From the Laboratoire de Génétique Moléculaire, Ecole Normale Supérieure, 46 rue d'Ulm, 75230 Paris Cedex 05, France
Received for publication, January 11, 2001, and in revised form, March 5, 2001
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ABSTRACT |
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DNA replication origins are located at random
with respect to DNA sequence in Xenopus early embryos and
on DNA replicated in Xenopus egg extracts. We have recently
shown that origins fire throughout the S phase in Xenopus
egg extracts. To study the temporal regulation of origin firing, we
have analyzed origin activation in sperm nuclei treated with the DNA
polymerase inhibitor aphidicolin. Sperm chromatin was incubated in
Xenopus egg extracts in the presence of aphidicolin and
transferred to a fresh extract, and digoxigenin-dUTP and biotin-dUTP
were added at various times after aphidicolin release to selectively
label early and late replicating DNA. Molecular combing analysis of
single DNA fibers showed that only a fraction of potential origins were
able to initiate in the presence of aphidicolin. After release from
aphidicolin, the remaining origins fired asynchronously throughout the
S phase. Therefore, initiation during the S phase depends on the normal
progression of replication forks assembled at earlier activated
origins. Caffeine, an inhibitor of the checkpoint kinases ATR and ATM,
did not relieve the aphidicolin-induced block to origin firing. We
conclude that a caffeine-insensitive intra-S phase checkpoint regulates
origin activation when DNA synthesis is inhibited in
Xenopus egg extracts.
DNA replication initiates from multiple origins in eukaryotes (1).
In most cells, replication initiates at specific sequences, and
distinct origins fire at different times in the S phase (2, 3). We are
interested in developmental transitions in origin usage and the
replication timing program in Xenopus. Studies using Drosophila and Xenopus have provided information
about the regulation of origin spacing and specificity. In the rapidly
cleaving early embryos of both organisms, origins are closely spaced
and lack any detectable sequence specificity (4-6). After the
midblastula transition
(MBT),1 when a critical
number of nuclei accumulate in the embryo (7), cell cycles become
slower, zygotic transcription is activated, and fewer but more specific
origins are used (8, 9). This restriction occurs in part by
inactivation of origins located within transcription units. Further
restriction of origin usage and specification of replication fork
pausing sites occur later in development (10).
Treatments that block the progression of the replication forks from the
early firing origins inhibit the firing of the late origins in yeast
and mammals. In Saccharomyces cerevisiae, the block to late
origin firing (also called the intra-S phase checkpoint) requires the
cell cycle checkpoint genes MEC1 and RAD53 (11, 12). In mammals, caffeine and 2-aminopurine were recently shown to
interfere with an intra-S checkpoint that prevents replication initiation within late replicating chromosomal domains in cells treated
with aphidicolin, an inhibitor of DNA polymerases (13). Caffeine and
2-aminopurine have been previously reported to abrogate a distinct,
so-called S/M checkpoint that normally prevents cells arrested with
inhibitors of DNA synthesis to enter mitosis (14). Targets of caffeine
are the checkpoint kinases ATM (ataxia-telangectasia-mutated) and ATR
(ATM and Rad3 related), which are the vertebrate homologues of Mec1
(S. cerevisiae), Rad3 (Schizosaccharomyces
pombe), and Tel1 (S. pombe or S. cerevisiae)
(15-17). In S. cerevisiae, the Mec1/Rad53 pathway also
contributes to maintain the inactivity of "dormant" origins, and it
has been speculated that the inactivation of replication origins in
Drosophila and Xenopus may be due to the
acquisition of an intra-S checkpoint at the MBT (18). Therefore, it
would be interesting to determine whether there is an intra-S checkpoint in Xenopus egg extracts, which are known to
initiate replication without detectable sequence specificity
(19-21).
It is not clear whether the acquisition of a replication timing program
is concomitant with the developmental restriction of origin usage.
Electron microscopy analysis of replicating DNA from
Drosophila embryos suggested that origins fire synchronously at the syncitial blastoderm stage but become asynchronous at
cellularization (MBT) (4, 22). Different results were obtained when the
replication of plasmid DNA or sperm nuclei in Xenopus egg
extracts was examined. Although small (<10 kb) plasmid molecules
support a single, randomly located initiation event in egg extracts
(19, 20), large plasmids undergo multiple initiation events, as shown
by two-dimensional gel and electron microscopy analyses (21). In
contrast to Drosophila embryos, these multiple initiation
events are not synchronous but occur throughout the S phase, despite
the dwindling length of the unreplicated DNA remaining available for
initiation as replication approaches completion (21). Similar
conclusions were obtained when the replication of sperm nuclei was
examined by molecular combing (23). These studies showed that the
frequency of initiation actually increases during the S phase, ensuring rapid and complete genome replication despite the lack of sequence specificity for initiation.
The evidence for asynchronous initiation in Xenopus egg
extracts raises the question of how the time of activation of any individual origin is determined. A simple model would be that potential
origins are abundant and equally competent to fire at any time during
the S phase. The frequency of initiation during the S phase would
increase because of the progressive concentration of a factor required
to trigger initiation at individual origins into nuclei (21).
Alternatively, a more complex temporal regulation could distinguish
"early" and "late" firing origins.
To gain further insight into the regulation of the S phase in
Xenopus egg extracts, we have examined the effect of
aphidicolin on origin usage in sperm nuclei replicating in egg
extracts. In the absence of an intra-S checkpoint, aphidicolin would
block fork progression but would not restrict the activation of all potential origins. Upon release from the inhibitor, elongation would
synchronously resume at all origins. However, we find that during the
aphidicolin block only a fraction of origins can fire, and upon release
from the block, the remaining origins still fire asynchronously. This
shows that an intra-S checkpoint is functional in Xenopus
egg extracts and argues against a regulation of origin firing solely
through the concentration of some origin-triggering factor into nuclei.
The potential implications of this checkpoint in ensuring rapid and
complete genome replication by controlling the number of simultaneously
active forks and its participation in a replication timing program are discussed.
Replication of Sperm Nuclei in Xenopus Egg
Extracts--
Replication-competent extracts from unfertilized
Xenopus eggs were prepared as described (24). Samples for
molecular combing analysis were prepared as follows. For control
reactions, sperm nuclei (final concentration, 2000/µl) were incubated
in fresh extracts in the presence of cycloheximide (250 µg/ml),
energy mix (7.5 mM creatine phosphate, 1 mM
ATP, 0.1 mM EGTA, pH 7.7, 1 mM
MgCl2), 20 µM digoxigenin-dUTP (added at
t = 0), and 20 µM biotin-dUTP (added at
t = 25, 40, and 50 min) (Roche Molecular Biochemicals).
Replication was allowed to continue for 2 h. For the aphidicolin
block and release experiments, sperm nuclei (final concentration,
2000/µl) were incubated in fresh extracts in the presence of
aphidicolin (100 µg/ml), energy mix, and cycloheximide. After 2 h nuclei were resuspended in 1 ml of washing buffer (50 mM
KCl, 50 mM Hepes KOH, pH 7.5, 5 mM
MgCl2, 1 mM dithiothreitol), pelleted at
1000 × g for 2 min, resuspended, and pelleted again (20). Washed nuclei were then added to fresh extract supplemented with
cycloheximide and energy mix and were allowed to continue replication
for 2 h in the presence of 20 µM digoxigenin-dUTP (added at t = 0) and 20 µM biotin-dUTP
(added at t = 10, 20, and 30 min) (Roche Molecular
Biochemicals). The kinetics of entry into the S phase (see Fig.
1A) was monitored by direct fluorescence microscopy of the
same nuclei in the same extracts supplemented with 20 µM
rhodamin-dUTP (Roche Molecular Biochemicals).
Molecular Combing and Detection by Fluorescent
Antibodies--
DNA was extracted and combed as described (23, 25).
The incorporated digoxigenin and biotin labels were simultaneously detected with fluorescent antibodies with FITC for the digoxigenin label and Texas Red for the biotin label, using five successive layers
of antibodies as described (23).
Measurements and Data Analysis--
Samples were viewed in a
fluorescence microscope (Leitz DMRB), and images of the combed DNA
molecules were acquired using a CCD camera (Hamamatsu C5985). The
fields of view were chosen at random. The size of most photographed
molecules was 120 kb. All of the photographed fibers showed a
continuous FITC label, indicating complete replication. Measurements
were made on each molecule using MAC BAS V2.5 software (Fuji).
Replication eyes were defined as regions replicated before addition of
biotin-dUTP and appeared as gaps of unstained DNA when viewed under the
Texas Red filter and as regions stained with FITC only on superimposed Texas Red + FITC images. Control experiments showed that gaps of
unstained DNA need to be >2 kb to be convincingly identified as
replication eyes, and smaller gaps were not considered significant. The
replication extent of each fiber was calculated as the sum of
replication eyes divided by the total length of the molecule. The
distances between the midpoints of adjacent nonstained segments (eye to
eye distances) were then separately determined. Measurements were
classified according to the extent that the molecule has undergone replication.
Alkaline-Agarose Gel Electrophoresis--
Sperm nuclei were
incubated in the presence of 100 µg/ml aphidicolin for 2 h and
released into fresh extract. At different time points after the release
1/12.5 volume of [ Visualizing Replication by Molecular Combing--
When sperm
chromatin is incubated in Xenopus egg extract, it is
assembled into normal interphase nuclei surrounded by a nuclear envelope and replicated semiconservatively (24). DNA replication starts
after a typical 15-30-min lag (Fig.
1A) immediately upon closure
of the nuclear membrane and is complete 30 min later. To visualize the
firing of the replication origins and the progression of the
replication forks in the Xenopus in vitro system, we used molecular combing of single DNA molecules. This technique (25, 26)
allows the efficient and reproducible stretching and irreversible fixation of DNA molecules onto silanized microscope glass slides. DNA
molecules prepared in this way are parallel, straight, and homogeneously stretched (1 µm = 2 kb), eliminating the need for arbitrary selection of appropriately spread fibers and internal length
calibration inherent to other fiber spreading techniques (25). Sperm
chromatin is replicated in Xenopus egg extracts in the
presence of digoxigenin-dUTP, and biotin-dUTP is added at different
time points to differentially label early (digoxigenin-dUTP only) and
late (digoxigenin-dUTP plus biotin-dUTP) replicating sequences. After a
2-h incubation, the fully replicated genomic DNA is purified and
combed, and the digoxigenin and biotin labels are revealed with
appropriate green (FITC) and red (Texas Red) fluorescing antibodies. It
is thereby possible to monitor the progression of replication eyes and
origin distances during the S phase on individual DNA molecules (23).
Here, we used this method to examine how the sequence of origin
activation in egg extracts is affected by aphidicolin block and release
of the replication forks (Fig. 1).
Aphidicolin Triggers a Block to Origin Firing in Xenopus Egg
Extracts--
Aphidicolin, an inhibitor of DNA polymerases
The resumption of DNA replication after washing and transfer of the
aphidicolin-treated nuclei into fresh extract without aphidicolin was
visualized in a control experiment by the incorporation of
rhodamin-dUTP and counting of labeled nuclei (Fig. 1A). All nuclei incorporated label within 2 min, whereas nuclei not treated with
aphidicolin asynchronously entered the S phase over a time window of
about 15 min. This clearly shows a synchronization of nuclei after
release from an aphidicolin block. To monitor complete DNA synthesis
digoxigenin-dUTP was added to the nuclei from the start of the release.
Biotin-dUTP was added at 10, 20, or 30 min, and replication was allowed
to proceed for up to 2 h (Fig. 1B). DNA of these three
samples was isolated and subjected to molecular combing (Fig.
1C). A total of 26 Mb of DNA was analyzed, and the measurements were performed as detailed under "Experimental
Procedures." Only molecules that were fully labeled with
digoxigenin-dUTP were selected. When the fibers were visualized for
biotin-dUTP using the Texas Red filter, eyes of early replicated DNA
appeared as nonstained regions between red-labeled stretches of late
replicated DNA (Fig. 1C). Eye lengths and eye to eye
distances (ETED; defined as the distances between the two midpoints of
two adjacent replication eyes; Fig. 1B) were measured, and
the replication extent was calculated for each fiber. The mean values
of these parameters were calculated separately for each time after
release. As expected, the overall replication extent of fibers (10 min,
23.7 ± 14.9%; 20 min, 32.1 ± 18.8%; and 30 min, 46.2 ± 21.8%) and the length of replication eyes (10 min, 5.5 ± 3.4 kb; 20 min, 9.1 ± 3.0 kb; and 30 min, 12.7 ± 12.1 kb)
increased with time after release because of S phase progression.
Individual DNA fibers of each time point were ranked according to their
replication extent, and eye lengths were plotted against the molecule
rank (Fig. 2). If aphidicolin did not
perturb origin activation, all of the potential origins would have
initiated during the 2-h aphidicolin block (the S phase is normally
completed in <1 h), and elongation would synchronously proceed from
these origins after release from the block. Consequently, eye lengths would be uniform, and the replication extents of individual fibers would show a fairly homogeneous increase with S phase progression. However, we observed that the earliest time point (10 min) contained fibers with small but broadly dispersed eye sizes (2-10 kb) and replication extents (15-50%) (Fig. 2A). Given that fork
speed has been estimated to 0.5 kb/min in this system, the few eyes larger than 10 kb at this point probably originate from a small number
of mergers. At 20 min (Fig. 2B) and 30 min (Fig.
2C), the distributions of eye lengths and replication
extents broadened further. Importantly, small eyes (i.e. new
initiation events) were still abundant even 20 and 30 min after release
from the replication block. These results suggest that some potential
origins were prevented from initiating in the presence of aphidicolin but retained the capacity to initiate at various times during the S
phase after the release of the block. An alternative interpretation could be that all origins fired during the aphidicolin block but that
elongation asynchronously resumed at preinitiated origins after
release. To test this hypothesis, nascent strand synthesis during the
aphidicolin block was examined by alkaline gel electrophoresis and
quantified by phosphorimager analysis or by trichloroacetic acid
precipitation. In the presence of 100 µg/ml aphidicolin DNA replication was reduced to 1% of the control, and nascent strand synthesis stalled after 300-400 nucleotide were polymerized
(data not shown). This leads to an estimation of only one activated origin every 30-40 kb, in agreement with the eye to eye distances observed by molecular combing (see below). Normal origin spacing has
been estimated to be about 10 kb in this system (6, 21). These data
suggest that only 25-33% of all origins actually fired in the
presence of aphidicolin.
The fact that at 30 min after release fibers showed a wide range
(3-83%) of replication extents confirms and extends the notion that
origins do not fire synchronously in all regions of the genome (taking
into consideration that the length of the photographed molecules is 120 kb). The distribution of eye lengths at different time points changed
even when fibers of similar replication extent were compared (Fig. 2).
Ten minutes after the release fibers mainly contained small replication
eyes, whereas at 20 and 30 min, fibers of similar replication extent
contained fewer but larger eyes. This demonstrates that fibers taken
from different stages of the S phase can show identical replication
extents because of different temporal sequences of origin activation.
However, the visual inspection of single 120-kb fibers (Fig.
1C) and of several randomly selected longer fibers (300-500
kb; Fig. 2D) did not suggest a marked local synchrony of
neighboring origins.
Control nuclei entered into the S phase much less synchronously than
nuclei released from an aphidicolin block (Fig. 1A). To
compare S phase progression in both conditions, the fibers from all
time points in the experiment above were pooled and compared with the
pooled fibers from nuclei replicated without aphidicolin pretreatment.
The fibers were ranked by replication extent, and the eye lengths of
each fiber were plotted against its replication extent (Fig.
3). A heterogeneous distribution of eye
lengths was observed at all replication extents in both samples. This
confirms that initiation occurs asynchronously during the S phase in
both conditions. However, after treatment with aphidicolin, the
distribution of eye lengths was shifted toward larger eyes, and the
mean eye length was 1.3-1.7-fold larger than in control fibers. This
could result from a lower rate of initiation, a higher rate of eye
fusion, or both. To further investigate this point, the mean density of new replication eyes (2-8 kb) relative to the amount of DNA remaining to be replicated was calculated for each replication class. This parameter measures the frequency of initiation according to replication extent. The frequency of initiation at identical replication extents was about 2.5-fold lower after aphidicolin release than in control nuclei (Fig. 4). Thus, a smaller fraction
of potential origins fired after release from the block. Nevertheless,
the frequency of initiation increased about 8-fold as replication
progressed, both in control nuclei and after aphidicolin release. We
conclude that (i) stalling replication forks assembled at early origins blocks the firing of later origins and (ii) once the block is relieved,
origins are still activated asynchronously as in the control nuclei,
albeit with reduced efficiency. These results suggest the existence of
an origin firing checkpoint in Xenopus egg extracts.
Nascent Strand Analysis Confirms Late Origin Firing in
Aphidicolin-treated Nuclei--
To confirm by a different method that
initiation occurs throughout the S phase after release from an
aphidicolin block, we developed a nascent strand assay using a short
radiolabel pulse. Sperm nuclei were released from an aphidicolin block,
equal amounts of nuclei were pulsed for 2 min with
[ Aphidicolin Does Not Induce Activation of Dormant
Origins--
Eukaryotic genomes often contain more potential origins
than are actually used during the S phase. In yeast, treatments that block fork progression can contribute to activate dormant origins (18).
To further investigate whether additional origins could initiate when
the replication forks are blocked by aphidicolin, we analyzed the
distribution of origins after release from the block. All fibers were
ranked by replication extent, and the ETED value of each fiber was
plotted against its replication extent in experiments with (Fig.
6A) or without aphidicolin
treatment (Fig. 6B). The mean ETED for each replication
class is plotted in Fig. 6C. In both experiments, ETED on
fibers with a low replication extent were large, showing that only few
initiations had occurred, and ETED decreased with replication extent,
because of more frequent initiations. Beyond 60-70% replication, ETED
increased, showing that fusion of adjacent eyes became predominant over
initiation of new eyes. ETED after release from an aphidicolin block
were slightly larger than in control nuclei at all replication extents (mean increase, 1.4-fold), as expected from the higher eye lengths (Figs. 2 and 3). These results were also consistent with the lower frequency of initiation and the extended S phase found by eye length
analysis (Figs. 4 and 5). Even at small replication extents, ETED were
larger in the aphidicolin sample than in the control. If cryptic
origins were specifically activated as a consequence of the aphidicolin
treatment, ETED should have been lower, and the S phase should have
been shorter. We conclude that no cryptic origins are activated and
that only a fraction of the early origins can fire in the presence of
aphidicolin in the Xenopus in vitro system.
Spacing of Replication Origins--
Origins must be spaced at no
more than 20-kb intervals to ensure completion of the S phase in the
absence of origin specificity in Xenopus. Experiments using
plasmid DNA (21) or sperm nuclei (23) did not reveal a striking regular
spacing of replication origins. However, merging of adjacent
replication eyes during the S phase may mask a regular spacing. We
reinvestigated this question with nuclei synchronized with aphidicolin.
The partial synchronization of the earliest origins and the reduced
frequency of initiation in these nuclei could increase the chance of
detecting a regular pattern of initiation at early replication stages,
before a significant number of mergers occur. We selectively analyzed fibers with a replication content of 0-35% that have undergone some
origin activation after aphidicolin release but still have a low
probability of fused replication eyes. The distribution of ETED of
these fibers was compared with the distribution expected for origins
spaced at random but with the same mean interval (21.5 kb), assuming
that no merger occurs (Fig. 7). Two
deviations of the observed distribution from the theoretical
distribution were detected. First, there was a deficit in small ETED.
Both mergers and the rejection of eyes <2 kb (see "Experimental
Procedures") probably led us to underestimate the frequency of small
ETED. Nevertheless, the exclusion of small ETED could also result from interference between closely spaced origins, as previously suggested for replication of plasmid DNA in egg extracts (21). Second, there was
an excess of ETED in the 11-23-kb range, with possible peaks at 11, 15, and 19 kb. This suggests some tendency to regular origin spacing. A
previous study reported similar shapes of ETED distributions (23).
Although the deviation from random spacing was previously unnoticed,
these distributions were consistent with an excess of ETED in the
10-22-kb range for early RIs and in the 6-13-kb range for late RIs
(see Fig. 3 in Ref. 23).
Caffeine Does Not Override the Block to Origin Firing in Xenopus
Egg Extracts--
In mammalian cells a block to late origin firing can
be abolished by caffeine (13). To test whether caffeine could also affect the block to late origin firing in the Xenopus in
vitro system, sperm nuclei were incubated in the presence of both
aphidicolin (100 µg/ml) and caffeine (5 mM) for 2 h,
washed, and released into fresh extract in the presence of dig-dUTP,
and biotin-dUTP was added after 10 min. DNA was isolated and subjected
to molecular combing as detailed before. 4.5 Mb of DNA was measured.
Replication eye lengths were plotted against the replication extent of
single fibers (Fig. 8). If caffeine was
able to override the intra-S checkpoint, more origins should initiate
during the aphidicolin block, and fewer or no initiations should occur
after release. However, we found that origin firing was mainly
unchanged in comparison with nuclei treated with aphidicolin only
(compare Fig. 8 with Fig. 2A). The eye lengths were similar
in both samples (aphidicolin alone, 5.5 ± 3.4 kb; aphidicolin
plus caffeine, 5.4 ± 3.4 kb). The ETED values were also similar
(aphidicolin alone, 15.7 ± 8.4 kb; aphidicolin plus caffeine,
16.3 ± 11.8 kb). In addition, neither the size nor the amount of
32P incorporated into stalled nascent strands during the
aphidicolin block was altered by caffeine treatment, as assessed by
phosphorimager analysis of alkaline gel electrophoresis and by
trichloroacetic acid precipitation (data not shown). We conclude that
the aphidicolin-induced block to origin firing is largely insensitive
to caffeine in Xenopus egg extracts.
Checkpoints are complex transduction pathways that recognize DNA
damage or DNA replication blocks and act in multiple ways to maintain
genome integrity (29, 30). It has been shown that the inhibition of DNA
synthesis prevents the firing of late replication origins in S. cerevisiae (11) and the initiation of replication within late
replicating chromosomal domains in mammalian cells (13). Here, we have
taken advantage of the molecular combing technique, a rapid and
reproducible method for stretching individual DNA fibers, to directly
visualize the activation of the replication origins on the whole genome
when the S phase is perturbed in the Xenopus cell-free
system. Our data show that aphidicolin, an inhibitor of elongation,
triggers a block to origin firing and that origins fire asynchronously
after the removal of the drug. In contrast to recent results in
mammalian cells (13), we find that the block to origin firing is not
suppressed by treatment with caffeine, a protein-kinase inhibitor known
to attenuate the S/M checkpoint in both mammalian cells (14) and
Xenopus egg extracts (31, 32).
An Intra-S Phase Checkpoint in Xenopus Egg Extracts--
Several
lines of evidence indicate that a large fraction of origins in sperm
nuclei are only activated after the release from aphidicolin and thus
have been prevented to fire in the presence of this drug. (i) Both
molecular combing and electrophoresis of pulse-labeled nascent strands
show the existence of small replication bubbles, i.e. new
initiation, throughout the S phase after aphidicolin release.
Alternatively, small bubbles late in the S phase could be due to some
forks progressing abnormally slowly after aphidicolin release. However,
no significant difference in fork speed between control and
aphidicolin-released nuclei is observed by nascent strand analysis
(data not shown). (ii) Eye to eye distances decrease with replication
extent. (iii) The density of small (2-8 kb) replication bubbles
relative to the length of the DNA remaining to be replicated increases
with replication extent. These data suggest that the frequency of
initiation increases with replication and that the small replication
bubbles are truly due to new initiations after release. (iv) Thirty
minutes after aphidicolin release, the DNA replication extent of single
120-kb DNA fibers varies broadly (3% to 83%), confirming that origins
do not fire synchronously in all regions of the genome after release
from a replication block. Taken together, our data provide evidence
that the replicational stress induced by aphidicolin inhibits the
activation of later firing origins and that these origins can only be
activated a variable time after release. In early Xenopus
embryos origins have been estimated to be spaced every 10 kb (6). Both
molecular combing and quantitative nascent strand analysis suggest that only 25-33% of all origins (one every 30-40 kb) can be activated before the intra-S phase checkpoint blocks further origin firing.
Although the mean density of new forks increases with replication
extent after the release from the replication block, our data reveal a
slight reduction of origin density in comparison with untreated nuclei.
The data discussed above suggest that this is not due to a slower fork
rate or to a delayed restart of stalled bubbles upon aphidicolin
release. This lower frequency of initiation may result from subtle
damage to the nuclei during washing and transfer (although controls
showed that membrane integrity was preserved; data not shown) or from
incomplete or slow reversal of the checkpoint response after release
into fresh extract. Further work is required to characterize the
pathways that activate and desactivate this checkpoint.
By analogy to the S/M checkpoint, our data suggest but do not prove
that the origin firing checkpoint is also operative in Xenopus early embryos. Aphidicolin blocks entry of cycling
egg extracts into mitosis if a sufficient concentration of nuclei is
used (31) but does not block the cell cycle in early embryos (33, 34).
Genetic evidence in Drosophila suggests that the S/M pathway
is only required at the MBT for complete termination of cleavage
divisions (35-37). The concentration of nuclei used in our
experiments (2000/µl) is below (but close to) that reached in the
embryo at the MBT. Direct examination of origin firing in embryos is
required to establish with certainty when the intra-S phase checkpoint
becomes active during normal development.
The Intra-S Checkpoint Involves a Caffeine-insensitive
Pathway--
Intra-S phase checkpoints that respond to stalled forks
have been described in yeast and mammalian cells. In S. cerevisiae, the early origins fire in the presence of hydroxyurea,
but the forks eventually stall because of the lack of nucleotide
precursors, and the late origins are inhibited in a MEC1-
and RAD53-dependent manner (11). A
caffeine-sensitive intra-S phase checkpoint was more recently described
in mammalian cells (13).
In this study, we find that the block to origin firing is unperturbed
by caffeine. This conclusion is based on the observation that
initiation continues to occur in a staggered fashion after removal of
aphidicolin and that small nascent strand synthesis in the presence of
aphidicolin is not increased by caffeine treatment. In mammalian cells
caffeine treatment not only relieves the block to late initiation but
also causes loss of replication proteins from arrested forks, rendering
them unable to resume elongation after aphidicolin removal ("fork
abandon") (13). Fork abandon in sperm nuclei treated with aphidicolin
plus caffeine may be consistent with some of our molecular combing
data. However, we did not detect a higher incorporation of radioactive
label into aphidicolin-stalled nascent strands in the presence of
caffeine, arguing against extensive additional initiation. We cannot
formally exclude the possibility that the latter result is due to
degradation of nascent strands following fork abandon. However, if
caffeine allowed most of the origins to fire in the presence of
aphidicolin, then few or no origins would remain available for
initiation after release, in contrast to our results. Therefore, we
favor the hypothesis that the origin firing checkpoint is largely
insensitive to caffeine in Xenopus egg extracts.
There is a precedent for a caffeine-insensitive checkpoint pathway in
Xenopus. Caffeine treatment compromises but does not completely abolish the S/M checkpoint in egg extracts (32), and
immunodepletion experiments have confirmed that both caffeine-sensitive (XATR- and XChk1-dependent) and caffeine-insensitive (XATR-
and XChk1-independent) pathways are involved (32, 38). It is possible that a related caffeine-insensitive pathway has a predominant role in
regulating the intra-S phase checkpoint described here.
A Regulated Order of Origin Activation in Xenopus Egg
Extracts--
The initiation of DNA replication in Xenopus
early embryos and egg extracts differs from that in yeast and mammals.
First, initiation occurs at specific sites or zones in yeast and
mammals, but no detectable sequence specificity has been observed in
Xenopus (reviewed in Ref. 2). Second, in somatic cells
patterns of replication foci change during the course of the S phase
(39-41), whereas during the rapid replication of sperm nuclei in egg
extract a single pattern of foci persists throughout the S phase (42). For a long time it has been unclear whether a temporal program of
replication similar to that seen in somatic cells also exists in the
Xenopus embryo. Our studies of origin activation on plasmid DNA and in sperm nuclei have recently provided evidence that origins fire asynchronously and that the frequency of initiation actually increases throughout the S phase in Xenopus egg extracts
(21, 23). Here, we find that the temporal sequence of origin firing is
essentially unchanged when replication resumes after release from an
aphidicolin block. These results demonstrate a regulated order of
origin activation in Xenopus egg extracts despite the lack
of sequence-specific initiation in this system.
It was suggested that the increase in the frequency of initiation
during the S phase could result from the progressive concentration of a
factor required to trigger initiation at individual origins into nuclei
(21). If so, nuclei preincubated for 2 h in the presence of
aphidicolin should accumulate a high concentration of this factor and
should undergo a high frequency of initiation immediately upon release
from the drug. However, initiation resumes at a low frequency upon
release and progressively increases thereafter. Therefore, the
frequency of initiation primarily depends on the amount of replicated
DNA and not on the time elapsed since assembly and closure of the
nuclear membrane. This suggests that mechanisms other than nuclear
import of an origin-triggering factor regulate the temporal order of
origin firing in Xenopus egg extracts.
The mechanisms regulating the temporal sequence of origin firing in
eukaryotes are not clearly known. Prereplication complexes are
assembled in late mitosis and the G1 phase and are
activated at various times during the S phase by two kinases, Cdc7/Dbf4 and cyclin-dependent kinases (Cdk2/cyclin E in
Xenopus) (reviewed in Refs. 3 and 43). Cdc7/Dbf4 and
cyclin-dependent kinases are both required to load Cdc45
onto origins, and Cdc45 is then required for loading polymerase Role of the Intra-S Checkpoint in Ensuring Complete Genome
Replication--
Replication origins lack any detectable sequence
specificity in Xenopus early embryos (6). However, a
completely random distribution of origins would generate some
interorigin distances too large to complete replication within a brief
S phase (49). The data reported here and in our previous studies (21,
23) suggest three potential mechanisms to resolve this paradox. First, the distribution of origins on plasmid DNA replicating in egg extracts
suggests that some "origin interference" prevents initiation from
occurring too close to a previous initiation event, potentially sparing replication fork components for more efficient use (21). Origin
interference could account for the deficit in small eye to eye
distances reported for sperm nuclei in this work, although this effect
is difficult to discern from the effect of mergers and the rejection of
small eyes. Second, we show here that origins are spaced more
frequently than expected by chance alone at intervals within the
11-23-kb range, a distance short enough to complete replication within
the normal duration of the S phase. The basis for this nonrandom
spacing is unclear, but a combination of "positive" and
"negative" origin interference acting over different distance ranges is one possibility. Additional evidence for nonrandom origin spacing was reported while this paper was under revision (50). Third,
the frequency of initiation on plasmid DNA and in sperm nuclei
increases throughout the S phase (21, 23), decreasing the mean origin
spacing in late S phase and speeding up replication of the last
remaining stretches of unreplicated DNA.
An interesting question is whether the intra-S phase
checkpoint has a role in regulating origin firing during an unperturbed S phase. In S. cerevisiae null mutations in MEC1
and RAD53 are lethal, and this may result from premature
origin activation causing excessive consumption of dNTPs (51). In
Xenopus egg extracts, dNTP levels do not appear limiting as
in yeast, but a function of the S phase checkpoint could be to
coordinate origin activation with levels of another limiting
replication component. We suggest that the checkpoint-controlled
recycling of an essential limiting factor required both for initiation
and as a component of the replication forks, such as Cdc45 (52), could
serve to keep the total number of simultaneously active forks
approximately constant during the S phase. This would maintain a fairly
constant rate of replication, despite the random fusion of replicons
and the dwindling length of unreplicated DNA remaining available for
initiation. This mechanism could explain the increase in the frequency
of initiation (relative to the remaining length of unreplicated DNA) during the S phase and the replication completion paradox.
In yeast and somatic cells, each origin has a characteristic firing
time that is probably determined by the higher order chromatin structure or by nuclear organization. Although we have shown that origins do not fire synchronously in Xenopus egg extracts,
it is still unclear whether specific sequences replicate at a preferred time in this system. Whereas in yeast and somatic cells the origin firing checkpoint ensures the correct execution of a sequence-specific replication program, its main role in Xenopus embryos may be
to control the number of simultaneously active forks and to ensure rapid genome replication in the absence of sequence-specific origins.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]dATP (3000 Ci/mmol) was added,
and replication was stopped 2 min later by the addition of DNAZOL
reagent (Life Technologies, Inc.). DNA was isolated according to the
manufacturer's protocol minimizing DNA strand breakage during sample
preparation. The pellets were resuspended and electrophoresed on
alkaline-agarose gels (1.1% agarose, 50 mM NaOH, 2 mM EDTA) at 2 V/cm for 17 h. The gels were fixed in
7% trichloroacetic acid, dried, exposed to Fuji BAS MP 2040S imaging
plates, and analyzed on a Fuji BAS 1000 phosphorimager.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (23K):
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Fig. 1.
Molecular combing of DNA of sperm nuclei
released from an aphidicolin block. A, kinetics of
entry into the S phase. Sperm nuclei (2000/µl) were either incubated
in a Xenopus egg extract in the presence of rhodamin-dUTP
(triangles) or incubated in the extract in the presence of
100 µg/ml aphidicolin for 2 h, washed, and released into fresh
extract in the presence of rhodamin-dUTP (squares). Aliquots
withdrawn at the indicated times were fixed, nuclei were stained with
Hoechst, and the percentage of rhodamine-positive nuclei was counted by
fluorescence microscopy. B, principle of molecular combing
analysis. Sperm nuclei released from a 2-h aphidicolin
(Aph.) block are transferred into fresh extract as above.
Digoxigenin-dUTP is added at t = 0, and biotin-dUTP is
added at t = 10, 20, or 30 min after the release. DNA
is isolated after 2 h and combed, and the two labels are
visualized by probing with fluorescent antibodies. Replication eye
lengths (EL) and ETED are measured on photographed
molecules. C, examples of 120-kb DNA fibers labeled with
biotin-dUTP at 10, 20, and 30 min after release and viewed under the
Texas Red filter. The eyes of early replicated DNA appear as nonstained
regions between biotin-labeled stretches of late replicated DNA.
and
, inhibits the processive elongation of DNA nascent strands but does
not prevent the initiation of DNA replication and the formation of short nascent strands (100-300 nucleotides) as analyzed by alkaline gel electrophoresis (Refs. 27 and 28 and data not shown). In a control
experiment, sperm nuclei (2000/µl) were incubated in
Xenopus egg extracts in the presence of 100 µg/ml
aphidicolin and biotin-dUTP for 2 h. The DNA was isolated, combed,
and stained with the general DNA stain YOYO-1 and with anti-biotin
antibodies. These DNA fibers did not contain any detectable
incorporation of biotin-dUTP (not shown). Alkaline gel electrophoresis
after [
-32P]dATP incorporation confirmed that
replication was inhibited by 99% and that the size of the nascent
strands was around 300 nucleotides, a size too small to visualize by
molecular combing. At lower aphidicolin concentrations (30 µg/ml)
fibers eventually contained stretches of labeled DNA, suggesting a less
complete replication block. Therefore, all of the following experiments were performed at 100 µg/ml aphidicolin.
View larger version (41K):
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Fig. 2.
Replication initiation resumes asynchronously
after release from an aphidicolin block. DNA of nuclei released
from an aphidicolin block was isolated and subjected to molecular
combing, and a total of 26 Mb of DNA was analyzed (for details see
"Experimental Procedures"). The length of each replication eye and
the global replication extent of each fiber were measured for each time
point after release: 10 min (A), 20 min (B), and
30 min (C). The eye lengths of each fiber were plotted
according to the replication extent of the fiber. Small eyes are seen
at all replication extents and at all times after release.
D, three long DNA fibers (panels a-c) from the
10-min time point. The arrows show overlaps of successive
fields of view.
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Fig. 3.
S phase progression with (A)
or without (B) prior synchronization of nuclei with
aphidicolin. Eye lengths for all time points of release shown in
Fig. 2 (total 26 Mb) were assembled and plotted against replication
extent of fibers (A). For comparison, 17 Mb of labeled DNA
of nuclei not synchronized with aphidicolin (see "Experimental
Procedures") was similarly analyzed (B). The mean eye
lengths values (curves with squares) were
calculated after grouping fibers into equal intervals according to
replication extent.
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Fig. 4.
The frequency of initiation after release
from aphidicolin is lower but increases during the S phase. The
number of replication eyes smaller than 8 kb/fiber was divided by the
length of DNA that remained to be replicated. This measures the
frequency of initiation according to the percentage of replication of
the fiber. Mean values for nonsynchronized nuclei (squares)
and synchronized nuclei (triangles) were calculated after
grouping fibers into equal intervals according to replication
extent.
-32P]dATP at various times after release, and DNA was
isolated and separated by alkaline-agarose gel electrophoresis (Fig.
5). Replication elongation started
immediately after the release as seen by nascent strands ranging
between 100 and 1000 nucleotides. As replication proceeded, larger
nascent strands became predominant, but small nascent strands
(100-1000 nucleotides) were still detectable at 20 and 30 min. After
30 min small nascent strands were no longer observed, although
incorporation of label in higher molecular weight products was still
detected at 45, 60, and 120 min after release. Nevertheless, when
nuclei were pulse-labeled with rhodamin-dUTP and observed by
fluorescence microscopy, a significant signal was seen up to 45-60 min
but not later (data not shown). This suggests that the S phase lasted
for 45-60 min in most nuclei and that the signal observed by
32P incorporation at 120 min was due to rare, residual DNA
synthesis. In a parallel control experiment, an equivalent sample of
continuously labeled DNA was analyzed (Fig. 5, lane C). No
small strands were detected. Thus, the small strands detected in Fig. 5
are true replication intermediates and not unspecific background
because of, for example, strand breakage during sample
manipulation.
View larger version (92K):
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Fig. 5.
Nascent strand analysis of synchronized
nuclei. Sperm nuclei were released from an aphidicolin block into
fresh extract. At the indicated times (0, 10, 20, 30, 45, 60, and 120 min) an aliquot was pulsed for 2 min with [ -32P]dATP.
The DNA was isolated and separated by alkaline-agarose gel
electrophoresis. Lane C, nuclei were continuously labeled
for 2 h with [
-32P]dATP, and an amount of labeled
DNA equivalent to the pulse-labeled samples was loaded.
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Fig. 6.
Origins are spaced at larger
intervals after release from aphidicolin. Eye to eye distances
were calculated (see Fig. 1 and "Experimental Procedures") from the
same fibers shown in Fig. 3 and plotted against the replication extent
of single fibers. A, nuclei released from aphidicolin.
B, nuclei not treated with aphidicolin. C, mean
values of eye to eye distances for control nuclei (squares)
and nuclei released from aphidicolin (triangles) were
calculated after grouping fibers into equal intervals according to
replication extent.
View larger version (19K):
[in a new window]
Fig. 7.
Distribution of eye to eye distances in
early S phase. Eye to eye distances (n = 265;
mean, 21.5 kb) of fibers with a replication extent of 0-35% were
grouped into 2-kb class intervals. The number of eye to eye distances
in each class interval was plotted against the middle of each size
interval (squares). The smooth curve shows the
theoretical distribution of origin distances expected for origins
spaced at random with the same mean distance (21.5 kb). Assuming that
the DNA consists of a lattice of potential origin sites spaced at
Z-kb intervals and that the probability of usage of each
site is m, the probability that the spacing between two
consecutive used sites is NZ kb is
P(N) = m (1 m)N
1. The curve shown is
assuming that Z = 2 kb and m = 2/21.5.
View larger version (15K):
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Fig. 8.
Caffeine does not affect the block to origin
firing. Sperm nuclei were incubated in the presence of aphidicolin
(100 µg/ml) and caffeine (5 mM) for 2 h and released
into fresh extract in the presence of digoxigenin-dUTP
(t = 0) and biotin-dUTP (t = 10 min)
for 2 h. DNA was isolated and combed, and replication eyes were
measured. Eye lengths were plotted against the replication extent of
single fibers.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
for the initiation of DNA replication. Experiments in S. cerevisiae and in mammals suggest that the replication timing
program is established during the G1 phase (41, 44) and
involves the differential loading of Cdc45 onto early and late origins
(45). Furthermore, activation of the S phase checkpoint inhibits
association of Cdc45 with late firing origins (45). In principle,
inhibition of either Cdc7/Dbf4 or cyclin-dependent kinases
could mediate checkpoint inhibition, and in Xenopus data
consistent with both possibilities have been presented (46-48). It is
possible that Cdc45 can only bind to a fraction of prereplication
complexes before S phase and must be released from completed early
replicons before it can bind to and mediate activation of later origins.
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ACKNOWLEDGEMENTS |
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We thank J.-F. Allemand (Ecole Normale Supérieure-Physique, Paris, France), B. Berge and Z. Gueroui (Ecole Normale Supérieure-Physique, Lyon, France), I. Lucas, M. Chevrier-Miller and M.-N. Prioleau (O. Hyrien's laboratory) for help and for discussions, and B. Miroux for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by the Association pour la Recherche sur le Cancer.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by fellowships from the Association pour la
Recherche sur le Cancer and the Fondation pour la Recherche
Médicale and by the CNRS.
§ To whom correspondence should be addressed. Tel.: 33-1-44-32-39-20; Fax: 33-1-44-32-39-41; E-mail: hyrien@wotan.ens.fr.
Published, JBC Papers in Press, March 6, 2001, DOI 10.1074/jbc.M100271200
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ABBREVIATIONS |
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The abbreviations used are: MBT, mid-blastula transition; FITC, fluoresceine isothiocyanate; ETED, eye to eye distances; Mb, megabase; kb, kilobase(s).
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