Aphidicolin Triggers a Block to Replication Origin Firing in Xenopus Egg Extracts*

Kathrin MarheinekeDagger and Olivier Hyrien§

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 [alpha -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

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).


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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.

Aphidicolin Triggers a Block to Origin Firing in Xenopus Egg Extracts-- Aphidicolin, an inhibitor of DNA polymerases alpha  and delta , 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 [alpha -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.

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.


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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.

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.


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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.

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 [alpha -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.


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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 [alpha -32P]dATP. The DNA was isolated and separated by alkaline-agarose gel electrophoresis. Lane C, nuclei were continuously labeled for 2 h with [alpha -32P]dATP, and an amount of labeled DNA equivalent to the pulse-labeled samples was loaded.

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.


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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.

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).


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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.

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.


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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

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 alpha  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.

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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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.

Dagger 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

    ABBREVIATIONS

The abbreviations used are: MBT, mid-blastula transition; FITC, fluoresceine isothiocyanate; ETED, eye to eye distances; Mb, megabase; kb, kilobase(s).

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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