Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Potters Bar, Herts EN6 3LD, United Kingdom
The replication licensing factor (RLF) is an essential initiation factor that is involved in preventing re-replication of chromosomal DNA in a single cell cycle. In Xenopus egg extracts, it can be separated into two components: RLF-M, a complex of MCM/P1 polypeptides, and RLF-B, which is currently unpurified. In this paper we investigate variations in RLF activity throughout the cell cycle. Total RLF activity is low in metaphase, due to a lack of RLF-B activity and the presence of an RLF inhibitor. RLF-B is rapidly activated on exit from metaphase, and then declines during interphase. The RLF inhibitor present in metaphase extracts is dependent on the activity of cyclin-dependent kinases (Cdks). Affinity depletion of Cdks from metaphase extracts removed the RLF inhibitor, while Cdc2/cyclin B directly inhibited RLF activity. In metaphase extracts treated with the protein kinase inhibitor 6-dimethylaminopurine (6-DMAP), both cyclin B and the RLF inhibitor were stabilized although the extracts morphologically entered interphase. These results are consistent with studies in other organisms that invoke a key role for Cdks in preventing re-replication of DNA in a single cell cycle.
During each S phase of the eukaryotic cell division
cycle, the entire genome is precisely duplicated.
To achieve this, many hundreds or thousands of
replication origins must each fire once and only once in
each S phase. Studies using cell-free systems derived from
Xenopus eggs have revealed that re-replication of chromosomal DNA in a single cell cycle is prevented by the action of two distinct replication signals (Blow and Laskey, 1988 The activation of RLF that occurs at the metaphase-
anaphase transition in Xenopus can be blocked by certain
protein kinase inhibitors, such as 6-dimethylaminopurine
(6-DMAP), that are known to inhibit cyclin-dependent kinases (Blow, 1993 MCM/P1 genes were originally identified in Saccharomyces cerevisiae in a screen for mutants unable to efficiently initiate replication at particular replication origins
(Maine et al., 1984 In this paper we analyze the cell cycle control of RLF
activity in Xenopus egg extracts. Total RLF activity is
sharply periodic in the cell cycle. An RLF inhibitor is
present in metaphase extracts and appears to be directly
dependent on the activity of cyclin-dependent kinases.
When the inhibitor is removed or diluted away, RLF-M, but not RLF-B, activity can be detected. RLF-B, but not
RLF-M, activity is unstable in interphase and decays over
a period of 60-120 min. However, the decay of RLF-B activity in interphase is not associated with activation of an
RLF inhibitor and does not depend on cyclin-dependent
kinases.
Preparation and Use of Xenopus Egg Extract
Metaphase-arrested and interphase Xenopus extract was prepared as
described (Blow, 1993 Suc1 depletion was performed essentially as described (Blow and
Nurse, 1990 Preparation of DNA Templates
Demembranated Xenopus sperm nuclei were prepared by lysolecithin
treatment as described (Blow and Laskey, 1986 Preparation of RLF-B and RLF-M
Polyethylene glycol (PEG)-fractionated RLF-B and RLF-M were prepared as described (Chong et al., 1995 Differential PEG precipitation was performed as follows (Chong et al.,
1995 RLF-M was further fractionated on Q Sepharose to produce RLFMinter as described (Chong et al., 1995 For the maturation promoting factor (MPF) inhibition experiment (see
Fig. 4 b), RLF-M from the Q Sepharose step was further purified by phenyl Sepharose chromatography and gel filtration, as previously described
(Chong et al., 1995
Licensing Assay
RLF was assayed in a two-step protocol as described (Chong et al., 1995 Alkaline Agarose Gel Electrophoresis
Reactions for agarose gel electrophoresis were stopped by digesting with
Stop-N (20 mM Tris-Cl, 200 mM NaCl, 5 mM EDTA, 0.5% SDS, pH 8.0)
containing 2 µg/ml RNase A and 200 µg/ml Proteinase K for 30 min at
37°C. DNA was extracted sequentially with phenol, phenol-chloroform,
and chloroform, and then precipitated with ethanol. Pellets were resuspended in alkali gel loading buffer (50 mM NaOH, 1 mM EDTA, 1.25%
Ficoll, 0.0125% bromocresol green). Alkaline gels were prepared by adding the required amount of agarose to 50 mM NaCl and 1 mM EDTA.
Once set, the gels were allowed to equilibrate for 1 h in alkaline gel running buffer (30 mM NaOH, 1 mM EDTA), and then were run at 2 V/cm.
After electrophoresis was complete, gels were fixed in 7% TCA, dried,
and autoradiographed.
Cip1 and Cdc2/Cyclin B
Glutathione-S-transferase (GST)-tagged Cip1 was prepared as described
(Strausfeld et al., 1994 Antibodies and Western Blotting
For Western blotting, chromatin was isolated from extract as described for
the preparation of 6-DMAP chromatin, except that the NIB buffer was
supplemented with 0.1% NP-40 (Chong et al., 1995 35S Labeling and Autoradiography
200 µCi [35S]methionine (Amersham Intl.) was dried under vacuum and
redissolved in 40 µl metaphase-arrested extract supplemented with phosphocreatine and creatine phosphokinase. The extract was incubated for 30 min at 23°C to label newly synthesized proteins, before the addition of 250 µg/ml cycloheximide. Extract was then supplemented with various concentrations of 6-DMAP and then with 0.3 mM CaCl2, before being incubated for an additional 30 min at 23°C to allow cyclin degradation to occur. Cyclin-cyclin-dependent kinase (Cdk) complexes were then collected
on p13suc1 beads at 4°C for 45 min. The beads were washed several times, and then were resuspended in sample loading buffer for electrophoresis and autoradiography according to standard techniques.
Quantification of RLF-M Required
for DNA Replication
RLF-M consists of a complex of MCM/P1 proteins, including XMcm3 (Chong et al., 1995
Fig. 1 b examines the RLF activity of chromatin licensed
with different dilutions of interphase extract. Chromatin
was licensed with serial dilutions of extract, and then
transferred to 6-DMAP-treated extract (lacking RLF activity) supplemented with [ Cell Cycle Variation in RLF Activity
The Xenopus egg is arrested at metaphase of meiosis II.
At fertilization, a calcium wave is released that overcomes
the metaphase arrest, and the early embryo embarks on a
series of 12 rapid cell cycles. Extracts prepared from unfertilized eggs maintain the metaphase arrest and can be released into interphase by addition of CaCl2 (Lohka and
Masui, 1984
The titrations of RLF activity in metaphase and interphase extracts were repeated in the presence of exogenous
RLF-B (Fig. 2, open circles) or RLF-M (Fig. 2, open squares)
to respectively determine the RLF-M and RLF-B activity
of the samples. Titrations of activity in interphase extract
under these conditions remained virtually unchanged (Fig.
2 b), suggesting that these extracts had approximately
equal activities of RLF-M and RLF-B. At high concentrations of metaphase extract, addition of exogenous RLF-B or RLF-M induced no RLF activity (Fig. 2 a). However,
when metaphase extracts were diluted down to 1/8-1/16,
significant RLF-M activity could be detected. No RLF-B
activity was observed at any dilution of metaphase extract.
The appearance of RLF-M activity after dilution of metaphase extract was unexpected and suggested the presence of an RLF inhibitor in these extracts that was being diluted away until it no longer inhibited the reaction. The
presence of an RLF inhibitor was demonstrated by mixing
serial dilutions of metaphase extract with a constant amount
of active RLF (RLF-B plus RLF-M; Fig. 2 a, triangles).
The licensing activity of exogenous RLF was inhibited by
high concentrations of metaphase extract. When metaphase extracts were diluted further, the inhibitory activity was titrated away, allowing exogenous RLF to license chromatin. The disappearance of the inhibitory activity upon
dilution corresponded to concentrations where RLF-M activity became assayable. In contrast, no RLF inhibitor was
observed in interphase extracts (Fig. 2 b, triangles). The
simplest explanation for the ability to assay RLF-M activity after dilution of metaphase extract is that the RLF inhibitor affects only RLF-B activity. Consistent with this interpretation, we could find no evidence for the inhibition
of RLF-M activity in metaphase (see below).
To quantify cell cycle variations of RLF activity, extract
was incubated for various times, and then diluted to 1/8,
where RLF activity should be in the linear range for the
assay (Fig. 3). In these extracts, chromatin decondensation
and nuclear assembly are complete by ~30-40 min after
CaCl2 addition, while S phase occurs at 40-90 min (Blow,
1993
Cdk Dependence of the RLF Inhibitor
The above results suggest the presence of an RLF inhibitor in metaphase extracts. Interphase Xenopus extracts
can be induced to enter metaphase by addition of MPF, a
Cdk activity that can be provided by either Cdc2/cyclin B
(Labbé et al., 1989a The Xenopus early embryo has at least three distinct cyclin-dependent kinases: Cdc2/cyclin A, Cdc2/cyclin B, and
Cdk2/cyclin E. Similar to the effect of p13suc1 depletion,
immunoprecipitation of Cdc2 from metaphase extracts blocked activation of RLF-B (data not shown) and prevented subsequent DNA replication (Blow and Nurse,
1990 We next performed a partial (~50-fold) purification
of RLF-M from metaphase and interphase extracts to
investigate whether active RLF-M can be found in a
metaphase-arrested extract (Fig. 5 a). RLF-M prepared
from metaphase extracts (RLF-Mmeta) showed virtually
identical activity to RLF-M prepared from interphase extracts (RLF-Minter). Mixing RLF-Mmeta and RLF-Minter together showed that the RLF-M had been purified away
from the metaphase-specific inhibitory activity. At this stage
we cannot be completely certain that RLF-M is fully active
in metaphase extracts, as it is possible that an inhibitory
modification (such as phosphorylation) might be reversed
during the purification protocol. In metaphase Xenopus
extracts, the XMcm4 protein, a component of RLF-M
(Chong et al., 1995
Since the RLF inhibitor present in metaphase extracts is
dependent on cyclin-dependent kinases, we next investigated whether the decline in RLF activity during interphase is also dependent on cyclin-dependent kinases. Both
cyclin A and cyclin B are abruptly degraded during late
mitosis, and must be resynthesized during the next cell cycle
before entry into mitosis. Therefore, treatment of Xenopus egg extracts with protein synthesis inhibitors such as
cycloheximide blocks the reappearance of these two cyclins. However, consistent with our previous results (Blow,
1993
Fig. 6 b shows the quantity of RLF-B and RLF-M activity in interphase extracts that had been incubated at 23°C
for 2 h. Total RLF activity was reduced approximately
eightfold over extract that had recently been released
from metaphase (compare Figs. 2 b and 6 b, filled circles).
This was almost exclusively due to a loss of RLF-B activity
(open circles), while RLF-M activity remained high (squares).
Despite the decline in RLF-B activity, no RLF inhibitor
could be detected (Fig. 6 b, triangles). Thus, Xenopus extracts can be defective in RLF activity in at least two distinct ways. In metaphase, a potent inhibitor of RLF activity is present, and this inhibitor is directly dependent on
the presence of Cdk activity. If this inhibitor is diluted
away, RLF-M but not RLF-B activity can be detected. In
contrast, when RLF-B activity decays during late interphase, this decay does not require Cdk activity and no
RLF inhibitor becomes activated.
Role of RLF in the Initiation of Replication
6-DMAP-treated extracts lack active RLF and therefore
cannot replicate double-stranded DNA (Blow, 1993
Effect of 6-DMAP on the Licensing System
When 6-DMAP is added to metaphase-arrested Xenopus
extracts, MPF activity is inhibited, and the extracts spontaneously exit from metaphase into interphase without the
need for CaCl2 addition (Zhang and Masui, 1992
To measure RLF-B, RLF-M, and RLF inhibitor activity
when 6-DMAP is added to metaphase-arrested extracts, we
performed serial dilutions of 6-DMAP-treated metaphase
extract using buffer supplemented with an additional 3 mM
6-DMAP (Fig. 8 b). The results of the titration were strikingly similar to those of untreated metaphase extracts
(compare Figs. 8 b and 2 a). RLF activity was not detected at any dilution (Fig. 8 b, filled circles), but an RLF inhibitor was detected at higher concentrations (triangles). Although no RLF-B could be detected at any dilution
(squares), significant RLF-M activity could be detected
once the inhibitor had been diluted away (open circles). It
therefore appears that although the 6-DMAP-treated extracts have morphologically exited from metaphase into
interphase, they still contain the RLF inhibitor normally
present in metaphase extracts.
Since the RLF inhibitor is dependent on the presence of
Cdk activity, we determined whether Cdc2/cyclin B is
present in 6-DMAP-treated extracts. Cyclins A and B are
normally degraded on exit from mitosis. Fig. 9 a shows an
autoradiograph of 35S-labeled cyclin B in extracts released
from metaphase by CaCl2 in the presence of different concentrations of 6-DMAP. Consistent with previous reports
(Felix et al., 1989
RLF is an essential initiation factor that ensures that chromosomal DNA is not re-replicated in a single cell cycle. In
this paper we measure the RLF activity present in Xenopus extracts and show that the ability of extracts to license
chromatin varies throughout the cell cycle. In metaphase
extract, RLF-M but not RLF-B activity can be assayed,
and, in addition, an RLF inhibitor is present. This RLF inhibitor is dependent on the presence of cyclin-dependent
kinases and is stabilized when cyclin B is stabilized by the
protein kinase inhibitor 6-DMAP. RLF activity is rapidly activated upon exit from metaphase, and then decays during late interphase. The decay of RLF activity in late interphase is mainly due to decay of RLF-B activity and is not
associated with reactivation of the RLF inhibitor.
RLF Activity of Xenopus Egg Extracts
Interphase Xenopus extract can license ~100-200 ng
DNA per µl extract for efficient replication. Titration of
RLF-M and RLF-B activity suggested that these two activities were present at approximately equal levels. Chromatin licensed at 100 ng DNA per µl extract could replicate
at rates typical of Xenopus egg extracts. Western blotting
of XMcm3, a component of RLF-M (Chong et al., 1995 Cell Cycle Variation of RLF Activity
RLF activity varies throughout the in vitro cell cycle. In
metaphase, RLF is largely inactive but is rapidly activated
after the metaphase-anaphase transition; during interphase, activity then gradually declines (Blow, 1993 The decline of RLF activity that occurs during interphase causes a different defect than is seen in metaphase
extracts. RLF-M activity remains high, but RLF-B activity
declines. Unlike metaphase extracts, the loss of RLF-B activity during interphase occurs without the appearance
of an RLF inhibitor. At present we cannot be certain
whether the decline of RLF-B activity during interphase is
of physiological relevance or whether it is an artifact of the
cell-free system. However, the decline of RLF-B activity is fairly specific, since extracts retain RLF-M activity and remain competent to replicate previously licensed chromatin
(Blow, 1993 Role of RLF in the Initiation of Replication
The initiation of chromosomal DNA replication is likely
to consist of several steps, including the unwinding of origin DNA, the synthesis of short DNA primers, and the establishment of a mature replication fork. On the basis of
immunofluorescence data showing that 6-DMAP-treated
extracts are able to support low levels of DNA synthesis, it
has been suggested that RLF is not required for the synthesis of DNA primers at replication origins, but it is required for correct assembly of replication forks (Yan and Newport, 1995 The RLF Inhibitor Depends on Cdk Activity
Cdks are key regulators of cell cycle progression (for review see Nigg, 1995 Inhibition of RLF function by Cdks is consistent with
experiments in other organisms that show, when Cdk activity is inhibited in G2 cells, re-replication of chromosomal DNA can occur without cells passing into mitosis.
This effect is plausibly caused by reactivation of RLF and
relicensing of replicated DNA. Re-replication of chromosomal DNA is seen in Saccharomyces pombe cells containing a temperature-sensitive cdc2 gene when they are
shifted to the nonpermissive temperature during G2 (Broek
et al., 1991 However, RLF regulation in the Xenopus system appears somewhat different from that in the other cell types.
First, loss of cytoplasmic RLF during interphase in Xenopus is not dependent on Cdk activity and is not associated
with an RLF inhibitor. Secondly, although relicensing of
DNA is dependent on nuclear envelope permeabilization in both Xenopus and Drosophila cell-free systems (Blow
and Laskey, 1988;
Chong et al., 1996
). The first signal, replication licensing
factor (RLF)1, "licenses" chromosomal DNA by putting
replication origins into an initiation-competent state. The
second signal, S-phase promoting factor (SPF), induces licensed origins to initiate and, in doing so, removes the licence. To achieve precise duplication of chromosomal DNA, the licensing and initiation signals must act on the
DNA sequentially, and never at the same time. This is
achieved in two different ways. First, active RLF cannot
cross the nuclear envelope, so it can only license DNA
when the nuclear envelope has broken down in mitosis
(Blow and Laskey, 1988
; Leno et al., 1992
; Coverley et al.,
1993
; Blow, 1993
); in contrast, SPF can only initiate DNA
replication on licensed DNA within an intact nucleus
(Blow and Watson, 1987
; Newport, 1987
; Sheehan et al., 1988
; Blow and Sleeman, 1990
). Secondly, both activities
are periodic in the cell cycle: RLF is abruptly activated after the metaphase-anaphase transition and decays during
interphase (Blow, 1993
), while SPF activity can only be detected during interphase (Blow and Nurse, 1990
). The spatial separation of RLF and SPF activities is thereby enhanced by a temporal regulation.
; Kubota and Takisawa, 1993
; Vesely et al.,
1994
). The licensing system has been subjected to biochemical and immunological analysis using extracts treated with these kinase inhibitors (Chong et al., 1995
; Kubota et
al., 1995
). RLF activity can be separated chromatographically into two essential components, RLF-M and RLF-B,
both of which are required for licensing (Chong et al.,
1995
). RLF-M has been purified to apparent homogeneity,
and it consists of a complex of all six currently identified
members of the Xenopus MCM/P1 family, XMcm2- XMcm7 (Chong et al., 1995
, 1996; Thömmes, P., Y. Kubota, H. Takisawa, and J.J. Blow, manuscript submitted
for publication). Anti-XMcm3 antibodies coprecipitated
all six Xenopus MCM/P1 proteins in a complex closely resembling the RLF-M complex (Kubota et al., 1995
; Madine et al., 1995a
,b; Thömmes, P., Y. Kubota, H. Takisawa, and J.J. Blow, manuscript submitted for publication) and
depleted the extracts of RLF-M activity (Chong et al.,
1995
).
), due to a failure of the initiation process (Maiti and Sinha, 1992
; Yan et al., 1993
). The proteins
show cell cycle-dependent changes in subnuclear localization, being observed within the nucleus only during late
mitosis and G1 (Hennessy et al., 1990
; Yan et al., 1993
;
Dalton and Whitbread, 1995
). Homologous MCM/P1 genes have been identified in a wide range of eukaryotes including insects, plants, amphibians, and mammals, where they
fall into six related groups designated MCM2-MCM7
(Chong et al., 1996
; Kearsey et al., 1996
). Consistent with
their role in yeast, MCM/P1 proteins in higher eukaryotes
are required for DNA replication (Kimura et al., 1994
;
Todorov et al., 1994
; Chong et al., 1995
; Kubota et al.,
1995
; Madine et al., 1995a
; Treisman et al., 1995
) and behave in accordance with their role as a central component
of the licensing system. They associate tightly with chromatin during late mitosis and G1, but are removed during
replication (Kimura et al., 1994
; Chong et al., 1995
; Kubota et al., 1995
; Madine et al., 1995a
,b; Todorov et al.,
1995
; Coué et al., 1996
). Reassociation of XMcm3 with
chromatin, which is required before an additional round of replication can take place, only occurs after permeabilization or breakdown of the nuclear envelope (Chong et al.,
1995
; Kubota et al., 1995
; Madine et al., 1995a
,b). The licensing of chromatin by RLF-M and RLF-B in Xenopus
requires the presence of the Xenopus origin recognition
complex (ORC) on the chromatin (Rowles et al., 1996
).
Materials and Methods
) and stored in 20-µl aliquots in liquid nitrogen.
Just before use, extract was thawed at room temperature and supplemented with 250 µg/ml cycloheximide, 25 mM phosphocreatine, and 15 µg/ml creatine phosphokinase. Metaphase-arrested extract was released
into interphase by the addition of 0.3 mM CaCl2. To block RLF activation,
metaphase-arrested extracts were supplemented with 3 mM 6-DMAP and
mixed thoroughly before CaCl2 addition (6-DMAP-treated extract). For
replication assays, extract was supplemented with 1/200 vol 10 mCi/ml
[
32P]dATP (Amersham Intl., Little Chalfont, UK). Extract incubations were performed at 23°C.
). Metaphase extract was mixed with an equal volume of
p13suc1-coupled Sepharose (10 µg Suc1/ml beads, washed in LFB1 [40 mM Hepes KOH, pH 8.0, 20 mM K2HPO4/KH2PO4, pH 8.0, 2 mM MgCl2; 1 mM EGTA, 2 mM DTT, 10% sucrose, 1 µg/ml each of leupeptin, pepstatin, and aprotinin] and incubated on a rotating wheel at 4°C for 60 min.
The supernatant was recovered by spinning the bead/extract slurry past a
glass ball wedged in a sawn-off yellow Gilson tip in an Eppendorf tube.
). They were assembled
into chromatin in 6-DMAP-treated extract, to form "6-DMAP chromatin" as follows (Chong et al., 1995
, 1997): 80 µl of a metaphase-arrested extract was supplemented with phosphocreatine, creatine phosphokinase, 3 mM 6-DMAP, and CaCl2 as described above, and then supplemented with 1.3 × 106 demembranated sperm nuclei (~50 ng DNA per µl extract). The mixture was incubated for 12 min at 23°C, and then diluted in 1 ml NIB (50 mM KCl; 50 mM Hepes KOH, pH 7.6; 5 mM MgCl2; 2 mM
DTT, 0.5 mM spermidine 3HCl; 0.15 mM spermine 4HCl; 1 µg/ml each leupeptin, aprotinin, and pepstatin) and underlayered with 100 µl NIB supplemented with 15% sucrose. The chromatin was isolated by centrifugation in a swing-out rotor at 6,200 g for 5 min at 4°C. The pellet was resuspended in 45 µl NIB (to a final concentration of 80 ng DNA/per µl)
and frozen in 5-µl aliquots in liquid nitrogen.
, 1997). Eggs were dejellied in 2%
cysteine solution, washed three times in Barth solution (88 mM NaCl, 2 mM
KCl, 1 mM MgCl2, 15 mM Tris HCl, pH 7.4, 0.5 mM CaCl2), and then incubated for 5-10 min in 100 ml Barth plus 20 µl 10 µg/ml calcium ionophore A23187. The activated eggs were then washed three times in Barth
solution at 23°C and three times in EB (50 mM KCl, 50 mM Hepes KOH,
pH 7.6, 5 mM MgCl2, 2 mM DTT) at 4°C. All additional steps were performed at 4°C or on ice. Eggs were packed by centrifuging at 800 g in a
swing-out rotor for 1 min. Excess buffer and sick eggs, which float to the
surface during this treatment, were then removed. Packed eggs were spincrushed by centrifugation at 10,000 g for 10 min in a swing-out rotor. The
cytoplasmic layer was taken, supplemented to a final concentration of 10 µg/ml cytochalasin B and 15% EDB-S (50 mM KCl, 50 mM Hepes KOH,
pH 7.6, 10% sucrose, 2 mM DTT, 0.4 mM MgCl2, 0.4 mM EGTA, 1 µg/ml
each of pepstatin, leupeptin, and aprotinin), and then diluted with 4 vol of
LFB1 containing 50 mM KCl and 0.5 mM freshly added PMSF. This diluted extract was spun at 60,000 g for 20 min at 4°C in a swing-out rotor.
The supernatant ("licensing factor extract") was decanted, drop frozen in
liquid nitrogen, and stored at
70°C. Since it is fivefold diluted over neat
extract, licensing factor extract is designated a concentration of 0.2×.
, 1997): licensing factor extract was supplemented with 0.075 vol of a
50% PEG solution (50% PEG 6000 [BDH Chemicals Ltd., Poole, UK] in
LFB1) to give a final concentration of 3.5% PEG. Extract was incubated
on ice for 30 min, and then centrifuged at 10,000 g for 10 min in a fixed-
angle rotor. The supernatant was decanted, and residual PEG solution
was removed after an additional brief spin. The pellet was then resuspended in LFB1 at 1/25 of the original volume of licensing factor extract
to give 5× concentrated RLF-B, and was stored in aliquots at
70°C. The
supernatant was supplemented with an additional 0.11 vol of a 50% PEG
solution, giving a final concentration of 9% PEG, incubated on ice, and pelleted as before. The pellet was resuspended in LFB1 at 1/25 of the original volume, to give 5× concentrated RLF-M, and was stored in aliquots
at
70°C.
, 1997). RLF-M produced by PEG precipitation was diluted to 1× in LFB1 plus 100 mM KCl and was adsorbed in batch for 30 min at 4°C onto an equal volume of loose Q
Sepharose (Pharmacia, Uppsala, Sweden) preequilibrated in LFB1 plus
100 mM KCl. The media was then packed into a column, washed with
LFB1 plus 100 mM KCl, and eluted as a step in LFB1 plus 325 mM KCl.
Eluted material was reprecipitated with PEG and resuspended in LFB1.
RLF-Mmeta was produced in the same way as RLF-Minter, except that the
starting material was metaphase-arrested extract.
, 1997). For this experiment, the RLF-B preparation
was also further purified, according to the protocol previously described
for Xenopus ORC (Rowles et al., 1996
; Chong et al., 1997
). Briefly, PEGfractionated RLF-B was bound to phosphocellulose equilibrated in LFB1
plus 150 mM KCl, and then step-eluted in LFB1 plus 500 mM KCl. Eluted
material, containing both RLF-B and Xenopus ORC activity, was precipitated with 40% ammonium sulfate, and the pellet was resuspended in
LFB1 for assay.
Fig. 4.
Evidence for a Cdk-dependent RLF inhibitor.
(a) Metaphase-arrested extracts were depleted of Cdks with an
equal volume of p13suc1-coupled Sepharose. Depleted extract was
subjected to serial dilution, and then mixed with an equal volume
of either buffer (
), crude RLF-B (
), crude RLF-M (
),
or a mixture of crude RLF-B and crude RLF-M (
). B, buffer
in place of diluted extract. Unlicensed chromatin was incubated
in the mixture (12 ng DNA/µl) for 15 min at 23°C to allow licensing, and then was transferred to 2.5 vol 6-DMAP-treated extract
containing [
32P]dATP. The total DNA synthesized after further
incubation for 90 min at 23°C was measured. (b) Preincubations
were performed for 15 min with different combinations of partially purified RLF-B, purified RLF-M, and Cdc2/cyclin B (MPF).
Unlicensed chromatin was then added, and the incubation was
continued for an additional 15 min to allow licensing to occur.
Samples were then transferred to 6-DMAP-treated extract containing [
32P]dATP, and the total DNA synthesized after further
incubation for 90 min at 23°C was measured.
[View Larger Version of this Image (26K GIF file)]
,
1997). 2-µl samples to be assayed were incubated with 0.3 µl 6-DMAP
chromatin (80 ng DNA/µl) for 15 min for the "licensing reaction." In assays for individual components, the 2-µl samples were typically composed
of 1-µl fraction of interest, plus 1-µl PEG-cut RLF-B and/or RLF-M diluted to 0.5× in LFB1, plus 50 mM KCl and 2.5 mM Mg-ATP. For extract
dilution experiments, dilutions were performed with LFB1 plus 50 mM KCl.
Once the licensing reaction was finished, 5.7 µl 6-DMAP-treated extract
containing cycloheximide, phosphocreatine, creatine phosphokinase,
6-DMAP, CaCl2 and [
32P]dATP were added. The incubation was continued for an additional 90 min at 23°C (the "replication reaction"). Replication reactions were terminated by the addition of 150 µl Stop-C (20 mM
Tris HCl, pH 7.5, 5 mM EDTA, 0.5% SDS) plus 0.2 µg/ml proteinase K
and incubated at 37°C for 15 min. TCA precipitation was performed as described (Blow and Laskey, 1986
; Chong et al., 1997
), and the total DNA synthesized (in ng DNA/µl) was calculated, assuming 50 µM endogenous dATP in the extract (Blow and Laskey, 1986
). Bromodeoxyuridine triphosphate (BrdUTP) density substitution was performed as described (Blow
and Laskey, 1986
).
). It was added to Xenopus extracts at 150 nM, an
amount sufficient to inhibit DNA replication by >95% (Strausfeld et al.,
1994
). Purified Cdc2/cyclin B was purchased from Upstate Biotechnology
Inc.(Lake Placid, NY) and was used in the licensing reaction at a final concentration of 1.25 ng/µl.
, 1997). Chromatin pellets were resuspended in gel loading buffer, electrophoresed, and Western
blotted by standard techniques. Anti-XMcm3 rabbit polyclonal antibodies
were raised against recombinant GST-tagged XMcm3 (amino acids 423-
798) produced from the construct described by Madine et al. (1995a).
Results
). To correlate the amount
of RLF-M activity in Xenopus extracts with the amount of
XMcm3 protein, we performed quantitative Western blotting on extracts and on chromatin assembled in these extracts (Fig. 1 a). An estimate of 75-150 µg XMcm3 protein per ml Xenopus extract (0.75-1.5 µM) was provided by a
number of duplicate blots (e.g., Fig. 1 a, lanes 1 and 2 and
17-19). Fig. 1 a also quantifies the association of XMcm3
with chromatin. Demembranated Xenopus sperm nuclei
(Fig. 1, lanes 10-16) or chromatin previously assembled in
6-DMAP-treated extract (6-DMAP chromatin; Fig. 1,
lanes 3-9) were briefly incubated with serial dilutions of
Xenopus egg extract. Chromatin was then isolated by centrifugation through sucrose and immunoblotted for
XMcm3. As more extract was incubated with a fixed quantity of chromatin, the amount of chromatin-associated XMcm3 increased. Only at the highest concentrations of
DNA (6-12 ng DNA per µl extract) was there any sign of
the chromatin becoming saturated with XMcm3. Comparison with the recombinant XMcm3 standard (Fig. 1, lanes 1 and 2) suggests that the extract can assemble in excess of
30 ng XMcm3 onto 300 ng DNA, equivalent to one
XMcm3 molecule bound to every 1.5 kb of DNA on average. Previous studies have estimated that the replicon size
of the early Xenopus embryo is 10-20 kb (Blow and Watson, 1987
; Mahbubani et al., 1992
; Hyrien and Méchali,
1993
), suggesting that there may be >10 XMcm3 molecules bound to each replicon.
Fig. 1.
Effect of serial dilution on XMcm3 assembled onto
chromatin and rate of replication. Interphase Xenopus extract
was subjected to serial dilution, and 30-µl aliquots (or 60-µl for a,
lanes 3 and 10) were incubated with equal quantities (360 ng
DNA) of unlicensed chromatin (a, lanes 3-9; b) or sperm nuclei
(a, lanes 10-16) for 15 min at 23°C. (a) Equal quantities of chromatin (~300 ng DNA) were isolated by centrifugation through
sucrose, run on a 7.5% polyacrylamide gel, and immunoblotted
with an anti-XMcm3 antibody. Dilutions were: (lanes 3 and 10)
undiluted (60 µl); (lanes 4 and 11) undiluted (30 µl); (lanes 5 and
12) 1/2; (lanes 6 and 13) 1/4; (lanes 7 and 14) 1/8; (lanes 8 and 15)
1/16; (lanes 9 and 16) 1/32. Samples of recombinant GST-tagged
XMcm3 (lane 1, 40 ng; lane 2, 20 ng) and interphase Xenopus egg
extract (lane 17, 0.125 µl; lane 18, 0.25 µl; lane 19, 0.5 µl) were
blotted in parallel. (b) Samples were transferred to 6-DMAP-
treated extract containing [32P]dATP. The total DNA synthesized at different times during an incubation at 23°C was measured. Samples corresponding to the blots in a are indicated with
identical symbols.
[View Larger Version of this Image (40K GIF file)]
32P]dATP. At different times
after transfer, the amount of DNA synthesis was measured. When incubated in extract at concentrations of
above 1/4-1/8 (corresponding to 100-200 ng DNA per µl
neat extract), chromatin was efficiently licensed, leading
to almost complete replication after an additional 90-min
incubation in 6-DMAP-treated extract. This is similar to
replication rates seen in untreated extract. When licensed
with lower amounts of extract (1/16 dilution and below),
the chromatin replicated at a lower rate, as though only a
fraction of the replication origins had initiated. Comparison of Fig. 1 a and 1 b suggests that maximal replication
rates were still seen with subsaturating quantities of
XMcm3 assembled onto the chromatin.
). RLF activity is low in such metaphase-
arrested extracts, but it rapidly increases upon CaCl2 addition (Blow, 1993
). We first determined the quantity of
RLF-B and RLF-M activity present in metaphase-arrested
and in interphase egg extracts (Fig. 2). Chromatin was licensed with serial dilutions of extract and transferred to
6-DMAP-treated extract, and the total DNA synthesized
over a 90-min incubation was measured (Fig. 2, filled circles). Consistent with the time courses shown in Fig. 1 b,
interphase extract efficiently licensed DNA synthesis after
dilution as low as 1/4-1/8 (Fig. 2 b). In contrast, no RLF activity was provided by the metaphase-arrested extract
(Fig. 2 a).
Fig. 2.
RLF activity in serial dilutions of metaphase and interphase extract. Metaphase arrested (a) or interphase (b) Xenopus
extracts were subjected to serial dilution, and then mixed with an
equal volume of either buffer (
), crude RLF-B (
), crude
RLF-M (
), or a mixture of crude RLF-B and crude RLF-M
(
). B, buffer in place of diluted extract. Chromatin was incubated in the mixture (12 ng DNA/µl) for 15 min at 23°C to allow
licensing, and then was transferred to 2.5 vol 6-DMAP-treated
extract containing [
32P]dATP. The total DNA synthesized after
further incubation for 90 min at 23°C was measured.
[View Larger Version of this Image (25K GIF file)]
). RLF levels in metaphase were low, but, within 15 min after release from the metaphase arrest by CaCl2 addition, maximum levels of total RLF activity were observed (Fig. 3 a, filled circles). Total RLF activity decayed
over 1-2 h (Fig. 3 b, filled circles), although the kinetics of
the decay varied from extract to extract. This decay of total RLF activity was almost entirely due to decay of RLF-B
activity (Fig. 3 b, open squares), while RLF-M activity remained fairly constant throughout the cell cycle (open circles).
Fig. 3.
Time course of RLF activity during the in vitro cell cycle. Interphase extract (b) or metaphase extract released into interphase with CaCl2 (a) was incubated at 23°C for various times.
Extract was then diluted eightfold and mixed with an equal volume of either buffer (
), crude RLF-B (
), or crude RLF-M
(
). Unlicensed chromatin was incubated in the mixture (12 ng
DNA/µl) for 15 min at 23°C to allow licensing, and then was transferred to 2.5 vol 6-DMAP-treated extract containing [
32P]dATP.
The total DNA synthesized after further incubation for 90 min at
23°C was measured. M, metaphase-arrested extract that was not
released with CaCl2.
[View Larger Version of this Image (23K GIF file)]
; Gautier et al., 1990
) or Cdc2/cyclin A
(Luca et al., 1991
; Roy et al., 1991
). We have previously
shown that the addition of high levels of cyclin A to interphase Xenopus extracts inactivates RLF (Blow, 1993
) and
blocks DNA replication (Strausfeld et al., 1996
). We therefore examined whether the RLF inhibitor present in
metaphase extracts was dependent on Cdk activity. Cyclin-dependent kinases can be affinity depleted from Xenopus extracts using p13suc1 coupled to Sepharose beads
(Dunphy et al., 1988
). When p13suc1 depletion is performed on metaphase-arrested Xenopus extracts, subsequent DNA synthesis is blocked (Blow and Nurse, 1990
).
Fig. 4 a (filled circles) shows that p13suc1 depleted metaphase extracts lack RLF activity. However, unlike the undepleted extracts (Fig. 2 a), RLF-M activity could be detected at the highest extract concentrations (Fig. 4 a, open
circles). Thus, no RLF inhibitor could be detected in these
extracts (Fig. 4 a, triangles), and the lack of RLF activity
was apparently due only to a lack of RLF-B activity (Fig. 4 a,
squares).
). To demonstrate a direct role for Cdc2 in inhibiting
RLF, we prepared purified RLF-M (Chong et al., 1995
)
and partially purified RLF-B (Rowles et al., 1996
; Chong
et al., 1997
). Incubation of recombinant Cdc2/cyclin B with these purified fractions caused strong inhibition of licensing (Fig. 4 b). This result strongly suggests that Cdc2/cyclin
B can directly inhibit the RLF system. Consistent with this
idea, we show below that stabilization of the RLF inhibitor
correlates well with stabilization of cyclin B.
; Thömmes, P., Y. Kubota, H. Takisawa, and J.J. Blow, manuscript submitted for publication), is phosphorylated and migrates slower on SDS gels
(Fig. 5 b, lanes 1 and 3; Coué et al., 1996
). This phosphorylation can be blocked with phosphatase inhibitor okadaic
acid (Fig. 5 b, lane 7; Coué et al., 1996
), but is lost during
the purification of the RLF-Mmeta fraction (Fig. 5 b, lane 2).
However, the presence of the slow migrating form of
XMcm4 does not correlate with the presence of the RLF
inhibitor, since XMcm4 is fast migrating in 6-DMAP-
treated extract (Fig. 5, lane 6) where the RLF inhibitor is
present (see below). Furthermore, XMcm4 is still slow migrating in metaphase extracts diluted to 1/8 and incubated
with RLF-B (Fig. 5 b, lanes 8 and 9), under which conditions the inhibitor has been titrated away (Fig. 2 a). Conversely, most of the XMcm4 is fast migrating in Suc1-
depleted extracts (Fig. 5 b, lane 5) where the inhibitor is
absent. Thus, we can find no evidence that mitotic phosphorylation of RLF-M inhibits its activity.
Fig. 5.
Features of RLF-M prepared from metaphase or interphase extract. (a) Unlicensed chromatin was incubated for 15 min
at 23°C with interphase extract, metaphase extract, or mixtures of
RLF-B and RLF-Mmeta (RLF-M prepared from metaphase extract)
and RLF-Minter (RLF-M prepared from interphase extract). Samples were then transferred to 6-DMAP-treated extract containing
[32P]dATP. The total DNA synthesized after further incubation for 90 min at 23°C was measured. (b) Protein samples electrophoresed on a 7.5% polyacrylamide gel and immunoblotted with an
anti-XMcm4 antibody. Samples: 1, metaphase extract; 2, RLFMmeta; 3, interphase extract; 4, RLF-Minter; 5, metaphase extract
depleted with p13suc1-Sepharose; 6, metaphase extract treated
with 3 mM 6-DMAP before CaCl2 release (6-DMAP-treated extract); 7, metaphase extract treated with 2 µM okadaic acid before
CaCl2 release; 8, metaphase extract diluted eightfold and incubated for 15 min at 23°C; 9, metaphase extract diluted eightfold,
mixed with crude RLF-B, and incubated for 15 min at 23°C.
[View Larger Version of this Image (29K GIF file)]
), cycloheximide only slightly delayed the decay of
RLF that normally occurs during interphase (Fig. 6 a, halffilled diamonds). In contrast to cyclins A and B, cyclin E
remains at approximately constant levels throughout the
early embryonic cell cycles (Rempel et al., 1995
; Chevalier et al., 1996
). Treatment of Xenopus extracts with the Cdk2
inhibitor Cip1 inhibits the SPF activity of Cdk2/cyclin E
and thus blocks the initiation of DNA replication (Strausfeld et al., 1994
; Jackson et al., 1995
). However, addition of
Cip1 to cycloheximide-treated extracts did not stabilize
RLF activity in the cell cycle (Fig. 6 a, filled diamonds).
Similarly, affinity depletion of Cdks from interphase extract using p13suc1-coupled Sepharose did not stabilize
RLF activity (data not shown). These results suggest that
the decay of RLF activity during interphase is independent of Cdk activity.
Fig. 6.
Effect of cycloheximide and Cip1 of RLF stability.
(a) Metaphase extract was released into interphase with CaCl2 either untreated (control) or in the presence of 100 µg/ml cycloheximide (+ CHX), or 100 µg/ml cycloheximide plus 150 nM p21Cip1
(+ CHX + Cip1). At the indicated times, samples were diluted eightfold and incubated for 15 min with unlicensed chromatin. Samples were then transferred to 6-DMAP-treated extract containing [32P]dATP. The total DNA synthesized after further incubation for 90 min at 23°C was measured. (b) Interphase extract
was incubated at 23°C for 2 h to allow RLF activity to decay. The
extract was subjected to serial dilution, and then mixed with an
equal volume of either buffer (
), crude RLF-B (
), crude
RLF-M (
), or a mixture of crude RLF-B and crude RLF-M
(). B, buffer in place of diluted extract. Unlicensed chromatin was incubated in the mixture (12 ng DNA/µl) for 15 min at
23°C to allow licensing, and then was transferred to 2.5 vol
6-DMAP-treated extract containing [
32P]dATP. The total
DNA synthesized after further incubation for 90 min at 23°C was
measured.
[View Larger Version of this Image (27K GIF file)]
). Despite this fact, they are still competent to support complementary strand synthesis on single-stranded DNA and to
elongate replication forks stalled with aphidicolin (Blow,
1993
). This suggests that RLF activity is required for the
initiation, but not the elongation, of replication forks. However, Yan and Newport (1995)
have suggested on the basis of immunofluorescence data that 6-DMAP-treated extracts are able to support the synthesis of DNA primers
at replication origins, but that these primers are not correctly processed into functional replication forks. We
therefore analyzed the residual DNA synthesis seen in
6-DMAP-treated extracts by alkaline gel electrophoresis (Fig. 7 a) and BrdUTP density substitution (Fig. 7, b and
c). 6-DMAP treatment of the metaphase-arrested extract
inhibited subsequent DNA synthesis by 95%. However,
BrdU density substitution showed that all of the residual
DNA synthesized in the 6-DMAP-treated extract (Fig. 7 b)
was of the same density as DNA synthesized in untreated
extract (Fig. 7 c), as expected of fully replicated heavy/light
DNA produced by semiconservative replication. This suggests that a small number of replicons escape the 6-DMAP
inhibition and produce relatively large tracts of fully replicated DNA. This conclusion was confirmed by analyzing
nascent strand length by alkaline agarose gel electrophoresis (Fig. 7 a). In both 6-DMAP-treated (lane 1) and
untreated extract (lane 9), virtually all of the nascent DNA
was in the form of high molecular weight DNA (>23,000 nt in size). In contrast, when replication forks are stalled
close to the origin by treating extracts with aphidicolin,
most of the nascent DNA is observed in a broad smear of
between 100 and 300 nt in length (Fig. 7 a, lanes 5-8).
These results strongly suggest that 6-DMAP blocks the
initiation of DNA replication at a stage before significant
primer synthesis has occurred. The few replication forks
that do initiate in 6-DMAP-treated extracts then proceed to synthesize extensive stretches of nascent DNA. No low
molecular weight products are observed when chromatin is
preincubated with RLF-B, RLF-M, or a mixture of both
before incubation in 6-DMAP-treated extract (Fig. 7 a, lanes
3-5), suggesting that both of these activities are required
for efficient initiation of DNA replication in 6-DMAP- treated extracts.
Fig. 7.
Analysis of the stage of replication blocked in 6-DMAP-
treated extracts. (a) Alkaline agarose gel of nascent DNA.
(Lanes 1-4) Chromatin was licensed for 15 min with either buffer
(lane 1), RLF-B (lane 2), RLF-M (lane 3), or RLF-B plus RLF-M
(lane 4), transferred to 6-DMAP-treated extract containing
[32P]dATP, and incubated for 90 min. (Lanes 5-9) Sperm nuclei
was incubated for 90 min in interphase Xenopus extract containing [
32P]dATP and various concentrations of aphidicolin (lane 5,
30 µg/ml; lane 6, 20 µg/ml; lane 7, 10 µg/ml; lane 8, 5 µg/ml; lane 9,
no aphidicolin). To compensate for the much higher incorporation of 32P in the reactions, the total DNA loaded in lane 4 was reduced to 20%, and the total DNA in lane 9 was reduced to 10%.
(b and c) BrdUTP density substitution of nascent DNA synthesized after incubation of sperm nuclei for 90 min in untreated (c)
or 6-DMAP-treated extract (b).
[View Larger Version of this Image (59K GIF file)]
; Blow,
1993
). These 6-DMAP-treated extracts assemble DNA
into normal nuclei and, with the exception of RLF, contain
all the activities required for complete chromosome replication (Blow, 1993
). Similar results have been reported for
two other kinase inhibitors: staurosporine (Blow, 1993
;
Kubota and Takisawa, 1993
) and olomoucine (Vesely et al.,
1994
). For all these inhibitors, there is a good correlation
between the inhibition of MPF activity (as judged by spontaneous assembly of interphase nuclei) and the inhibition
of RLF activity. To cause inhibition of the licensing system, the kinase inhibitors must be added to Xenopus extracts before exit from metaphase arrest (Blow, 1993
; Kubota and Takisawa, 1993
). This suggests that the protein
kinase inhibitors block the RLF activation that normally
occurs upon exit from metaphase, rather than inhibiting
the action of active RLF. This conclusion is confirmed in
Fig. 8 a. Active RLF-M and RLF-B fractions prepared from interphase Xenopus extract by differential PEG precipitation were resuspended in buffers plus or minus 3 mM
6-DMAP. This concentration of 6-DMAP is sufficient to
block RLF activation in metaphase extracts (Blow, 1993
).
Efficient licensing of DNA replication required both RLFB and RLF-M, but it was not significantly affected by the
presence of 6-DMAP.
Fig. 8.
Effect of 6-DMAP on RLF activity. (a) Unlicensed
chromatin was incubated for 15 min plus or minus 3 mM 6-DMAP
with crude RLF-B, crude RLF-M, or a mixture of the two. Samples were then transferred to 6-DMAP-treated extract containing [32P]dATP. The total DNA synthesized after further incubation for 90 min at 23°C was measured. (b) Metaphase-arrested
Xenopus extract was treated with 3 mM 6-DMAP and 0.3 mM
CaCl2, followed by serial dilution in buffer containing 3 mM
6-DMAP. Samples were then mixed with equal volumes of either
buffer (
), crude RLF-B (
), crude RLF-M (
), or a mixture of crude RLF-B and crude RLF-M (
). B, buffer in place of
diluted extract. Chromatin was incubated in the mixture (12 ng
DNA/µl) for 15 min at 23°C to allow licensing, and then was transferred to 2.5 vol 6-DMAP-treated extract containing [
32P]dATP.
The total DNA synthesized after further incubation for 90 min at
23°C was measured.
[View Larger Version of this Image (33K GIF file)]
; Luca and Ruderman, 1989
), 6-DMAP at
concentrations of above 2 mM blocked cyclin B degradation that normally occurs after CaCl2 addition (Fig. 9 a,
lanes 5-8). However, these extracts all entered interphase as judged by the assembly of DNA into interphase nuclei,
despite the stability of cyclin B at higher concentrations
(Fig. 9 b; +CaCl2). Above 1 mM, 6-DMAP could inhibit
MPF and cause metaphase extract to spontaneously enter
interphase (as judged by the assembly of DNA into interphase nuclei) in the absence of added CaCl2 (Fig. 9 b,
CaCl2; Zhang and Masui, 1992
). The concentrations of
6-DMAP just sufficient to block cyclin B degradation correlated well with concentrations at which RLF became stably inhibited (Fig. 9 c). It therefore seems most likely that
while high levels of Cdc2 kinase are required to trigger cyclin B degradation (Felix et al., 1989
), lower levels of kinase sufficient to inhibit RLF activity are still present in
extracts treated with 3 mM 6-DMAP.
Fig. 9.
Titration of 6-DMAP into metaphase extract. (a) Metaphase extract was labeled for 20 min with [35S]methionine, and
then was supplemented with 100 µg/ml cycloheximide and the indicated concentrations of 6-DMAP. Extract was then further supplemented plus (lanes 2-8) or minus (lane 1) 0.3 mM CaCl2. After incubation at 23°C for 30 min, cyclin-Cdk complexes were collected on p13suc1 beads, electrophoresed on polyacrylamide
gels, and autoradiographed. The migration of cyclin B is indicated. (b) Metaphase extract was supplemented with sperm nuclei (3 ng DNA/µl) and various concentrations of 6-DMAP, plus
(lower panel) or minus (upper panel) 0.3 mM CaCl2. The morphology of chromatin after 1.5 h at 23°C was assessed by microscopy: (), condensed chromosomes; (
), interphase nuclei; (
),
partial nuclear assembly. (c) Metaphase extract was supplemented with sperm nuclei (3 ng DNA/µl), 0.3 mM CaCl2,
[
32P]dATP, and various concentrations of 6-DMAP. The total
DNA synthesized after 3 h at 23°C was measured.
[View Larger Version of this Image (23K GIF file)]
Discussion
), suggests that, at 100 ng DNA per µl extract, one XMcm3
molecule is assembled on average onto every 5-10 kb
DNA. This represents on average one to three XMcm3
molecules per replicon. However, at lower concentrations
of DNA, more XMcm3 can be assembled onto chromatin
(up to one XMcm3 molecule per 1.5 kb, or 10 XMcm3
molecules per replicon) without significantly affecting the
overall replication rate. This higher density of XMcm3 is
more likely to reflect conditions in vivo, since up to the
midblastula transition (when zygotic transcription starts),
the concentration of chromosomal DNA is <25 ng/µl. A
similar excess of Mcm3 over replication origins has been
reported in human cells (Burkhart et al., 1995
) and budding yeast (Lei et al., 1996
).
). We
have analyzed some of the basic causes of this variation. In
metaphase an RLF inhibitor was present that inhibited licensing by exogenous RLF. When this metaphase extract
was diluted so that the inhibitor no longer interfered with
the licensing assay, levels of RLF-M were detected comparable with those seen in interphase extract. In contrast, no
RLF-B activity was detected. Partially purified RLF-M
prepared from metaphase extracts (RLF-Mmeta) was also
fully active. XMcm4, a component of RLF-M (Thömmes, P., Y. Kubota, H. Takisawa, and J.J. Blow, manuscript
submitted for publication), was phosphorylated during
metaphase (Coué et al., 1996
), but this phosphorylated
XMcm4 was still associated with active RLF-M. The simplest explanation for these results is that the target of the
RLF inhibitor is RLF-B, not RLF-M, although we cannot
completely rule out an inhibitory modification to RLF-M that we have been unable to stabilize.
).
). We have examined this point in detail to
determine whether RLF is required for the synthesis of
DNA primers early in initiation. Alkaline gel electrophoresis of nascent strands showed that in 6-DMAP-
treated extracts lacking RLF, no short DNA primers could
be observed. In contrast, short nascent strands could be
readily detected when replication forks were blocked by
the polymerase inhibitor aphidicolin. Both RLF-B and
RLF-M were required for efficient DNA replication, but
in the presence of both these activities, nascent strands
were rapidly extended into high molecular weight DNA.
We therefore conclude that RLF is required for the initiation of DNA replication before the synthesis of DNA
primers.
). MPF is an activity present in metaphase cells capable of inducing entry into mitosis. It consists of a Cdc2/cyclin B heterodimer (Dunphy et al., 1988
;
Gautier et al., 1988
, 1990; Labbé et al., 1989a
,b). Cdc2/cyclin A, which is also active at this stage of the cell cycle,
can also generate MPF activity (Luca et al., 1991
; Roy et al.,
1991
; Strausfeld et al., 1996
). The RLF inhibitor present in
metaphase extracts appears to be directly dependent on
Cdks active at this time. When Cdks were affinity depleted
from metaphase extracts using p13suc1, the RLF inhibitor
was removed. Inhibition of RLF function by kinase inhibitors such as 6-DMAP caused persistence into interphase state of the RLF inhibitor normally only present during
metaphase. This also correlated with inhibition of MPF activity (Blow, 1993
; Kubota and Takisawa, 1993
; Vesely et al.,
1994
) and stabilization of cyclin B. Active RLF was also
inhibited by the activation of Cdks. When recombinant
cyclin A was added to interphase extract, RLF activity
abruptly decayed (Blow, 1993
). Furthermore, purified Cdc2/cyclin B directly inhibited licensing activity provided
by purified RLF-M and partially purified RLF-B. These
results strongly suggest a direct inhibition of the licensing
system by Cdc2 kinase. Although MCM/P1 polypeptides
are expected to be good substrates for this kinase (Coué
et al., 1996
), the results discussed above suggest that inhibition of licensing is unlikely to be mediated by inhibition
of RLF-M function. Instead, RLF-B appears to be a more
likely target of Cdk inhibition.
). Repeated rounds of DNA synthesis are seen
in S. pombe cells lacking the cdc13 (cyclin B) gene (Hayles
et al., 1994
) or cells that overexpress the Cdc2 inhibitor
Rum1 (Moreno and Nurse, 1994
). In S. cerevisiae, the establishment of a pre-replicative footprint over origins of
replication that probably corresponds to the licensed state can only occur in late mitosis and G1 when Cdk levels are
low (Diffley et al., 1994
; Piatti et al., 1996
). Furthermore,
inhibition of Cdks in G2/M-phase S. cerevisiae cells is sufficient to induce a pre-replicative footprint over origins of
replication (Dahmann et al., 1995
). Treatment of mammalian cells with certain protein kinases that inhibit Cdks was
sufficient to allow re-replication of DNA in the absence of
mitosis (Usui et al., 1991
). Similar treatments allow mammalian G2 nuclei to become relicensed for replication in
Xenopus egg extracts (Coverley et al., 1996
). A requirement for Cdks to prevent re-replication of DNA late in the
cell cycle therefore appears widespread throughout the
eukaryotic kingdom and is plausibly mediated by an RLF
inhibitor that prevents relicensing of replicated DNA. This
is consistent with the results presented here demonstrating
a Cdk-dependent RLF inhibitor present in metaphase extract.
; Crevel and Cotterill, 1991
), this does
not seem necessary in other cell types. One attractive explanation for these apparent differences is that in Xenopus, relicensing of DNA in replicated nuclei is prevented
by a Cdk-dependent inhibitor that is active only within the
nucleus. Permeabilization of the nuclear envelope would
then release the inhibitor and permit relicensing of the DNA by RLF-B and RLF-M.
P. Thömmes' present address is Glaxo Wellcome Virology Unit, Gunnels Wood Road, Stevenage, Herts SG1 2NY, United Kingdom.
.
J.J. Blow is a Lister Institute Research fellow. We thank Noel Lowndes and John Diffley for comments on the manuscript.BrdUTP, bromodeoxyuridine triphosphate; Cdk, cyclin-dependent kinase; 6-DMAP, 6-dimethyl aminopurine; GST, glutathione-S-transferase; MPF, maturation promoting factor; ORC, origin recognition complex; PEG, polyethylene glycol; RLF, replication licensing factor; SPF, S-phase promoting factor.