Cell, Vol. 62, 655-662, September 7, 1990, Copyright 0 1990 by Cell Press
A cdc2-like Protein Is Involved in the Initiation
of DNA Replication in Xenopus Egg Extracts
J. Julian Blow and Paul Nurse
lCRF Cell Cycle Group
Microbiology Unit
Department of Biochemistry
University of Oxford
South Parks Road
Oxford OX1 3QU
England
Summary
Extracts of Xenopus eggs efficiently initiate and complete chromosomal
DNA replication in vitro, under
normal cell cycle controls. Such extracts can be depleted of Xenopus p34-,
either by affinity depletion
using the protein p13w” or by epeciffc immunodepletion. Depleted extracts am incapable of inltiating DNA
replication, atthough they efficiently elongate tepllcation forks initiated in undepleted extracte. Deptetlon
of p34does not prevent nuclear aesembly, which
is requited for the Initiation of DNA mpllcation in thls
system. Activity can be restored to depleted extracts
by readdltion of ~13”” eluates enriched for ~34~.
Thesamsults ~thatp34~,oraveryclosely related protein, Is involved in the initiation of chromosomal DNA replication in the cell cycle of higher
eukryotes.
Introduction
Although DNA replication is a central feature of the cell division cycle, very little is known about how it is controlled
in eukaryotes. One of the major limitations to the analysis
of this process has been the lack of cell-free systems that
support the initiation of DNA replication from chromosomal (nonviral) origins of replication. However, using a
cell-free extract of Xenopus eggs originally described by
Lohka and Masui (1983) the efficient initiation and completion of chromosomal DNA replication has been demonstrated in vitro (Blow and Laskey, 1986; Blow and Watson,
1987). Replication in this system appears to be under the
same cell cycle controls that exist in vivo, namely: DNA is
replicated precisely once in extracts that are blocked in interphase (Blow and Laskey, 1986; Blow and Watson, 1987)
and must pass through mitosis before a second round of
replication can be initiated (Hutchison et al., 1987, 1988;
Blow and Laskey, 1988; Minshull et al., 1989); the initiation
of DNA replication only occurs in interphase extracts and
not in extracts blocked in mitosis (Blow and Sleeman,
1990); and the initiation of DNA replication is dependent
on the template DNA being assembled into an interphase
nucleus (Blow and Laskey, 1988; Blow and Watson, 1981;
Newport, 1987; Sheehan et al., 1988; Blow and Sleeman,
1990). Xenopus egg extracts therefore represent a good
model system for the biochemical analysis ef proteins involved in controlling the initiation of chromosomal DNA
replication in eukaryotes (for a review see Blow et al.,
1989).
Proteins with potentially important roles in the control of
DNA replication have been defined by genetic analysis
of the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae. The CDC28 gene from S. cerevisiae
(Hartwell et al., 1973; Lorincz and Reed, 1984) and its
homolog c&2 from S. pombe (Nurse et al., 1976; Hindley
and Phear, 1984) are particularly interesting. Their function has been highly conserved through evolution, as the
S. cerevisiae and S. pombe genes can be substituted for
one another (Beach et al., 1982; Booher and Beach,
1986). In addition, a homologous gene from human cells
is capable of completely substituting for the c&2 gene in
S. pombe (Lee and Nurse, 1987) and for the CDC28 gene
in S. cerevisiae (Wittenberg and Reed, 1989). The c&2
gene is also unusual in that it is required for two distinct
transitions in the yeast cell cycle. It is required in Gl for
progress through “Start” just prior to the initiation of DNA
replication, and is required again in late G2 for the initiation of mitosis (Nurse and Bissett, 1981; Piggott et al.,
1982).
A homolog of the cdc2 gene product, p34CdC2,has
been identified in Xenopus eggs as a component of the
mitotic inducer maturation-promoting factor (MPF) (Gautier et al., 1988; Dunphy et al., 1988). Active MPF is a complex of ~34~~~ and cyclin B (Lohka et al., 1988; Draetta et
al., 1989; Labbe et al., 1989; Gautier et al., 1999). Cyclins
are rapidly degraded as cells exit from metwhase (Evans
et al., 1983) and therefore must be synthesized anew during interphase of each cell cycle in order to allow cells to
generate MPF and progress into mitosis (Minshull et al.,
1989; Murray and Kirschner, 1989). ~34~~~~ also complexes with another protein, ~13~~~’(Hayles et al., 1986;
Brizuela et al., 1987). Addition of ~13~~~’to Xenopus egg
extracts inhibits MPF activity (Dunphy et al., 1988), and
agarose beads with ~13~~~’covalently linked to them
(sucl beads) can deplete Xenopus egg extracts of MPF
activity (Dunphy et al., 1988). This shows that ~34~~~~
plays an important role in controlling the G2/M transition
in higher eukaryotes.
A requirement for p34Hc2 in the Gl/S transition has not
been demonstrated thus far in higher eukaryotic cells. Human p34cdc2 undergoes changes in protein level and
phosphorylation near the Gl/S boundary (Lee et al., 1988)
and displays a weak kinase activity during interphase
(Giordano et al., 1989; C. Norbury, personal communication). However, neither of these observations directly
demonstrates a Gl/S role for ~34~~~~.In a cell-free System
that supports sV40 DNA replication in vitro, the viral initiation protein T antigen is inactive when prepared as an underphosphorylated form from Escherichia coli. Phosphorylation of T antigen by the mitotic form of ~34~~~
from human cells restores its competence to initiate viral
DNA replication in vitro (McVey et al., 1989). Although interesting, this experiment does not address the potential
role of p34*2 in chromosomal DNA replication, as it de-
Cdl
856
a
40
0
10
20
30
40
t---*
controlbeads
volume of beads ( % voI:voI )
c.wWalbesds
o--m
suc1baads
-
"Ol"rn'e0Of
beaz
( % "CZOI
)
0
wc1be2ds
4
-’
sucl aided (r&/ml)
pends on initiation from the viral origin of replication
driven by a viral initiation protein, whose function is at
least in part to release viral DNA replication from normal
cell cycle controls. Furthermore, the form of ~34~~~ used
was the active M phase kinase, rather than the interphase
form presumably required for chromosomal DNA replication.
Indeed, the only biochemical experiments performed
thus far to investigate the possible role of ~34~~~’ in
higher eukaryotes have failed to find a role for it in chromosomal DNA replication (Riabowol et al., 1989). Although microinjection of antisera raised against ~34~d~*
prevented cultured rat cells from progressing from interphase into mitosis, they did not prevent such cells from initiating DNA replication. This does not rigorously exclude
a GllS role for p34Cdc2, since the GUS form of ~34~dC*
may have different antigenic availability or may be required at a lower activity than the G2/M form.
In this paper we have depleted Xenopus egg extracts of
p34cdc2, by using either sucl beads or an antibody directed against ~34~~~. Depleted extracts cannot initiate
DNA replication, but remain capable of assembling sperm
chromatin into interphase nuclei and elongating replication forks initiated in undepleted extracts. This demonstrates that p34cdc2, or a very closely related protein, is involved in the initiation stage of DNA replication in a higher
eukaryotic cell cycle. We also show that the cdc2 protein
completes its S phase function soon after the egg exits
from metaphase, before the initiation of replication actually takes place.
Results
A cdcBlike Proteln Is Required for
DNA Replication
SUCT Bead Depletion
Extracts of Xenopus eggs were incubated with sucl beads
to deplete them of ~34~~~~.They were then assayed for
their ability to replicate sperm chromatin (Figure 1). Two
different types of egg extract were used in these experiments. Mature Xenopus eggs are arrested in meiotic
metaphase II, and if extracts are prepared in the presence
Figure 1. Inhibition of DNA Synthesis by Depletion of Extracts with sucl Beads
Mitotic (A) or interphase extracts (B) were
treated with either sucl beads (open squares),
control beads (solid diamonds), or sucl protein
(open circles). Mitotic extracts were then activated by Ca2+ addition. Extracts were assayed
for their subsequent ability to replicate sperm
chromatin, as measured by the incorporation
of [a32P]dATPinto DNA. The volume of beads
added Is expressed as a percentage of the extract volume to which they were subsequently
added. Thus, 30% v/v means an addition in the
ratio of 3 pl of sucl beads to IO pl of extract.
sucl protein was added to give final concentrations over the range of 0.254 mg/ml. DNA synthesis is expressed as the percentage incorporation compared with an untreated control.
of EGTA, they are maintained in the metaphase state
(Lohka and Masui, 1985). Addition of Ca*+ to such extracts causes them to exit from metaphase into interphase,
after which, nuclear assembly and DNA synthesis take
place (Lohka and Masui, 1985; Blow and Sleeman, 1990).
Alternatively, eggs can be activated in vivo to cause exit
from metaphase prior to extract preparation, so that they
are already in interphase. They are therefore capable of
assembling nuclei and replicating DNA without further exposure to Ca2+ (Lohka and Masui, 1983; Blow and Laskey,
1986). Addition of 30%-40% v/v of sucl beads to either
metaphase (Figure 1A) or interphase extracts (Figure 1B)
caused an almost complete inhibition of subsequent DNA
synthesis (Figure 1, open squares). Treatment with control
beads (agarose beads without sucl cross-linked to them)
did not cause this inhibition (Figure 1, solid diamonds).
Inhibition of DNA synthesis was not simply due to the
addition of sucl protein, since addition of high concentrations of free ~135”~~(up to 3 mglml) did not significantly
reduce DNA synthesis (Figure lA, open circles). This provides a biochemical distinction between the GllS and
G2/M transitions involving cdc2. Dunphy et al. (1988) have
demonstrated that the MPF activity of Xenopus egg extracts can be completely inhibited by addition of 0.03
mglml sucl protein. In contrast, DNA synthesis is unaffected by 100 times this concentration of sucl (Figure 1).
Although p138uc1binds to p34cdc2, it is important to determine that the inhibition of DNA synthesis actually results from ~34~~~~depletion. Western blotting with “4711”
antiserum, raised against yeast p34&* (Gould and Nurse,
1989), identifies a strong upper band and a weaker lower
one in the 34 kd region that are both specifically bound
to sucl beads (Figure 2A, lanes 1 and 2). The same two
bands are also recognized by “PSKAIR” antiserum raised
against a conserved peptide in the cdc2 protein (Lee and
Nurse, 1987) and by two other antisera raised against
yeast cdc2 protein (data not shown). Specific interaction
with sucl beads and recognition by anticdc2 antisera
identifies these proteins as Xenopus p34a
homologs.
The differing electrophoretic mobilities of these two bands
may be due to phosphorylation, as has been observed in
other cell types (Draetta and Beach, 1988).
cdc2 and Xenopus DNA Replication
857
1
2
3
1
2
3
4
A
92.5
69
depleted
Figure 2. Binding of ~34~~~”to sucl Beads and Its Elution with Excess sucl Protein
Extracts were treated with sucl beads or control beads. Beads were
washed and then incuL@d with 30 mg/ml sucl protein to specifically
elute p34cdd. At different stages samples were run on 10% polyacrylamide gels.
(A) Gels were Western blotted with 4711, an antiserum raised against
~34~~. Lane 1, material bound to control beads; lane 2, material
bound to sucl beads; lane 3, material eluted from sucl beads by 30
mg/ml sucl.
(B) Gels were silver stained to show all proteins present in the samples.
Lane 1, material bound to control beads; lane 2, material bound to sucl
beads; lane 3, material bound to sucl beads and stained at higher sensitivity, equivalent to that of lane 4; lane 4, material eluted from sucl
beads by 30 mglml sucl, and stained at the same sensitivity as lane 3.
Lane8 1 and 2 in Figure 26 show a silver-stained gel of
proteins bound to sucl beads and control beads. It can be
seen that many proteins become bound, and only minor
differences can be seen between sucl and control beads.
Similar results have been reported by others (Dunphy et
al., 1988; Labbe et al., 1989). In particular, the 34 kd region
of the gel is obliterated by strong bands binding to the control beads. This indicates that not all proteins of around 34
kd that bind to sucl beads actually represent p34dc2.
However, treatment of extract with 30% by volume of sucl
beads, sufficient to almost completely abolish DNA replication, removes 80%-80% of p34cdc2, as detected by
Western blotting (data not shown).
To improve the specificity of the system, we have modified a procedure described by Labbe et al. (1989) for
purifying p34 &C2, by specifically eluting it from sucl
beads with high concentrations of sucl protein. Western
blotting with 4711 antiserum shows that approximately
50% of the Xenopus ~34~~ bound to sucl beads can be
eluted using this procedure (Figure 2A, lane 3; data not
shown). This material is also recognized by the PSTAIR
antiserum (Lee and Nurse, 1987; data not shown). When
the eluate is examined on a silver-stained gel, it seems to
be highly enriched in two bands that comigrate with Xenopus p34cdc2 (Figure 28, lanes 3 and 4). Although this onestep enrichment using highly concentrated Xenopus extracts fails to eliminate all contaminants from the ~34~~~
preparation, it is largely in agreement with the results
presented by Labbe et al. (1989). A band at about 45 kd
is present, as expected of cyclin B, which is bound to
under these conditions (Lohka et al., 1988; DraP34etta et al., 1989; Labbe et al., 1989; Gautier et al., 1990).
depleted
+ S”Cl
depleted
+ eluate
Figure 3. Recovery of Replication Competence by Supplementing
Depleted Extracts with Material Eluted from sucl Beads
Extracts were depleted with sucl beads. Aliquots were assayed
directly, or were supplemented with sucl at 3 mg/ml, or were supplemented with one-tenth their volume of material eluted from sucl
beads by 30 mg/ml sucl, and then assayed. Extracts were assayed for
their ability to replicate sperm chromatin. DNA synthesis is expressed
as percentage of the control, which was extract treated with control
beads. Each type of bar represents a different experiment. In the first
experiment (solid bars), no sucl addition was made.
A contaminating band at about 30 kd and minor high molecular weight contaminants are also present at the corresponding stage of the procedure described by Labbe et
al. (1989).
When eluate enriched in p34CdC2is added back to egg
extract depleted with sucl beads, it partially restores the
ability to replicate DNA (Figure 3). This restoration is not
due to the high levels of p13*uC1in the eluate, since pure
sucl protein cannot restore replicative activity on its own.
Since Labbe et al. (1989) have shown that the elution is
specific for p34dc2, these experiments suggest that the
inhibition of DNA synthesis caused by sucl bead depletion is due to depletion of ~34~~~~.
lmmunodepletion
To confirm the involvement of p34Cdc2in DNA replication
in this system, we used 4711 antiserum, raised against
bacterially produced yeast p34cdC2 (Gould and Nurse,
1989), to immunodeplete the Xenopus egg extract. As well
as Western blotting p34cdc2, 4711 antiserum immunoprecipitates two bands of the size expected of ~34~~~ (Figure 4A, lane 2). Silver staining of the immunoprecipitates
shows a single protein of about 34 kd being specifically
brought down by 4711 antibody in the 25-50 kd size range
(data not shown). Proteins outside this size range are obscured by the IgG chains. lmmunodepletion of ~34~~~
using IgG purified from 4711 antiserum render8 the egg
extract incapable of supporting DNA replication (Figure
46, open squares). In contrast, treatment with preimmune
IgG from the same rabbit only slightly inhibits subsequent
DNA replication (Figure 4B, solid diamonds). When 8 cll
of immune IgG is added to 10 PI of extract, which almost
totally abolishes DNA replication, approximately 400/o-80%
of the p34cdc2 detected by Western blotting is removed
(data not shown). This suggests that ~34~~~~is required
for DNA replication in this system.
To determine that the same component is removed from
the extract by sucl bead treatment and immunodepletion,
mixing experiments were performed (Table 1). Both SUCK
bead depletion and immunodepletion abolish the ability of
6
IgG added (~01%)
Figure 4. Inhibition of DNA Synthesis by lmmunodepletion of p&P2
(A) Metaphase-arrested egg extracts were labeled with [35S]methionine for 45 min, after which time proteins were immunoprecipitated,
electrophoresed on 10% polyacrylamide gels, and autoradiographed.
lane 1, immunoprecipitation with preimmune 4711 serum; lane 2, immunoprecipitation with immune 47ll serum. In addition to the two
bands comigrating with ~64~~ in lane 2, another band appears at
around 45 kd. This comigrates with cyclin B, which is expected to complex with pWdC2 under the conditions used.
(8) Extracts were immunodepleted with either preimmune 47ll IgG
(solid diamonds) or immune 47ll IgG (open squares). Extracts were
assayed for their subsequent ability to replicate sperm chromatin. DNA
synthesis is expressed as a percentage of that attained when buffer
was added instead of serum. The volume of IgG added is expressed
as a percentage of the volume of extract to which it was subsequently
added. Thus, 60 ~01% means an addition in the ratio of 6 ul of IgG to
IO ul of extract.
the extract to support DNA replication. In both cases, mixing an equal volume of depleted and undepleted extract
efficiently restores replicative capacity. This shows that
both types of depleted extract fail to replicate DNA through
having lost an essential replication factor rather than
through having gained an inhibitory activity. In these
mixes, the ~34~~~~concentration is restored to approximately 80% of that in untreated extract. However, a mixture of equal volumes of sucl bead-depleted and immunodepleted extract is still incapable of supporting DNA
replication. This demonstrates that sucl bead depletion
and immunodepletion both remove the same essential
replication factor from Xenopus egg extracts. Given the
apparent specificity of both these reagents (Figures 2 and
4) this is almost certainly ~34~~~~.
Table 1. lmmunodepleted and Affinity-Depleted Extracts
Cannot Complement One Another
Extract Treated
DNA Svnthesis
Untreated
sucl bead-depleted
Antibody-depleted
Untreated + sucl bead-depleted
Untreated + antibody-depleted
sucl bead-depleted + antibody-depleted
100
2
15
61
65
11
Extracts were depleted with either 40% by volume sucl beads or 60%
by volume 4711 immune IgG, or were left untreated. Aliquots were combined to produce mixes containing equal volumes of extract treated
in different ways. They were then assayed for their ability to replicate
sperm chromatin. DNA synthesis is expressed as a percentage of that
attained by untreated extract alone.
Figure 5. Nuclear Formation in ExtractsTreatedwith Control and sucl
Beads
Sperm nuclei were incubated for 1.5 hr in extracts treated with either
sucl beads (a-f) or control beads (g-l). DNA was stained with Hoechst
66258, and nuclei were viewed wet under UV fluorescence (a-c and
g-i) or under phase-contrast optics (d-f and j-l). Scale bar = 6 urn.
cdc2 Is Involved in the Initiation Stage
of DNA Replication
Three stages can be distinguished in the replication of
DNA in Xenopus egg extract. The first of these is assembly of the template DNA into a fully functional interphase
nucleus surrounded by a nuclear envelope. This must be
completed before the initiation of DNA replication occurs
(Blow and Laskey, 1988; Blow and Watson, 1987; Newport,
1987; Sheehan et al., 1988; Blow and Sleeman, 1990). The
second stage is the coordinate initiation of thousands of
replication forks within individual interphase nuclei (Blow
and Watson, 1987; Blow, 1988). Finally, in the elongation
stage, replication forks progress semidiscontinuously to
fully replicate the template DNA (Blow and Laskey, 1988;
Blow and Watson, 1987).
We have investigated at which of these stages the cdc2
function is required. Treatment of extracts with sucl beads
does not prevent the decondensation of sperm chromatin
and its subsequent assembly into interphase nuclei (Figure 5). Nuclei assembled in depleted extracts are clearly
surrounded by a phase-dense nuclear envelope (Figures
5a-5f) and are indistinguishable under the light microscope from nuclei assembled in extracts treated with control beads (Figures 5g-51). However, addition of either
control beads or sucl beads to the egg extract causes a
certain reduction in size of nuclei assembled from sperm
chromatin (data not shown).
Egg extra&depleted
with sucl beads are fully capable
of supporting the elongation stage of DNA replication (Fig-
cd@ and Xenopus DNA Replication
659
123456
4SIOf
am
qhmw
0’: 0
I
5
10
15
Time of sucl bead depletion
(mins after Ca++ addition)
Figure 6. Effect of sucl Bead Depletion on Replication of Different
DNA Templates
Extracts were treated with either control beads (lanes 1, 3. and 5) or
sucl beads (lanes 2,4, and 6). They were then assayed for their ability
to replicate demembranated sperm nuclei (lanes 1 and 2), aphidicolinblocked sperm nuclei (lanes 3 and 4) or single-stranded Ml3 DNA
(lanes 5 and 6). Aliquots of the incubation were TCA precipitated or
electrophoresed on a 0.6% agarose gel and autoradiographed. Arrowheads show the slot, the migration of high molecular weight linear DNA
(hmw). and double-stranded Ml3 form I (I). TCA precipitation gave the
ratio of DNA synthesis in sucl bead-treated extracts over control
bead-treated extracts as 29% for sperm nuclei, 127% for aphidicolinblocked nuclei, and 121% for single-stranded M13.
ure 6). Aphidicolin is a specific inhibitor of eukaryotic DNA
polymerases a and 6 and blocks DNA synthesis in Xenopus egg extracts without affecting nuclear formation (Blow
and Laskey, 1966; Blow and Watson, 1967). Most of the
events occurring during the initiation stage of DNA replication can still probably occur in the presence of aphidicolin (Decker et al., 1986). When nuclei that have been
assembled in egg extract containing aphidicolin are subsequently transferred to sucl bead-depleted extract, they
are efficiently replicated (Figure 6, lanes 3 and 4). Similarly, single-stranded Ml3 DNA can be fully replicated in
Xenopus egg extracts unable to initiate DNA replication on
double-stranded DNA (MQhali and Harland, 1982; Blow
and La8key, 1986). Lanes 5 and 6 in Figure 6 show that
single-stranded Ml3 DNA can be fully replicated, ligated,
and assembled into chromatin by sucl bead-depleted extracts. These experiments show that the cdc2 activity is
not required for the elongation stage of DNA replication
and must therefore be involved in the initiation of DNA
replication.
cdc2 Acts Soon After Exit from Metaphase
When Ca2+ is added to a me&phase extract, it enters interphase and starts the Series of steps leading to the initiation of DNA replication. The extract can be depleted with
sucl beads at various times after Ca2+ release in order to
determine the time at which th8 cdc2-requiring step has
been COmpl8ted. Figure 7 (solid squares) shows that after
only 15 min in interphase, sucl bead depletion has become totally incapable of inhibiting subsequent DNA synthesis. Fifteen minutes into interphase is well before the
initiation of DNA replication, which does not start until
about 45 min after Ca*+ release (Blow and Laskey, 1986;
data not shown). It is also before the first signs of nuclear
envelope formation, which are not visible until about 30
Figure 7. Effect of sud Bead Depletion of Extracts at Different Times
After Exit from Me&phase
Sperm nuclei were incubated in mitotic extracts with [assP)dATPfor
various times after Ca2+ release from metaphase. sucl beads (closed
squares and diamond) or control beads (open square and diamond)
were added at various times after this and allowed to deplete the extract for QOmin at 4% before the incubation was resumed at 25% for
3 hr. TCA precipitation was used to assess the final extent of DNA synthesis. Closed squares and open square: DNA and Ca2+ were added
to extract at the same time. Closed diamond and open diamond: DNA
was preincubated in mitotic extract for 20 min at 25% before Ca* addition and treatment with beads.
min after Ca*+ release. Western blotting shows that the
sucl beads can still efficiently bind ~34~~~ 15 min after
Ca*+ release (data not shown). Interestingly, sucl beads
are capable of inhibiting subsequent DNA synthesis 8ven
if they are not removed from the extract by centrifugation
(Figure 7, solid symbols).
However, the cdc2-requiring step Cannot b8 performed
during metaphase. If DNA is incubated in mitotic extract
for 20 min before depletion with sucl beads and Ca* release, subsequent DNA synthesis is efficiently inhibited
(Figure 7, solid diamond). These results demonstrate that
th8 cd&requiring
step is normally executed very soon after the Xenopus egg exits from meiotic metaphase into th8
first mitotic interphase.
Discussion
We have depleted Xenopus egg extracts of ~34~~~ both
by immunodepl8tton and by affinity depletion using pl3”“c1.
Both treatments leave extracts unable to replicate sperm
nuclei added to them. Mixing experiments show that immunodepleted and sucl bead-depleted extracts cannot
complement 8aCh other, d8mOnStrating that they have
been depleted of at least one essential common factor.
Apart from p34cdc2, no Other common factor can b8 readily observed (Figures 2 and 4). In particular, the antibody
used does not immunoblot a 32 kd protein kinas with se
quence similarity to p34cdfi that also binds to sucl beads
(8. Gabrielli and J. Maker, personal communication). However, we Cannot eXClUd8 the possibility that the loss of
DNA replicative capacity is due to d8pl8tiOn of a hitherto
unknown protein that shares immunological features and
sucl binding ability with p34cdc2. It is also possible that
depletion of ~34~ abolishes subsequent DNA replication through simultaneous depletion of another essential
component (such as a Gl cyclin) with which it is com-
c&2 in MPF (metaphase-inducing)form.
DNA licensed for replication in next cell cycle.
I MPF inactivation
Assembly of
interphase nucleus.
cdc2 performs
GVS function.
initiation of DNA Replication
Figure 8. Schematic Representation of the Involvement of cdd in the
initiation of DNA Replication
MPF, an activitycapableof inducingthe metaphaaestate in Xanopus
eggs and oocytes,is a complexof f~%~* and cyciinB. When MPF is
inactivatedat the end of mitosis,two distincteventsdccurthat are both
required for the subsequent initiation of DNA replication: chromosomes decondense and are assembled into an interphase nucleus,
and cdc2 can execute its GllS function. See text for further details.
plexed. However, we can conclude that ~34~~ or a very
closely related protein is involved in the initiation of DNA
replication in Xenopus eggs.
Analysis of depleted extracts shows that they are specifically unable to initiate DNA replication. However, they can
still assemble template DNA into interphase nuclei, which
is an early stage required before the initiation of replication can normally take place (Blow and Laskey, 1988; Newport, 1987; Sheehan et al., 1988; Blow and Sleeman,
1990). In addition, depleted extracts are fully competent to
elongate replication forks that have been initiated in undepleted extracts. This shows that depletion of cdc2
causes a specific block to the initiation of DNA replication.
Xenopus p34CdC2has previously been identified as a
component of the mitotic activator MPF (Gautier et al.,
1988; Dunphy et al., 1988) which is required for progression from interphase into mitosis. Taken together with our
current results, these observations suggest that ~34~~~~
is required at two distinct stages of the Xenopus cell cycle:
for both the Gl/S transition and for the G2/M transition.
This’ is consistent with genetic analysis in yeast, which
give it distinct roles at these two separate transitions in
both S. pombe and S. cerevisiae (Nurse and Bissett, 1981;
Piggott et al., 1982). Furthermore, the human CDC2 gene
is capable of rescuing both Gl/S and G2/M deficiencies
in S. pombe cdc2 mutants (Lee and Nurse, 1987) and in
S. cerevisiae CDC28 mutants (Wittenberg and Reed,
1989). These results suggest that the cdc2 gene function
at both points in the cell cycle has been extremely well
conserved throughout eukaryotic evolution.
The involvement of cdc2 in these two different cell cycle
transitions suggests that it controls DNA replication in the
Xenopus egg extract in at least two distinct ways (Figure
8). First, replicated nuclei must be disassembled in mitosis under the action of MPF in order for them to be
licensed for a single round of replication in the next cell
cycle (Blow and Laskey, 1988; Minshull et al., 1989). However, the initiation of replication depends on the subsequent assembly of template DNA into an interphase nu-
cteus (Slow and Sleeman, 1990). This requires that the
metaphase-inducing MPF activity of ~34~~~~ be turned
off. Second, we demonstrate here that after release from
metaphase, p34cdc2, or a very closely related protein, is
required prior to the initiation of DNA replication, even in
the presence of apparently normal nuclear assembly. The
nature of this interphase activity of ~34~~~ is Currently
unknown. Although the two different pathways leading to
nuclear assembly and the execution of the GllS function
of p34CdC2can apparently occur independently of one another, both are required before DNA replication can be initiated (Figure 8).
The transition point of cdc2 for DNA replication in Xenopus egg extracts is also similar to that observed in the
yeast S. pombe (Nurse and Bissett, 1981; Novak and
Mitchison, 1989). In both these cell types, which normally
display only a short Gl phase, the cdc2 step is performed
soon after exit from mitosis. In contrast, the yeast S.
cerevisiae, which usually has a longer Gl phase, may perform its CDC28 (equivalent to cdc2) step at some later
time during the Gl phase, once its size requirement for division has been achieved (Hartwell and Unger, 1977;
Johnston et al., 1977). However, in neither of the two
yeasts can the cdc2KDC28 step be executed before exit
from mitosis, in agreement with the results presented here.
Our experiments show that it is possible to identify a
functionally important homolog of a yeast cell cycle protein required for DNA synthesis in the Xenopus cell-free
system. Depletion of the frog homolog creates an extract
directly comparable with a yeast cell displaying a loss-offunction mutation in this gene. This suggests that further
homologs of yeast cell cycle proteins can be depleted and
studied biochemically by a similar approach.
Experimental Procedures
Pqswatlon of Egg Extmcte
Interphase extracts were prepared as described by Blow and Sleeman
ww.
Mitotic extracts were prepared by a modification of previous extraction procedures (Lohka and Masui, lW5; Blow and Sieeman, 1990).
Eggs were collected in high salt Barb solution (110 m M NaCi, 15
mhl Tris-HCI[pi-i 7.4),2 m M KCi, 2 m M NaHCQ, 1 m M Mg&& 05 m M
NazHP04)and ware washed in high salt Barb containing 2 mM
EGTA.Any degeneratedor activatedaggawara removedat this stage.
Eggawere dejaitii in 2% cyateineHCI(pH 74,2 mM EGTA.bajeiiiad
eggs were washed first in high salt Bart’scontaining 2 m M EGTA,and
then in extraction buffer (50 m M KCI, 50 m M HEPES KOH [pH 7.61,5
m M MgCis, 5 m M EGTA,2 m M 5-mercaptoethanoi) containing 5 m M
EGTAat 4%. Eggawere packed into 15 ml Corex tubes by centrifugation in a Beckman JS-13 rotor at 2VO0r-pmfor 1 min. Ail excess buffer
was then removed, along with any degenerated eggs, which float to the
surface under these spin conditions. Eggs ware spincrushad at
12,000 rpm for 10 min at 4% in the JS-13 rotor. The entire cytopiasmic
layer was taken with a Pasteur pipette and was supplemented with
cytdchaiasinB to a final concentrationof 10 P@ml.‘The extractWB~
raaPunat 40,000 rpm in a Beckman SW50 rotor at 4% for IO min. This
separates the cytopiasm into a golden fraction above a loose yellow
vesicular fraction. The golden fraction was removed with a Pasteur pipette, avoiding the yellow vesicular fraction, which is inhibitory to extract function. The extract was then made 1% with respect to glycerol
and was frozenby dropping20 PI aliqudtadiractiyinto liquid nitrogen.
After treatment with beads, extracts were supplemented with 25 m M
phosphucreatine.5 @ml creatine phosphokinase, 250 pa/ml cycioheximide, and 1 Fi of [assPMATP.Demembranated sperm nuclei
were added to give a final sperm DNA concentration of 50 ng of DNA
cdc2 and Xenopus DNA Replication
661
per ul of extract (Blow and Laskey, 1966). Metaphase extracts were
released into interphase by the addition of 0.3 mM Cat&.
TCA precipitation, agarose gel electrophoresis, and autoradiography were performed as described (Blow and Laskey, 1966).
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby
marked *a&ertisement” in accordance with 16 U.S.C. Section 1734
solely to indicate this fact.
sucl Bead Deptetton
Bacterially produced sucl protein (a kind gift of Kevin Crawford and
Jacky Haytes) was attached to CNBractivated agarose beads (Sigma)
at 10 mglml, according to the manufacturer’s instructions. sucl beads
were stored in extraction buffer supplemented with 0.1% sodium azide.
Control beads were treated in the same way except without addition
of sucl protein. Prior to use, beads were washed three times in extraction buffer. Measured volumes were added to egg extract, and the mixture was incubated for 90 min on a rotating wheel at 4%. Extracts were
then centrifuged for 5 s in a microfuge, and the supernatant was taken.
In experiments where DNA was added to the extract prior to sucl bead
depletion, centrifugation was omitted, and the incubation was allowed
to proceed in the presence of sud beads.
Received February 7, 1990; revised June 16, 1990.
Elutlon of p34*
from sucl Beads
P34”dc2was eluted from sucl beads by a modification of published
procedures (Labbe et al., 1969). sucl beads were incubated in egg extract as described above, and then washed six times in either NP40
buffer (150 mM NaCI, 50 mM NaF, 6 mM NasHPO,, 4 mM NaH2P04,
2 mM EDNA,0.1 mM sodium vanadate, 1% NP40, 5 ug/ml leupeptin),
extraction buffer, or extraction buffer containing 5 mM EGTA. Best
results were obtained using NP40 buffer. Beads were then washed
once in extraction buffer, transferred to 0.5 ml Eppendorf tubes, and
centrifuged to remove all excess buffer. The beads were then mixed
with one-third of their own volume of 30 mg/ml sucl protein (made up
in extraction buffer), and incubated on a rotary wheel for 10 min at room
temperature. Beads were again pelleted in a microfuge, and the supernatant was used as a highly enriched source of ~34~‘.
Immunodepte4tion
4711 rabbit antiserum was raised against bacterially produced yeast
p34cdc2protein (Gould and Nurse, 1969) and was a kind gift of Kathy
Gould. IgG was purified from both immune and preimmune serum
by ammonium sulphate precipitation followed by binding to protein
A-Sepharose CL-tB (Pharmacia). After elution at low pH, IgG was
concentrated on Centriprep and Centricon columns (Amicon) to give
a final protein concentration of 40 mg/ml.
Extracts were incubated with immune and preimmune IgG for 60
min at 4%. To remove IgG, extracts were supplemented with a volume
of packed protein A-Sepharose equal to twice the volume of added
IgG, and were incubated for 30 min on a rotary wheel at 4%. Protein
A-Sepharose was removed by centrifugation for 5 s in a microfuge.
Preparatton of DNA Tempt&s
Demembmnated Xenopus sperm nuclei were prepared as described
(Blow and Laskey, 1966).
Ml3 single-stranded DNA was prepared as described (Maniatis et
al., 1962).
Aphidicolin-blocked nuclei were prepared by incubating sperm
nuclei for 2 hr in extract supplemented with 20 &ml aphidicolin.
Nuclei were then resuspended in 1 ml of extmction buffer, and pelleted
at 500 x g for 2 min. Ail overlying buffer was removed, and nuclei were
resuspended in 4 ul of extraction buffer for transfer to fresh extract.
Prow&t Datectlon
Samples for protein analysis were electrophoresed on 10% polyacrylamide minigets. Silver staining was performed as described (Morrissey, 1961). Samples for Western blotting were transferred to ImmobiionP (Miliipore) and processed according to the manufacturer’s
instructions, using an alkaline phosphatase detection system.
We thank Kevin Crawford and Jacky Hayles for providing sucl protein,
and Kathy Gould for 4711 antiserum. We also thank Rachel Bartlett,
Sue Dorrtngton, Susan Forsburg, Chris Norbury. and Shelley Sazer for
critical reading of the manuscript. This work was supported by the
Medical Research Council and the imperial Cancer Research Fund.
References
Beach, D., Durkacz, B., and Nurse, F?(1962). Functionally homologous
cell cycle control genes in budding and fission yeast. Nature 300,
662-667.
Blow, J. J. (1966). Eukaryotic DNA replication reconstituted outside the
cell. Bioessays 8, 149-152.
Blow, J. J., and Laskey, R. A. (1966). Initiation of DNA replication in
nuclei and purified DNA by a ceil-free extract of Xenopus eggs. Cell
47, 577-567.
Blow, J. J., and Laskey, R. A. (1966). A role for the nuclear envelope
in controlling DNA replication within the cell cycle. Nature 332, 546
540.
Blow, J. J., and Sleeman, A. M. (1990). Replication of purified DNA in
Xenopus egg extract is dependent on nuclear assembly. J. Ceil Sci. 95,
363-391.
Blow, J. J., and Watson, J. V (1967). Nuclei act as independent and integrated units of replication in a Xenopus cell-free system. EMBO J. 6,
1997-2002.
Blow, J. J., Sheehan, M. A., Watson, J. V., and Laskey. R. A. (1969).
Nuclear structure and the control of DNA replication in the Xenopus
embryo. J. Cell Sci. (Suppl.) 72, 163-195.
Booher, R., and Beach, D. (1966). Site specific mutagenesis of cdc2+
a cell cycle control gene of the fission yeast Schizosaccharomyces
pombe. Mol. Cell. Biol. 6, 3523-3530.
Brizuela, L., Draetta, G., and Beach, D. (1967). ~13~~’acts in the fission yeast cell cycle as a component of the ~34~~ protein kinase.
EMBO J. 6, 3507-3514.
Decker, R. S.. Yamaguchi, M., Possenti, R., and DeF’amphilis, M. L.
(1966). Initiation of simian virus 40 DNA in vitro: aphidicdin causes acof the initial direction of DNA synthesis. Mol. Cell. Biol. 6, 3615-3625.
Draetta, G., and Beach, D. (1966). Activation of c&2 protein kinase
during mitosis in human cells: ceil cycle-dependent phosphorylation
and subunit rearrangement. Cell 54, 17-26.
Draetta, G., Luca, F., Westendorf, J., Brizuela, L., Ruderman, J., and
Beach, D. (1969). cdc2 protein kinase is complexed with both cyclin A
and 8: evidence for proteolytic inactivation of MPF Cell 56, 629-636.
Dunphy, W. G., Brizueia, L., Beach, D., and Newport, J. (1966). The
Xenopus cdc2 protein is a component of MPF, a cytoplasmic regulator
of mitosis. Cell 54, 423431.
Evans, T., Rosenthal, E. T., Youngblom, J., Distel, D., and Hunt, T
(1963). Cyclin: a protein specified by maternal mRNA in sea urchin
eggs that is destroyed at each cleavage division. Cell 33, 369-396.
Gautier, J., Norbury, C., Lohka, M., Nurse, f?. and Mailer, J. (1966). Purified maturation-promoting factor contains the product of a Xenopus
homolog of the fission yeast cell cycle control gene cd&‘+. Cell 54,
433-439.
Gautier, J., Minshuil, J., Lohka, M., Glotzer, M., Hunt, T., and Mailer,
J. L. (1990). Cyclin is a component of maturation-promoting factor from
Xenopus. Ceil 60, 467-494.
Giordano, A., Whyte, P., Hertow, E., Franza, B. R., Jr., Beach, D., and
Draetta, G. (1969). A 60 kd cdc2associated polypeptide complexes
with the EIA proteins in adenovirus-infected cells. Cell 56, 961-990.
Gould, K. L., and Nurse, P (1969). Tyrosine phosphorylatiin of the fission yeast cdc2+ protein kinase regulates entry into mitosis. Nature
342, 3945.
Hartwell, L. H., and Unger, M. W. (1977). Unequal division in Saccharomyces cerevisiae and its implications for the control of cell division. J. Cell Biol. 75, 422-435.
Hartwell, L. H., Mortimer, R. K., Culotti, J., and Culotti. M. (1973).
Cdl
882
Genetic control of the cell division cycle in yeast: V. Genetic analysis
of cdc mutants. Genetics 74, 267.
Haylea, J., Beach, D., Durkacz, B., and Nurse, P (1986). The fission
yeast cell cycle control gene cd& isolation of a sequence sucl that
suppresses cdc2 mutant function. Mol. Gen. Genet. 202, 291-293.
Hindley, J., and Phear. G. A. (1964). Sequence of the cell cycle division
gene CDCP from Schizosacchammyces pombe; patterns of splicing
and homology to protein kinases. Gene 37, 129-134.
Hutchison, C. J., Cox, R., Drepaul, R. S., Gomperts, M., and Ford,
C. C. (1987). Periodic DNA synthesis in cell-free extracts of Xenopus
eggs. EMBO J. 6, 2003-2010.
Hutchison, C. J., Cox, Ft.. and Ford, C. C. (1988). The control of DNA
replication in a cell-free extract that recapitulates a basic cell-cycle in
vitro. Development 703, 553-568.
Johnston, G. C.. Pringle, J. R., and Hartwell, L. H. (1977).Coordination
of growth with cell division in the yeast Saccharomyces cerevisiae.
Exp. Cell Res. 105, 79-98.
Labbe, J.. Capony, J., Caput, D., Cavadore, J.. Derancourt. J., Kaghad,
M., Lelias, J., Picard, A., and Dome, M. (1989). MPF fmm starfish oocytes at first m&tic me&phase is a hetemdimer containing one molecule of cdc2 and one molecule of cyclin 8. EMBO J. 8, 3053-3058.
Lee, M. G., and Nurse, R (1967).Complementation used to clone a human homologue of the fission yeast cell cycle control gene cdc2. Nature 327, 31-35.
Lee, M. G.. Norbury, C. J., Spurr, N. K., and Nurse, P (1988). Regulated expression and phosphorylation of a possible mammalian cellcycle control protein. Nature 333, 876-679.
Lohka, M. J., and Masui, Y. (1983). Formation in vitro of sperm
pronuclei and mitotic chromosomes induced by amphibian ooplasmic
contents. Science 220, 7l9-721.
Lohka, M. J., and Masui, Y. (1965). Effect of Ca2+ ions on the formation of metaphase chromosomes and sperm pronuclei in cell-free
preparations from unactivated Rana pipens eggs. Dev. Biol. 103,
434-442.
Lohka. hf. J., Hayes, M. K., and Mailer, J. L. (1988). Purification of
maturation-promoting factor, an intracellular regulator of early mitotic
events. Proc. Natl. Acad. Sci. USA 85. 3009-3013.
Lorincz, A. T., and Reed, S. I. (1984). Primary structure homology between the product of yeast cell division gene CDC28 and vertebrate
oncogenes. Nature 307. 183-185.
Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982). Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, New York: Cold Spring
Harbor Laboratory).
McVey. D., Brizuela. L., Mohr, I., Marshak, D. R., Gluzman, Y., and
Beach, D. (1989). Phosphorylation of large tumour antigen by cdc2
stimulates SV40 DNA replication. Nature 341, 503-507.
MBchali, M., and Harland, Ft. M. (1982). DNA synthesis in a cellfree system from Xenopus eggs: priming and elongation on singlestranded DNA in vitro. Cell 30, 93-101.
Minshull. J., Blow, J. J., and Hunt, T. (1989). Translation of cyclin mRNA
is necessary for extracts of activated Xenopus eggs to enter mitosis.
Cell 56, 947-956.
Morrissey, J. H. (1981).Silver stain for proteins in polyacrylamide gels:
a modified procedure with enhanced uniform sensitivity. Anal. Biothem. 117,307-310.
Murray, A. W., and Kirachner, M. W. (1989). Cyclin synthesis drives the
early embryonic cell cycle. Nature 339, 275-280.
Newport, J. (1987). Nuclear reconstitution in vitm: stages of assembly
around protein-free DNA. Cell 48, 205-217.
Novak, B., and Mitch/son, J. M. (1969). The first transition point of the
mutant cdc2.33 in the fission yeast Schizosacchammyces pornbe. J.
Cell Sci. 94, 857-662.
Nurse, l?, and Bissett, Y. (1981).Gene required in Gl for commitment
to the cell cycle and in 62 for control of mitosis in fission yeast. Nature
292,558-580.
Nurse, f?, Thuriaux, P, and Nasmyth, K. (1978).Genetic control of the
cell division cycle in the fission yeast Schizosacchammyces pombe.
Mol. Gen. Genet. 746, 167-178.
Piggott, J. R., Rai, R., and Carter, 8. L. A. (1982). A.bifunctional gene
product involved in two phases of the yeast cell cycle. Nature 298,
391-394.
Riabowol, K., Draetta, G., Brizuela, L., Vandre, D., and Beach, D
(1989). The cdc2 kinase is a nuclear protein that is essential for mitosis
in mammalian cells. Cell 57, 393-401.
Sheehan, M. A., Mills, A. D., Sleeman, A. M., Laskey, Ft. A., and Blow,
J. J. (1988). Steps in the assembly of replication-competent nuclei in
a cell-free system from Xenopus eggs. J. Cell Biol. 106, 1-12.
Wittenberg, C., and Reed, S. I. (1989). Conservation of function and
regulation within the CDC28/cdc2 protein kinase family: characterization of the human CDC2Hs protein kinase in Sacchammyces cerevisiae. Mol. Cell. Biol. 9, 4064-4068.