Cipl blocks the initiation of DNA replication
in Xenopus extracts by inhibition of
cyclin-dependent kinases
Ulrich P.Strausfeld*, Mike Howell*, Rachel Rempel t , James L. Mallert,
Tim Hunt* and J.Julian Blow*
*ICRF Clare Hall Laboratories, South Mimms, Potters Bar, Herts EN6 3LD, UK and tHoward Hughes Medical Institute,
4200 East Ninth Avenue, Denver, Colorado 80262, USA.
Background: Cipl is a 21 kD protein that interacts with
and inhibits cyclin-dependent kinases (cdks). Expression of
Cipl is induced by the tumour suppressor p53, and
tumour cells have greatly reduced levels of Cipl. As cdks
are required for normal progression through the cell cycle,
their inhibition by Cipl may mediate the ability of p53 to
block cell proliferation. Cipl has also been shown to inhibit the DNA polymerase auxiliary factor PCNA (proliferating cell nuclear antigen), which is required for
replication-fork elongation, and this could be an alternative mechanism by which p53-induced Cipl blocks cell
proliferation.
Results: We have investigated the effect of Cipl protein
on chromosomal DNA replication, using cell-free extracts
of Xenopus eggs that initiate and complete chromosome
replication under normal cell-cycle control. Cipl protein
strongly inhibited an early stage of DNA replication in this
system, and this inhibition was not complemented by
extracts that had been affinity-depleted of cdks. In contrast,
Cipl did not inhibit the elongation of replication forks that
had accumulated in the presence of aphidicolin. Cipl inhibition of DNA replication was fully rescued by addition of
cyclins A or E, but not cyclin B, cdk2 or PCNA.
Conclusions: Our results suggest that Cipl specifically
blocks the initiation of DNA replication by inhibition of a
cyclin-dependent kinase (cdk2), but has no major effect
on the elongation of preassembled replication forks. The
ability of cyclin A or cyclin E to rescue the Cipl inhibition suggests that these cyclins may play a direct role in
the initiation of replication in the Xenopus system.
Current Biology 1994, 4:876-883
Background
Cyclin-dependent kinases (cdks) are required for a
number of key cell-cycle transitions in eukaryotic cells.
In particular, in a number of cell types, cdks are
necessary for chromosome replication and progression
from the G1 phase of the cell cycle into S phase
(reviewed in [1-3]). We have been using cell-free
extracts of Xenopus eggs that recapitulate basic cell-cycle
events in vitro to study this requirement for cdks.
Xenopus extracts can assemble exogenous DNA into
interphase nuclei, and then initiate and complete a
single round of DNA replication in vitro before passing
into mitosis [4-8]. Previous work has identified a role
for cdks in DNA replication in this system. Extracts
affinity-depleted of cdks by pl13 sUC were unable to
replicate added DNA, but could assemble nuclei and
support replication-fork elongation [9]. Replication
could be restored to depleted extracts by the addition of
fractions enriched for active cdks. A similar block to
replication was obtained when antibodies directed
specifically against Xenopus cdk2 were used to immunodeplete extracts that were already in interphase [10,11].
These results suggest that cdk2, probably in conjunction
with a G1 cyclin, is required for an early event in
chromosome replication.
A certain proportion of cdks are found in quaternary
complexes consisting of a cdk, a cyclin, PCNA (proliferating cell nuclear antigen, the DNA polymerase 8
auxiliary factor) and a 21 kD protein [12] that has
recently been identified as Cipl, a specific inhibitor of
cyclin-dependent kinase activity [13-16]. Cipl is also
identical to a protein called Wafl that is induced by
wild-type p53 [17]. The anti-oncogene p53 is required
for cells to delay DNA replication if their DNA has been
damaged by ionizing radiation [18,19]. After y-irradiation in G1, cells arrest prior to S phase with their
cdk2-cyclin E complexes inhibited by Cipl [16]. It has
therefore been suggested that the p53-dependent G1
checkpoint is mediated by Cipl-inhibition of cdks that
are required for entry into S phase.
It has also been suggested that Cipl may inhibit PCNA,
the DNA polymerase 8 auxiliary factor [20]. PCNA
greatly enhances the processivity of DNA polymerase 8,
and is required for the replication of SV40 DNA in vitro
[21-23]. Cipl inhibited SV40 DNA replication in crude
cell lysates, as well as SV40 DNA replication reconstituted with purified components [20]. This inhibition
could be relieved by addition of excess PCNA. On
mixing purified PCNA and Cipl, a proportion of the
Cipl could be found in a complex that sedimented with
Correspondence to: J.Julian Blow.
876
© Current Biology 1994, Vol 4 No 10
RESEARCH PAPER
Cipl inhibition of DNA replication Strausfeld et al.
Cipi inhibition of DNA replication Strausfeld et al.
PCNA on glycerol gradients. These results suggest that
Cipl might inhibit chromosome replication by inhibiting
PCNA and blocking replication fork elongation.
RESEARCH PAPER
(a)
30
E
25-
x
We have investigated the ability of Cipl protein to block
chromosomal DNA replication in Xenopus egg extracts.
Cipl protein strongly inhibited an early event in DNA
replication in this system. This inhibition could
be rescued by adding cyclins A or E, but not cyclin B,
cdk2 or PCNA. Our results suggest that Cipl blocks
cell-cycle progression by specifically inhibiting a cyclindependent kinase activity required for chromosomal
DNA replication.
0
20-
-_
15-
.5
u
10-
E
o +Cycl0in
0 +Cyclin E
a)
A -Cyclin
5*
,
_
b
,
b
00
(b)
1
10
100
1000
Cip1 concentration (nM)
10 000
2.5-
P
Inhibition of DNA synthesis by Cipi
We purified glutathione-S transferase (GST)- and
histidine-tagged versions of human Cipl from Escherichia
coli. As expected from earlier studies [13-15], the tagged
proteins inhibited the histone H1 kinase activity induced
by addition of cyclins A and E to interphase Xenopus
extracts supplemented with cdk2 (Fig. la). The inhibition was rapid, being essentially complete within a
minute (data not shown). We then tested whether Cipl
inhibits the replication of sperm chromatin in Xenopus
egg extracts. DNA normally undergoes one complete
round of DNA replication in such extracts, but the
addition of either GST- or histidine-tagged Cipl at concentrations above 100 nM inhibited DNA replication by
more than 95 % (Fig. lb and data not shown).
Figure c shows the kinetics of Cipl inhibition of DNA
replication: when sperm chromatin is added to Xenopus
extracts, it decondenses and assembles into an interphase
nucleus after about 30-40 minutes. The initiation of
DNA replication occurs only after nuclear assembly is
complete [5,6,8,24,25]. Replication is typically complete
by 90-180 minutes (Fig. c, filled triangles). At different
times during incubation in vitro, aliquots were taken,
mixed with Cipl protein, and the total DNA replication
over a 180 minute incubation was measured (Fig. 1c, open
circles). Cipl protein completely blocked subsequent
DNA synthesis only when added during the pre-synthesis
lag, and the inhibition rapidly declined once DNA replication had started. This suggests that Cipl specifically
inhibits an early stage in the replication process.
The morphology of nuclei assembled in Cipl-treated
extracts was essentially indistinguishable from that of
controls (Fig. 2). Whether or not Cipl was added, the
chromatin decondensed (Fig. 2a,d,g,j) and acquired an
intact, phase-dense nuclear envelope (Fig. 2b,e,h,k).
Nuclei in the presence of Cipl were slightly smaller and
more multi-lobed than in control extracts, though this is
not normally associated with a failure to replicate DNA.
Nuclei assembled in Cipl-treated extracts were capable
of selectively accumulating proteins with a nuclear
localization signal (Fig. 2c,f,i,l).
No Cip
o
a
Results
0 2 .0-
,
U
u
C
added
1.5-
E
u
6.
1.0-
D
U
8_1
v
C)
0.5-
z
10
(rc
100
1 000
Cipl concentration (nM)
10( )00
3.0'
No Cip] added
o
2.5
o
.
2.0
a 1.5
1.0
.5
c5 0.5·
7
I
u
0
20io 40
60
80
100
12o
Time after DNA addition (minutes)
Fig. 1. Inhibition of histone HI kinase activity and DNA replication by Cipl protein. (a) GST-Cipl was added to interphase
Xenopus extracts supplemented with 1.2 pM human GST-cdk2
and either 800 nM cyclin A1-AN56 (circles), 500 nM cyclin E6His (squares), or no added cyclins (triangles). Samples were
incubated at 23 °C for 45 minutes, and assayed for histone Hi
kinase activity. Filled symbols show H1 kinase activity in the
absence of added Cip. (b) GST-Cipl was added to interphase
Xenopus extract supplemented with sperm chromatin (3 ng DNA
per pl of extract) and a 32 P-dATP. After 3 hours at 23 °C, DNA
synthesis was assessed by trichloroacetic acid (TCA) precipitation. DNA synthesis is expressed as the percentage of total a32PdATP incorporated into TCA-insoluble material. (c) Interphase
Xenopus extract was supplemented with sperm chromatin (3 ng
DNA per pl extract) and a32P-dATP, and incubated at 23 °C. At
the indicated times, aliquots were taken, supplemented with
160 nM GST-Cipl and incubated for a total of 3 hours at 23 °C
before TCA precipitation (open circles). A time course of DNA
synthesis in extract without Cipl was also performed, showing
the extent of DNA synthesis that has occurred at the indicated
times (filled triangles). DNA synthesis is expressed as the percentage of total a32P-dATP incorporated into TCA-insoluble material.
877
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Current Biology 1994, Vol 4 No 10
pl3suc1-depleted extract ('sucl nuclei'); this is consistent
with Cipl inhibiting cdk2 function. We also examined
the effect of Cipl on DNA templates that do not require
the initiation of replication forks to support DNA
synthesis. 'Aphidicolin-blocked nuclei' are isolated from
extracts supplemented with aphidicolin, a competitive
inhibitor of replicative DNA polymerases; singlestranded M13 DNA can undergo complementary strand
synthesis under the action of DNA primase and DNA
polymerase. Although the replication of both aphidicolin-blocked nuclei and M13 DNA was slightly
inhibited in the presence of Cipl, this inhibition was
much less than was seen with sperm chromatin or sucl
nuclei, which require the initiation of replication forks
(Figure 3b).
Fig. 2. Morphology of nuclei assembled in control and Cipltreated Xenopus egg extracts. Sperm chromatin was incubated
with (a-f) or without (g-I) 80 nM GST-Cipl in extract supplemented with allophycocyanin coupled to a nuclear localization
sequence. After 90 minutes at 23 C, samples were stained with
Hoechst 33258 and viewed by fluorescence microscopy. (a,d,g,j)
UV fluorescence for DNA; (b,e,h,k) phase contrast; (c,f,i,l) allophycocyanin fluorescence for nuclear transport. Scale bar (for all
fields) = 10 pm.
Analysis of Cipi block point
It has previously been shown that DNA replication in
Xenopus egg extracts requires cdk2 activity [9-11], which
is therefore a likely candidate for being the target of the
Cipl inhibition. Figure 3a shows the ability of extracts
supplemented with different concentrations of Cipl to
rescue extracts depleted of cdks using pl 3 suc1 beads.
Extract supplemented with more than 160 nM Cipl was
incapable of rescuing a sucl-depleted extract. The similarity between the levels of Cipl required to block the
replication of sperm chromatin (Fig. lb) and those
required to abolish rescue of sucl-depleted extracts
(Fig. 3a) suggests that the inhibition of DNA replication
by Cipl is mediated by inhibition of cdk activity.
Although a certain proportion of cdks are found in a
quaternary complex containing PCNA [12], sucldepletion did not significantly lower the endogenous
PCNA pool (inset to Fig 3a; see below).
We next examined the ability of the Cipl-inhibited
extracts to replicate different DNA templates (Fig. 3b).
Cipl severely inhibited the replication of both
sperm chromatin and nuclei previously assembled in
In order to see whether Cipl affected the elongation rate
of replication forks that had been initiated in the
presence of aphidicolin, we analyzed the nascent DNA
on denaturing agarose gels (Fig. 4). Aphidicolin-blocked
nuclei were isolated and transferred to extracts supplemented with a32P-dATP, with or without 160 nM
Cipl. At different times after transfer, aliquots were
removed and assayed by trichloroacetic acid (TCA) precipitation (Fig. 4a) or by alkaline agarose gel electrophoresis (Fig. 4b,c). Immediately after transfer, nascent
strands were seen elongating at about 16 nucleotides per
second, whether or not Cipl was present (Fig. 4b,c).
After about 15 minutes, the rate of synthesis dropped
(Fig. 4a), presumably due to termination of most of the
replication forks [26]. In the absence of Cipl, however,
further synthesis occurred; this probably resulted from
late initiation events. During this later period, short
replicative intermediates did not accumulate, as would be
expected if Cipl were inhibiting chain elongation at this
stage (Fig. 4c). In addition, short nascent strands did not
accumulate when sperm chromatin was incubated in
extracts with Cipl (Fig. 4c, lane 11). Therefore, we
could not detect any sign of inhibition of the elongation
stage of DNA synthesis by Cipl.
Rescue by cyclins A and E
Cipl can be found in quaternary complexes containing
cdks, cyclins and PCNA [12], and Cipl has recently
been shown to inhibit the ability of PCNA to activate
DNA polymerase
in a highly purified SV40 DNA
replication assay [20]. Cipl is unlikely to block replication by interaction with PCNA in Xenopus extracts,
however, as PCNA is present at about 4-8 liM in
Xenopus oocytes [27] and egg extracts (inset to Fig. 5a);
this level exceeds the concentration of Cipl required for
inhibition (Fig. lb). Nevertheless, we tested whether
purified PCNA could restore DNA replication in Cipltreated extracts. Figure 5a shows that even the addition
of 360 nM PCNA did not rescue DNA replication in
extracts treated with 73 nM Cipl.
The experiments described above suggest that Cipl
inhibition of DNA replication was mediated by cdk
inhibition, and so we next investigated the ability of
Cipl inhibition of DNA replication Strausfeld et al.
Cipi inhibition of DNA replication Strausfeld et a!.
recombinant Xenopus cdk2 [28] to rescue DNA replication in Cipl-treated extracts (Fig. 5b). Only at very high
cdk2 concentrations (4 t.M) was there any detectable rescue of DNA synthesis in Cipl-treated extracts. In Xenopus
eggs, however, the activity of cycin-dependent kinases is
largely dependent on the concentration of cyclins, rather
than on the concentration of cdks (which are about
600 nM in the case of cdc2, and about 60 nM in the case
of cdk2; [29] and data not shown). We therefore tested
whether purified Xenopus cyclins A, B or E could rescue
DNA replication in Cipl-treated extracts (Fig. 6).
When increasing amounts of cyclin A were added to
Xenopus extract without added Cipl, histone H1 kinase
Fig. 3. Characteristics of Cipl inhibition of DNA replication. (a)
Extracts were supplemented with the indicated concentrations of
GST-Cipl and mixed with p135ucl-depleted extracts in a 1:5
ratio. These mixtures were assayed for the replication of sperm
chromatin over a 3 hour incubation. DNA synthesis is expressed
as the percentage of total &c32P-dATPincorporated into TCAinsoluble material. The inset shows extract before (lanes 1 and 3)
and after (lanes 2 and 4) sucl-depletion, Western blotted for
cdks (lanes 1 and 2) and PCNA (lanes 3 and 4). (b) Different
DNA templates were incubated in interphase Xenopus extract,
with (white) or without (grey) 160 nM GST-Cipl. Total DNA
synthesis after 2 hours at 23 C was measured, and is expressed
as the percentage of total 32P-dATP incorporated into TCAinsoluble material. Sperm chromatin, de-membranated Xenopus
sperm nuclei; sucl nuclei, nuclei isolated after incubation of
sperm chromatin in p13Sucl-depleted Xenopus extract; aphidicolin-blocked, nuclei isolated after incubation of sperm
chromatin in Xenopus extract supplemented with 30 pg ml-1
aphidicolin; ssM13 DNA, single-stranded M13 DNA. All DNA
templates were added at approximately 3 ng DNA per p extract.
RESEARCH PAPER
RESEARCH PAPER
activity was induced (Fig. 6a, open squares), causing
mitotic events such as nuclear envelope breakdown and
chromatin condensation. As expected, this led to the
inhibition of DNA synthesis (Fig. 6a, closed circles), as
the initiation of DNA replication does not occur during
mitosis in the Xenopus system [25]. In the presence of
80 nM GST-Cipl, the histone H1 kinase activity
induced by cyclin A was inhibited (Fig. 6b, open squares;
Fig. la), and so normal nuclear assembly was maintained
in these extracts. Under these conditions, the inhibition
of DNA replication caused by 80 nM Cipl was fully
rescued by cyclin A at concentrations above 200 nM
(Fig. 6b, filled circles). BrdUTP density substitution
confirmed that this synthesis consisted entirely of a single
complete round of semiconservative DNA replication
(data not shown).
Fig. 4. Effect of Cipl on replication of aphidicolin-blocked
nuclei. Aphidicolin-blocked nuclei were prepared by incubating
sperm chromatin, for 90 minutes at 23 C, in Xenopus extract
supplemented with 30 pg ml-' aphidicolin. After isolation, equal
aliquots of nuclei were incubated in interphase Xenopus extract
containing a32P-dATP, with or without 160 nM GST-Cipl. At
different times, aliquots of the reaction were taken and assayed.
(a) TCA-precipitation assay. Open squares, reaction without
added Cipl; filled circles, reaction with added 160 nM GSTCipl. DNA synthesis is expressed as the percentage of total
a32 P-dATP incorporated into TCA-insoluble material. (b,c)
Aliquots were electrophoresed on a 0.9 % alkaline agarose gel
without (b) and with (c) added 160 nM GST-Cipl. Lanes 1-10
show samples taken at 1, 2, 3, 4, 6, 10, 20, 30, 60, 90 minutes
after transfer, respectively. Lane 11 shows sperm chromatin
incubated in the same extracts for 90 minutes. M, end-labelled
lambda/Hindlll markers, with molecular weights indicated in kb.
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Current Biology 1994, Vol 4 No 10
In the absence of added Cipl, Xenopus cyclin B also
induced high levels of H1 kinase activity (Fig. 6c, open
squares) and hence inhibited DNA synthesis (Fig. 6c,
filled circles). In contrast to cyclin A, cyclin B caused no
observable rescue of DNA synthesis in the presence of
added Cipl (Fig. 6d, filled circles). Histone H1 kinase
activity induced by cyclin B was efficiently inhibited by
Cipl (Fig. 6d, open squares), and so premature entry
into mitosis cannot account for the failure of cyclin B to
rescue DNA replication.
Xenopus cyclin E behaved differently from both cyclins A
and B (Fig. 6e and f). High histone H1 kinase levels
were not induced by addition of Xenopus cyclin E to
control extracts (Fig. 6e, open squares), and DNA
synthesis remained correspondingly high in the absence
of Cipl at cyclin E concentrations up to 300 nM
(Fig. 6e, filled circles). This cyclin E construct was
nevertheless capable of inducing high H1 kinase levels
when recombinant cdk2 was also added (Fig. la; manuscript in preparation). Despite its inability to induce H1
kinase activity, 300 nM cyclin E efficiently rescued DNA
synthesis in Cipl-inhibited extract (Fig. 6f, filled circles).
Discussion
Cipl inhibition of replication mediated by cdk inhibition
After affinity-depletion of cdks with p13sucl [9], or
immunodepletion of cdk2 [10,11], Xenopus egg extracts
are unable to support the replication of added sperm
chromatin. Analysis of the depleted extracts suggested
that they were specifically unable to support the initiation of DNA replication. Unlike the situation in somatic
cells, progression from mitosis through S phase in
Xenopus eggs and egg extracts requires no new protein
synthesis [30,31], although cyclin B translation is then
required for cells to progress from G2 back into mitosis
[32,33]. This means that the function of cdk2 in supporting DNA replication in Xenopus egg extracts must be
mediated solely by post-translational modification, presumably by phosphorylating and activating essential
replication proteins. Furthermore, any cyclins required to
activate cdk2 for this function must be preformed and
stable from the time of mitosis.
We have explored the role of cdk2 in controlling DNA
replication in the Xenopus system using recombinant
Cipl protein, a recently identified inhibitor of cyclindependent kinases [13-16]. Cipl inhibited DNA replication in the Xenopus cell-free system at concentrations
comparable to the concentration of endogenous cdk2.
Cipl-treated and cdk-depleted extracts did not crosscomplement one another, suggesting that they both lack
the same essential cdk2 activity required for chromosomal DNA replication. Similarly, nuclei assembled in
extracts that had been depleted of cdks using p13sucI did
not replicate on transfer to extracts supplemented with
Cipl. Furthermore, the ability of purified cyclins to
rescue the Cipl inhibition in full suggests that Cipl
blocks DNA replication via inhibition of cdks. The
activity of cyclin-dependent kinases in Xenopus eggs is
probably limited by the concentration of cyclins, rather
than by the concentration of cdks. Presumably recombinant cyclins, alone or in association with previously
uncomplexed cdks, titrate out the inhibiting Cipl.
Fig. 5. Neither PCNA or cdk2 can rescue Cipl-treated extracts.
Extracts were incubated with various concentrations of (a)
human PCNA or (b) human GST-tagged cdk2, for 3 hours at
23 C, with sperm chromatin (3 ng DNA per pl extract) and
a32P-dATP with or without added GST-Cipl (73 nM in (a);
160 nM in (b)). DNA synthesis is expressed as the percentage of
total &c32P-dATPincorporated into TCA-insoluble material. The
inset in (a)shows a Western blot with anti-PCNA antibody (mAb
p10) against purified human PCNA and interphase Xenopus
extract. Lanes 1-4: 112, 22, 4.5 and 0.9 ng of purified human
PCNA; lanes 5,6: 0.4 and 0.1 pl interphase Xenopus extract.
Using the highly fractionated SV40 DNA replication
system derived from somatic cells, Waga et al. [20] have
recently shown that Cipl can block replication by
inhibiting PCNA, an essential DNA polymerase
auxiliary factor. Cipl and PCNA are found together in
quaternary complexes of cdks, cyclins, PCNA and Cipl
[12]. However, PCNA is an abundant protein in
Xenopus eggs and egg extracts at 4-8 pIM (Fig. 5a; [27]),
meaning that only a small fraction of the total PCNA is
found in these quaternary complexes (Fig 3a).
Inhibition of DNA replication in Xenopus egg extracts
RESEARCH PAPER
Cipl inhibition of DNA replication Strausfeld et al.
Cipi inhibition of DNA replication Strausfeld et al.
-Cip
4 (a)
RESEARCH PAPER
new replication forks, and is similar to results obtained
with extracts affinity-depleted of cdks with pl3 suc1 [9].
+Cipl
Cyclin A (b)
CyclinA 40
3
30
2
20
I
10
L6f
(C)
0
Cyclin B 40
Cyclin B (d)
0 3
2
E
,
30
/
20
I
0 100 200 300 400 0 100 200 300 400
Cyclin
concentratio
(nM) n
Cyclin
concentration (nM)
Fig. 6. Cyclins A and Ecan rescue DNA synthesis in Cipl -treated
extract, but cyclin B cannot. Interphase Xenopus extract was
supplemented with sperm chromatin (3 ng DNA per pi extract)
without (ac,e) or with (b,d,f) 80 nM GST-Cipl protein and
various concentrations of bacterially produced Xenopus cyclin
A1-AN56 (a,b), cyclin B-MBP (c,d), or cyclin E-6His (e,f), and
was incubated at 23 C. After 45 minutes at 23 C, aliquots were
assayed for histone H1 kinase activity (open squares). DNA
synthesis (filled circles) is expressed as the percentage of total
a32P-dATP incorporated into TCA-insoluble material during a
3 hour incubation.
In order to examine this point in more detail, aphidicolin-blocked nuclei were replicated in Cipl-treated
extracts, and the nascent DNA synthesized was analysed
on denaturing gels. Immediately after transfer of aphidicolin-blocked nuclei into fresh extract, nascent strands
grew at a rate of about 16 nucleotides per second, close
to the rate expected in vivo [26,34], whether or not Cipl
was present. At later times, some inhibition by Cipl
occurred, which is likely to result from the inhibition of
late initiation events, as no accumulation of short nascent
strands was observed. As cdk inhibition by Cipl is rapid,
and as Cipl can apparently pass in and out of intact
nuclei (data not shown), the block to replication caused
by Cipl in this system is therefore unlikely to be
mediated by inhibition of replication fork elongation.
Although it is hard to rule out any more subtle effects on
elongation, these results suggest that the major point of
Cipl inhibition is over the initiation of new replication
forks, consistent with the observation that Cipl only
inhibited replication of sperm chromatin when Cipl was
added prior to the end of the pre-synthesis lag phase
(Fig. c).
by 100 nM Cipl is therefore unlikely to result from the
direct inhibition of PCNA by Cipl. This interpretation
is supported by the inability of 360 nM human PCNA
to rescue the inhibition of DNA replication caused by
73 nM Cipl. When Xenopus extract was fractionated
in order to lower the PCNA concentration approximately 100-fold, Cipl inhibition of PCNA function in
DNA synthesis was observed (M.K.K. Shivji, S.J. Grey,
U.P.S., R.D. Wood and JJ.B., manuscript submitted).
However, the high level of endogenous PCNA in
unfractionated Xenopus extract means that PCNA inhibition by nanomolar concentrations of Cipl is unlikely
to be significant.
Identity of cyclins required for DNA replication
In order to promote entry into S phase, cdk2 is presumably activated by one or more cyclin partners. We have
found that either cyclin A or E can rescue DNA replication in Cipl-treated Xenopus extracts. Cyclin B,
however, which is required for the G2-to-M transition,
did not rescue the Cipl inhibition. Both cyclin A
[35-37] and cyclin E [38,39] have previously been implicated in S-phase control in other cell types. In somatic
cells, cdk2 associates with these cyclins during G1 and S
phases [36,40-42,]. In the rapid, early-cleavage cell
cycles of Xenopus, however, cyclin E and cdk2 levels are
constant, whereas cyclin A undergoes periodic degradation at the end of mitosis [43]. The insensitivity of DNA
replication in the Xenopus system to protein synthesis
inhibitors tends to argue against a role for endogenous
cyclin A in S-phase control in this system. Furthermore,
anti-sense ablation of cyclin Al mRNA in Xenopus
extracts did not directly block DNA synthesis, but
instead affected checkpoint control of entry into mitosis
[44]. In agreement with this interpretation, in the
Drosophila embryo, cyclin A mutants show defective
mitotic entry but no gross S-phase defect [45,46].
Arrest point of Cipl-treated extracts
The initiation of new replication forks is likely to be the
major point at which chromosome replication is controlled. Cipl-treated extracts were unable to support the
replication of sperm chromatin, but still remained
competent to elongate replication forks accumulated in
the presence of aphidicolin, and to perform complementary strand synthesis on single-stranded DNA
(Figs 3 and 4). This result suggests that the main effect of
Cipl on DNA replication is to block the initiation of
Cyclin E thus appears to be a better candidate for the
cyclin required for the initiation of DNA replication in
the Xenopus embryo. Cyclin A associates principally with
p3 4 cdc2 in the Xenopus egg, and associates with p33 cdk2
only later in development, whereas cyclin E associates
exclusively with p33 cdk2 in the egg ([43] and data not
shown). It is also noteworthy that the rescue of Cipltreated extracts by cyclins A and E shown here occurred
in the absence of significant histone H1 kinase activity.
This may indicate that the active kinase has a much
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Current Biology 1994, Vol 4 No 10
higher affinity for its physiological substrate than it does
for histone H1. Indeed, the high levels of H1 kinase
activity induced by cyclin A (and cyclin B) were in fact
inhibitory to DNA replication, as they drove extracts
into mitosis, when the initiation of replication cannot
occur [25]. Both cyclins A and E could rescue replication in Cipl-treated extracts: our results thus neither
prove a direct role for cyclin E in DNA replication nor
rule out a role for cyclin A. In extracts that were
depleted of cdks by affinity binding, cycin A was significantly better than cyclin E at rescuing DNA synthesis
(manuscript in preparation), suggesting that cyclin E
alone may not be sufficient to promote full DNA replication. The precise identification of the proteins present
in the cdk2 complex required for DNA replication
should help to resolve these questions.
Conclusions
The cdk inhibitor Cipl blocks an early stage of DNA
replication in Xenopus egg extracts at concentrations
close to those of endogenous cdk2. No significant inhibition of the elongation stage of DNA replication could
be observed. Cipl-treated extracts did not cross-complement DNA replication in extracts depleted of cdks by
pl3suc1. Cipl-treated extract could, however, be fully
rescued with Xenopus cyclins A and E, but not by
cyclin B, PCNA or cdk2. Our results suggest that Cipl
specifically inhibits the initiation of DNA replication by
inhibiting a cdk2-G1 cyclin activity required for this
process.
Preparation of Cip 1, cyclins and PCNA
The polymerase chain reaction (PCR) was used to isolate
human Cipl cDNA from a human cDNA library [13]
provided by S. Elledge, using the primers 3'-CGGGTCGACCATGGGCCTCTTGGAGAAGT and 5'-GCCGGATCCATGGCAGAACCGGGGGAT. The PCR product was cut
with NcoI and ligated with pGEX-KG and pET21d DNA.
Bacterially expressed protein was produced as described [49].
The purified Cipl fusion proteins were both able to inhibit
purified cyclinA-cdk2 kinase (R.Y.C. Poon, M.H.,
K. Yamashita and R.T. H., manuscript in preparation).
Full-length Xenopus cyclin E (Genbank accession number
L23857) was cloned into pRSTC (Invitrogen, San Diego,
California). The recombinant cyclin E protein has 15 additional amino acids, including a tag of six histidine residues, at
the amino terminus. Protein was expressed in BL21(DE3) after
induction with 0.1 mM IPTG (isopropyl-p-D-thiogalactoside)
at 23 °C for 4 hours. Soluble cyclin E was partially purified on
nickel resin (Ni 2 +-NTA (nitrilo-tri-acetic acid) agarose,
Qiagen, Studio City, California), eluted with 250 mM
imidazole, dialyzed and concentrated using a Centricon-30
microconcentrator (Amicon, Beverly, Massachusetts). The
protein concentration was estimated using bovine serum
albumin as a standard.
Xenopus cyclin Al was produced as described [49]. The
construct for bacterial expression of Xenopus cyclin B1 tagged
with maltose-binding protein was originally constructed by
M.-A. Flix (EMBL, Heidelberg) and was provided by
K. Yamashita. Human PCNA was prepared as described
[50,51] and was a gift of M. Shivji. It was fully active in a
PCNA-dependent DNA repair reaction [51]. GST-tagged
human cdk2 was prepared as described [49].
Acknowledgements: J.J.B. is a Lister-Jenner research Fellow. We
would like to thank Gary Martin for his excellent maintenance of
the Xenopus colony.
References
Materials and methods
3.
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Xenopus egg extract for 15 minutes at 23 C with half its
volume of Sepharose beads coupled with p13sucl at
10 mg ml-t; Sepharose beads were removed by centrifuging
briefly in a microfuge, followed by filtration. Before use,
extracts were supplemented with 100 ,ug ml-1 cycloheximide,
5 g ml-' creatine kinase and 25 mM phosphocreatine.
Replication reactions were performed at 23 C. DNA
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H1 kinase was assayed as described [48]. The allophycocyanin
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from S. Grey.
Nuclei for transfer experiments were prepared after incubation
for 90 minutes in either sucl-depleted extract or in extract supplemented with 30 g ml-' aphidicolin, and were isolated by
centrifuging twice through 20 % sucrose, as described [9,47].
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Accepted: 30 August 1994.
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