in vivo (reviewed in Ref. 12). These observations strongly
suggest that MCMs are positive targets of cdc7 phosphorylation. In
contrast to cdc7/dbf4, little is known about what component(s) of the
replication machinery must be phosphorylated by
cyclin-dependent kinases to stimulate initiation of DNA replication.
This paper examines the roles of Xenopus cdc7 and cdk2 in
the initiation of DNA replication. A Xenopus
laevis cdc7 homolog (xCdc7) has been isolated (15) and shown
by antibody interference experiments to be required for DNA replication
in Xenopus egg extracts (16). cdk2/cyclin E is the only cdk
with significant activity in interphase Xenopus egg
extracts, and it appears to be the only cdk necessary to support DNA
replication in this system (17, 18). To facilitate the analysis of
these kinases, we are using a modification of Xenopus
nuclear assembly extracts in which DNA replication occurs in the
absence of nuclei (19). In this system, replication is initiated in two
steps. First, demembranated sperm chromatin is incubated with
membrane-free egg cytosol to form pre-RCs. Second, a nucleoplasmic
extract (NPE) is added, which initiates replication, and a complete
round of semi-conservative DNA replication takes place (19). This
system has been used to further define the role of xCdc7 in replication initiation and to understand its functional relationship to cdk2.
DNA Replication and Chromatin Binding Assays--
Extract
preparation and replication assays were carried out as described (19).
To inhibit initiation, purified glutatione S-transferase-tagged p27Kip (20, 21) was
preincubated with extracts for 10 min at a final concentration of 1 µM. To isolate chromatin (22), up to 12 µl of extract
containing 10,000 sperm/µl was mixed with 70 µl of egg lysis buffer
(ELB; 250 mM sucrose, 2.5 mM MgCl2,
50 mM KCl, 10 mM Hepes, pH 7.7) containing
0.2% Triton X-100 and then centrifuged through a sucrose cushion. The
chromatin pellet was washed with 200 µl of ELB.
Immunological Methods--
To deplete proteins from egg cytosol
or NPE, 1 volume of extract was incubated for 3 h with 0.2 volumes
of protein A-Sepharose fast flow (Amersham Pharmacia Biotech) that had
been bound to 0.6 volumes of xCdc7 antiserum (6), xMcm7 antiserum (6), or the appropriate preimmune serum. Extract was harvested, and the
procedure was repeated once. Western blotting was performed using
antisera at 1:5000 dilution against xCdc7 (6), xMcm3 (23), xOrc2 (22),
xCdc45 (6), and the 34-kDa subsunit of xRpa (6).
The Rate of DNA Replication Is Regulated by the Concentration of
cdc7--
We previously showed that immunodepletion of egg cytosol and
NPE with an antibody raised against xCdc7 protein prevented replication initiation (6). To better understand how xCdc7 contributes to
replication initiation, egg cytosol and NPE were immunoblotted with
anti-xCdc7 antibody. The concentration of xCdc7 in NPE was 10-20 times
higher than in egg cytosol (Fig.
1A, compare lane 1 with lanes 2-4), indicating that xCdc7 localizes to the
nucleus.
It was of interest to determine whether xCdc7 must be supplied by egg
cytosol, NPE, or both to drive replication. When both extracts were
depleted of xCdc7 (Fig. 1A, lanes 5 and
6), replication was eliminated relative to control extracts
that were depleted with preimmune serum (Fig. 1B, compare
squares and diamonds). When egg cytosol but not
NPE was depleted of xCdc7, replication was normal (Fig. 1B,
circles). However, when NPE but not egg cytosol was depleted
of xCdc7, the rate of replication was reduced 5-6-fold (Fig.
1B, triangles). Therefore, although egg
cytosol-derived xCdc7 can support significant levels of DNA
replication, NPE-derived xCdc7 is essential to achieve rates of DNA
replication that are similar to what is observed in nuclei and in embryos.
To ask whether the high concentration of xCdc7 present in NPE is
important for efficient DNA replication, we incubated sperm chromatin
in mock depleted egg cytosol to form a pre-RC. We then added mock
depleted and cdc7-depleted NPE in different ratios to determine what
concentration of nuclear xCdc7 is necessary to stimulate efficient DNA
replication. Reactions in which the xCdc7-depleted NPE was mixed in a
10:1 or 1:1 ratio with mock depleted NPE (Fig. 1C,
diamonds and circles) replicated better than
reactions containing only cdc7-depleted NPE (Fig. 1C,
squares). However, the most efficient replication was
obtained when only mock depleted NPE was used (Fig. 1C,
triangles). Therefore, a full complement of NPE-derived
xCdc7 is necessary to achieve efficient DNA replication, indicating
that this protein kinase must be present at high concentration to
function efficiently in Xenopus egg extracts.
MCMs Are Required to Recruit xCdc7 to Chromatin--
cdc7 and dbf4
have been shown to bind chromatin in budding yeast (24, 25). We
therefore tested whether xCdc7 binds to chromatin
in egg extracts. Very low levels of xCdc7
bound to chromatin in egg cytosol (Fig. 2, lane 1; see also
Fig. 3A, lane 1),
and much higher amounts bound within 5 min after addition of NPE (Fig. 2, lane 2, and Fig. 3A, lane 5). As
replication proceeded, xCdc7 was gradually lost from the chromatin with
similar kinetics as xMcm3 (Fig. 2, lanes 2-5, compare xCdc7
and xMcm3 panels). When DNA replication was inhibited with aphidicolin,
neither MCM nor cdc7 were displaced (Fig. 2, lanes 10-13).
When initiation of replication was blocked by the cdk2 inhibitor
p27Kip (20, 21), MCM again was not displaced (Fig. 2,
lanes 6-9). In the presence of p27Kip, the
amount of xCdc7 that loaded onto chromatin at the G1/S
transition was slightly less, and interestingly, its mobility became
retarded over time. The significance of this observation is not clear
at present. Together, the above results show that there is a dramatic increase in the level of cdc7 binding to chromatin upon addition of NPE
but before initiation occurs, and xCdc7 is displaced from chromatin
during S phase in a process that appears to require movement of the
replication fork. It is noteworthy that the amount of RPA that loads
onto chromatin is dramatically enhanced in the presence of aphidicolin
(Fig. 2, compare lanes 2-5 with 10-13), consistent with our previous observation that aphidicolin induces massive DNA unwinding (6).
MCMs are likely targets of cdc7 phosphorylation (12) and are therefore
attractive candidates for proteins that might recruit cdc7 to
chromatin. To test this, the replication inhibitor geminin was used
because it blocks the loading of MCMs onto chromatin without affecting
ORC or cdc6 binding (26). Low levels of xCdc7 were bound in egg
cytosol, and higher levels were bound after addition of NPE (Fig.
3A, lanes 1 and 5). In the presence of
geminin, binding of both xMcm3 and xCdc7 to chromatin was almost
completely abolished (Fig. 3A, lanes 3 and
7), whereas binding of ORC was enhanced, as previously
reported (Fig. 3A, lanes 3 and 7)
(26). Identical results were obtained when NPE containing
p27Kip was used to block replication initiation (data not
shown). Because it is not known whether geminin affects the binding of
MCMs directly or through another factor such as cdt1 that is required
for MCM binding (27), we used antibodies against xMcm7 to remove the MCM complex from egg cytosol. Depletion was at least 95% efficient (Fig. 3B, compare lanes 1 and 2).
Sperm chromatin was incubated with MCM depleted egg cytosol for 30 min
and was then supplemented with undepleted NPE. Because NPE contains
inhibitors of MCM binding (19), it was not necessary to deplete MCMs
from NPE. Indeed, even after the addition of undepleted NPE, there was
no MCM bound to chromatin (Fig. 3C, lane 2).
Under these conditions, xCdc7 binding to chromatin was much less than
the control, which contained chromatin-bound MCM (Fig. 3C,
compare lanes 1 and 2). Together, these data
indicate that the MCM complex is essential to recruit xCdc7 to chromatin.
The stability of chromatin-bound xCdc7 was also examined (Fig.
3D). Sperm chromatin was incubated in egg cytosol for 30 min followed by an additional 30 min incubation with NPE.
p27Kip was included with the NPE to prevent removal of cdc7
from the chromatin by replication. Like ORC (28), xCdc7 was almost
completely eluted from chromatin by 250 mM KCl (Fig.
3D, compare lanes 1 and 3). A small
amount of xCdc7 remained bound to chromatin under these washing
conditions (Fig. 3D, lane 3, dark exposure).
However, this xCdc7 remained bound even after exposure of the chromatin to 650 mM KCl, a treatment that displaces MCM from
chromatin (Fig. 3D, lane 5). Because xCdc7
recruitment to chromatin is MCM-dependent (Fig.
3C), the residual xCdc7 bound to chromatin after exposure to
high salt is likely bound to nonorigin DNA and therefore nonfunctional. The salt displacement of xCdc7 is important for the interpretation of
chromatin-transfer experiments shown below.
xCdc7 Is Required to Load xCdc45 onto Chromatin--
We previously
showed that xCdc7 and xCdc45 are required for origin unwinding (6). To
determine whether xCdc7 is required for binding of xCdc45 to DNA at the
G1/S transition, sperm chromatin was incubated sequentially
with xCdc7-depleted egg cytosol and xCdc7-depleted NPE, isolated, and
blotted for xCdc45. Compared with mock depletion, xCdc7 depletion
caused a dramatic reduction in the amount of xCdc45 that loaded onto
chromatin (Fig. 4, compare lanes
1 and 3). The reduction is equivalent to that observed
when cdk2 is inactivated by p27Kip (Fig. 4, lane
2, and Refs. 5 and 6). Thus, both cdk2 and xCdc7 are required to
recruit xCdc45 to origins at the G1/S transition. As
expected, given its requirement for unwinding (6), xCdc7 is also
necessary to load RPA onto the chromatin (Fig. 4, lane 3).
The xCdc7 and cdk-dependent Initiation Steps Are
Separable--
As seen from the data in Fig. 4, the execution points
of cdk2 and xCdc7 are identical, because neither kinase is required for
pre-RC assembly, and both are needed to recruit xCdc45. The question
arises of whether xCdc7 and cdk2 exert their effects on pre-RCs in a
defined order, and if so, which kinase acts first. It was first tested
whether xCdc7 could exert its function before cdk2 (Fig.
5A, sequence 2; see
Fig. 5B for schematic representations of experiments
performed in Fig. 5A). To allow the
xCdc7-dependent step to occur in the absence of cdk2
activity, sperm chromatin was incubated for 30 min with egg cytosol
containing p27Kip and then for another 30 min following the
addition of NPE containing p27Kip. It was necessary to use
both egg cytosol and NPE to execute the cdc7 step because xCdc7 is only
maximally active when supplied by NPE (Fig. 1B). The
chromatin was then isolated through a sucrose cushion in the presence
of 250 mM KCl and incubated in xCdc7-depleted NPE lacking
p27Kip to supply cdk2 activity in the absence of xCdc7.
[ Evidence That cdk2 Cannot Execute Its Function before cdc7--
We
next asked whether replication occurs when chromatin is exposed to the
two protein kinases in the reverse order (cdk2 first, cdc7 second). To
supply cdk2 activity in the absence of xCdc7, sperm chromatin was
incubated for 30 min in xCdc7-depleted egg cytosol and then for a
further 30 min after addition of xCdc7-depleted NPE. The incubation in
xCdc7-depleted NPE was necessary to execute the cdk2 step because like
xCdc7, cdk2/cyclin E must be supplied by NPE
(19).2 After the
incubations with cdk2, the chromatin was isolated in the presence of
250 mM KCl and supplemented with mock depleted NPE
containing p27Kip and [
An alternative explanation for the absence of replication in Fig. 5
(sequence 4) was that the product of cdk2 phosphorylation cannot withstand the relatively stringent washing procedure employed during the chromatin transfer. To address this possibility, the same
experiment was performed, but the chromatin was isolated in the absence
of detergent and using 50 mM instead of 250 mM KCl. Again, no DNA replication was observed (data not shown). To
further reduce the possibility of disrupting products of cdk2 phosphorylation, we performed the experiment without the chromatin isolation step (Fig. 6, sequence
2). Thus, sperm chromatin was incubated with xCdc7-depleted egg
cytosol followed by xCdc7-depleted NPE to allow the cdk2 step to occur.
After 20 min in the NPE, p27Kip was added to inactivate
cdk2, and after a further 5 min, undepleted NPE containing
p27Kip was added to supply xCdc7 activity. This procedure
exposed chromatin to cdk2 before xCdc7 without a chromatin isolation
step, but there was still no significant DNA replication when compared
with a similar sequence in which both kinases are present together
(Fig. 6, compare sequences 2 and 1). Indeed, most
of the small amount of DNA replication that is observed in sequence 2 is accounted for by residual replication that occurred because of
incomplete cdc7 depletion (Fig. 6, sequence 3).
In summary, chromatin replicates well when both kinases are active
simultaneously or when xCdc7 is allowed to act before cdk2 but not when
chromatin is exposed to cdk2 before xCdc7. Taken together, the data
strongly suggest that xCdc7 must exert its function before cdk2 during
replication initiation in Xenopus egg extracts.
We previously speculated that in Xenopus nuclear
assembly extracts, the nucleus stimulates DNA replication by acting as
a concentrating device for one or more replication factors that do not
function at the concentrations present in egg cytosol (19). In
agreement with this model, this paper shows that the high concentration of xCdc7 that is present in NPE is essential for efficient DNA replication. However, we cannot at present rule out the possibility that, in addition, nuclear xCdc7 is modified in some fashion that renders it fully active.
Our data show that the recruitment of xCdc7 to chromatin requires the
presence of MCM on chromatin. This is consistent with evidence that
MCMs are the physiological targets of cdc7 phosphorylation (1), and it
suggests that the binding of xCdc7 to chromatin is essential for its
function during DNA replication. In further support of this idea, xCdc7
loads onto the DNA before initiation has occurred. This can be seen by
the fact that loading is not inhibited when initiation is blocked with
p27Kip, and loading of xCdc7 precedes the binding of RPA
(Fig. 2, compare lanes 2 and 3 or 10 and 11), indicating that it occurs before the origin is
unwound (6). In contrast, in budding yeast, cdc7 and dbf4 recruitment
to the DNA does not require MCM, but rather depends on ORC (29).
Moreover, in yeast, cdc7 must be present during pre-RC formation (29).
This differs from our observation that efficient replication is
achieved when NPE is mixed with pre-RCs that were assembled in egg
extracts lacking xCdc7 (Fig. 1B, circles).
Therefore, the mechanism by which cdc7 is recruited to chromatin in
yeast and metazoans appears to be different.
cdc45 is an initiation factor whose recruitment to chromatin at the
G1/S transition requires cdk2 (2, 5, 6, 30). Initially,
experiments in budding yeast suggested that cdc45 binds chromatin
independently of cdc7 (2), but later experiments in the same organism
suggested that stable association of cdc45 does require cdc7 (3). This
report shows that cdc7 is required for the stable association of cdc45
with chromatin in a metazoan organism. Importantly, we find that like
cdk2, xCdc7 is not required for the chromatin binding of MCMs to form
pre-RCs (Fig. 3B). Based on these criteria, the execution
points of xCdc7 and cdk2 during replication initiation are identical.
To ask whether xCdc7 and cdk2 carry out their functions in a defined
order, chromatin was transferred between extracts deficient in one or
the other kinase. Chromatin exposed first to extract containing only
xCdc7 and then to extract containing only cdk2 activity replicated as
well as chromatin exposed to extract in which both kinases were active.
These results indicate that the ability of xCdc7 to execute its
function during replication does not require the simultaneous presence
of cdk2 activity or vice versa, and this is consistent with
observations in yeast (4). The fact that the xCdc7- and
cdk2-dependent initiation steps can be separated
experimentally further argues that a stable initiation intermediate
exists between the pre-RC and the preinitiation complex, but the nature
of this intermediate is not clear at present. It is conceivable that in
our experiments, some component of the replication machinery, or xCdc7
itself (31), has been preactivated by cdks before the cdc7 step.
However, even if this were to be the case, there is clearly an
additional requirement for cdk2 after the xCdc7 step has occurred.
Critically, when chromatin was exposed first to cdk2 activity,
isolated, and then incubated with extract containing xCdc7, there was
no DNA replication. We performed several experiments to address whether
this result is due to the product of cdk2 phosphorylation being
destroyed during transfer of chromatin between extracts. Most
importantly, a sequence in which chromatin is exposed to cdk2 followed
by xCdc7 was performed in the absence of chromatin isolation, and still
no DNA replication was observed. Although we cannot unequivocally
exclude the possibility that in this experiment the cdk2 product was
still destroyed, the simplest interpretation of the results is that
there is a defined order in which kinases must act during replication
initiation in Xenopus egg extracts. In this view, xCdc7 must
exert its function on pre-RCs before cdk2 can act.
While this work was in progress, Jares and Blow (32) reported an
overlapping set of observations. Using nucleus-dependent Xenopus egg extracts, these authors found that xCdc7 binds
chromatin in an MCM-dependent fashion and that xCdc7 is
necessary for cdc45 binding to chromatin. Importantly, although these
authors showed that cdc7 can exert its function before cdk2,
we have provided evidence that it must do so.
Our proposal that cdc7 must act before cdk2 in egg extracts has
important implications for the regulation of S phase. In metazoans and
in yeast, origins initiate replication following a defined temporal
order, with some origins firing early in the S phase and others firing
late (reviewed in Ref. 33). In yeast, cdc7 and cdks are required for
firing of early and late origins (34-36), and this is likely also the
case in metazoans. When replication is blocked in the middle of S
phase, firing of additional origins is prevented by an "intra-S
phase" checkpoint (37-39). It is not known whether sperm chromatin
replicating in Xenopus extracts obeys an early-late program
of origin firing. However, when the activity of cdk2 is inhibited after
the first set of initiations has occurred, replication is still
extremely efficient (22), indicating that these early initiations
support a rapid and complete round of replication. Therefore, it is not
unexpected that restricting xCdc7 activity to the beginning of S phase
as was done here also results in efficient replication. We further
suggest that an initiation mechanism in which cdc7 acts before cdk2 is
not incompatible with an intra-S phase checkpoint, even in somatic
cells. It is currently not known why some origins do not fire until
late in S phase, but it likely involves higher order chromatin
structure or nuclear organization that renders late firing origins
refractory to the action of cdk and cdc7 until the appropriate time in
S phase. In this view, when a checkpoint is induced during S phase,
inhibiting either cdc7 or cdk2 would be sufficient to prevent further
origins from firing.
The mechanism and regulation of replication in yeast and metazoans is
highly conserved (1, 40). Nevertheless, our findings appear to differ
from those made in yeast. Nougarede et al. (4) used
temperature shift experiments to examine the order in which cdc7 and
cdk exert their effects during replication initiation in
Saccharomyces cerevisiae. They found that cells replicated their DNA when cdk was present before cdc7 but not when cdc7 was present before cdk. This finding is also consistent with earlier observations in S. cerevisiae that cdc45 and cdc7 must be
present at the same time for replication to initiate (41). However, a
recent paper reported that structural changes at yeast origins that are
normally dependent on cdc7 can occur in G1 phase arrested cells that harbor the cdc7-bypass mutation
mcm5/cdc46-bob1 (42). This observation is
interesting because it suggests that in S. cerevisiae, there
is no absolute cdk requirement for at least some of the structural
changes at origins that are normally catalyzed by cdc7. More work will
be needed to fully resolve to what extent the mechanism of cdc7 action
and its relationship to cdk differs between yeast and metazoans.
The gift of purified geminin from James
Wohlschlegel and Anindya Dutta is gratefully acknowledged.
*
This work was supported by a Giovanni-Armenise Fellowship
and a Burroughs Wellcome Career Award in the Biomedical Sciences.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Published, JBC Papers in Press, September 25, 2000, DOI 10.1074/jbc.M008107200
2
J. Walter, unpublished results.
The abbreviations used are:
pre-RC, prereplication complex;
ORC, origin recognition complex;
MCM, minichromosome maintenance;
NPE, nucleoplasmic extract;
cdk, cyclin-dependent kinase;
ELB, egg lysis
buffer.
Evidence for Sequential Action of cdc7 and cdk2 Protein Kinases
during Initiation of DNA Replication in Xenopus Egg Extracts*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, the presumptive initiating DNA polymerase, is recruited to the
origin, and DNA synthesis can begin (6, 7, 43).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The high concentration of xCdc7 present in
NPE is essential for efficient DNA replication. A,
xCdc7 is ~20 times more concentrated in NPE than in egg cytosol. 0.75 µl of mock depleted egg cytosol (EC) (lane
1), 0.75, 0.075, and 0.0375 µl of mock depleted NPE (lanes
2-4, respectively), 0.75 µl of xCdc7-depleted egg cytosol
(lane 5), or 0.75 µl of xCdc7-depleted NPE (lane
6) were separated through a 10% SDS-polyacrylamide gel and
immunoblotted with xCdc7 antiserum. IgG, contamination from
depletion procedure; *, cross-reacting protein. B, sperm
chromatin (final concentration, 10,000/µl) was incubated for 30 min
with mock depleted (squares and triangles) or
xCdc7-depleted egg cytosol (circles and diamonds)
and then supplemented with 2 volumes mock depleted (circles
and squares) or xCdc7-depleted NPE (triangles and
diamonds) containing [ -32P]dATP, and
replication was measured 20, 48, and 70 min after NPE addition.
C, sperm chromatin was incubated for 30 min with
xCdc7-depleted (filled circles) or mock depleted egg cytosol
(open triangles, circles, diamonds,
and squares) and then supplemented with 2 volumes total of
mock depleted and xCdc7-depleted NPE mixed in different ratios as
indicated in the figure. The NPE also contained
[-32P]dATP, and replication was measured 20, 40, and
60 min after NPE addition.
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Fig. 2.
xCdc7 binds to chromatin before replication
initiation, and it is displaced from chromatin during S phase.
Sperm chromatin was incubated with egg cytosol (EC,
lane 1) or egg cytosol followed by NPE (lanes
2-5), NPE containing p27Kip (lanes 6-9),
or NPE containing 50 µg/ml aphidicolin (lanes 10-13).
After the indicated time, chromatin was isolated through a sucrose
cushion and blotted with antibodies against xMCM3, xOrc2, xCdc7, or
xRPA.
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Fig. 3.
XCdc7 binding to chromatin is
MCM-dependent and salt-sensitive. A, sperm
chromatin was incubated with egg cytosol (EC) containing
(lanes 3, 4, 7, and 8) or
lacking 100 nM his-tagged human geminin (lanes
1, 2, 5, and 6). Chromatin was
isolated after 30 min (lanes 1-4) or after a further 30 min
incubation with NPE (lanes 5-8) and blotted with antibodies
against xMcm3, xOrc2, and xCdc7. Controls lacking sperm were included
(lanes 2, 4, 6, and 8)
B, 0.5 µl of egg cytosol depleted with anti-xMcm7 antibody
(lane 2), and 0.5 µl (lane 3) or 0.025 µl
(lane 1) of mock depleted egg cytosol were immunoblotted
with anti-xMcm3 antibodies. C, sperm chromatin was incubated
with mock depleted egg cytosol followed by NPE containing
p27Kip to prevent replication-mediated removal of MCM
(lane 1) or xMcm7-depleted egg cytosol followed by NPE
(lane 2). After 30 min in NPE, chromatin was isolated and
blotted for xMCM3, xOrc2, or xCdc7. D, sperm chromatin was
incubated in egg cytosol for 30 min and then supplemented with NPE
containing p27Kip. Chromatin was diluted with ELB
containing 0.2% Triton X-100 (ELB/TX; lane 1) or ELB/TX
containing 250 mM final concentration of KCl (lanes
2-5) and centrifuged through a sucrose cushion. The chromatin
pellet was washed with ELB (lanes 1-3) or ELB/TX containing
650 mM (final concentration) of KCl (lanes 4 and
5). Samples 4 and 5 were washed once more with ELB. The
pellet was immunoblotted with antibodies against xMcm3, xOrc2, or
xCdc7. The lower cCdc7 panel was exposed for a long
time.
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Fig. 4.
XCdc7 is required for recruitment of xCdc45
to chromatin. Sperm chromatin was incubated with mock depleted
(lanes 1 and 2) or xCdc7-depleted egg cytosol
(EC, lanes 3 and 4). After 30 min,
mock depleted (lanes 1 and 2) or xCdc7-depleted
NPE supplemented with 50 µg/ml aphidicolin and containing
(lanes 2 and 4) or lacking (lanes 1 and 3) p27Kip was added. After 30 min, chromatin
was isolated and immunoblotted with xMcm3, xCdc45, or xRPA
antibodies.
-32P]dATP was included in this second incubation and
replication was measured after 60 min. This chromatin replicated (Fig.
5A, sequence 2), and the efficiency of
replication was at least as good as that of chromatin which went
through a similar set of manipulations but where both kinases were
allowed to function at the same time (Fig. 5A,
sequence 1). The replication observed in sequence 2 is
unlikely because of chromatin-mediated transfer of xCdc7 from the first
to the second extract because the chromatin was isolated in the
presence of 250 mM KCl, which reduces chromatin-bound xCdc7
to background levels (Fig. 3D). Furthermore, controls show that xCdc7 was functionally depleted from extracts (Fig. 5,
sequence 5) and that when p27Kip was present in
all incubations, no replication took place (Fig. 5, sequence
3). These results strongly argue that in Xenopus egg extracts, the xCdc7 and cdk2 initiation steps are separable and that
xCdc7 can exert its function before cdk2.
View larger version (39K):
[in a new window]
Fig. 5.
The xCdc7- and cdk2-dependent
replication initiation steps can be experimentally separated.
A, sperm was incubated for 30 min with 2 µl of
xCdc7-depleted (sequences 4 and 5) or mock
depleted egg cytosol containing p27Kip (sequences
1-3). After 30 min, 4 µl of xCdc7-depleted NPE (sequences
4 and 5) or mock depleted NPE containing
p27Kip (sequences 1-3) was added. After 30 min,
the reaction was diluted with ELB containing 250 mM KCl and
0.2% Triton X-100 and isolated. The pellet was washed with ELB and
then supplemented with 5 µl mock depleted NPE (sequence
1), xCdc7-depleted NPE (sequences 2 and 5),
xCdc7-depleted NPE containing p27Kip (sequence
3), or mock depleted NPE containing p27Kip
(sequence 4), and replication was measured in the presence
of [ -32P]dATP for 60 min. As observed previously (19),
maximum replication efficiency of isolated chromatin was typically
~30%. B, schematic representation of experiments shown in
A. EC, egg cytosol.
-32P]dATP. This
second incubation was intended to allow the cdc7-dependent step to occur in the absence of cdk2. This series of incubations did
not result in any significant DNA replication (Fig. 5, sequence 4). The absence of replication in sequence 4 was not due to an effect of p27Kip on elongation because replication of sperm
chromatin that had previously initiated replication in the presence of
aphidicolin was not affected by the addition of p27Kip
(data not shown). Together, these results suggested the possibility that cdk2 cannot exert its function before cdc7.
View larger version (31K):
[in a new window]
Fig. 6.
DNA replication does not occur when chromatin
is exposed to cdk2 and then cdc7, even in the absence of chromatin
isolation. Sperm chromatin (final concentration, 5000/µl) was
incubated with 2.5 µl of cdc7-depleted egg cytosol for 30 min and
then mixed with 2 volumes of cdc7-depleted NPE. Sequence 1,
after 25 min, 4 µl of undepleted NPE containing
[ -32P]dATP was added to supply cdc7, and replication
was measured after further 60 min incubation. Sequence 2,
the same as sequence 1 except that p27Kip was added 5 min
before the addition of undepleted NPE, which also contained
p27Kip. Sequence 3, the cdc7-depleted NPE
contained [-32P]dATP, and replication was measured 60 min after its addition. In each case, the amount of replication per
sperm was plotted.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Dept. of Biological
Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Ave., Boston, MA 02115. Tel.: 617-432-4799; Fax: 617-738-0516; E-mail: johannes_walter@hms.harvard.edu.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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