Replication of php locus is not scheduled
To follow the replication of php
gene encoding a subtilisin-like protease, we carried out kinetic analyses. Plasmodia were harvested at specific time points through the 3 h of S phase. DNA samples embedded in agarose plugs were digested by restriction endonucleases, resolved in 2D-gel electrophoresis and hybridized with a specific cDNA probe. Surprisingly, in disagreement with our previous reports (21
), we found that the 6.7-kb EcoRV-EcoRI fragment encompassing php
gene exhibited prominent Replication Intermediates (RIs) during the first hour of S phase (A). A transition from a bubble arc to a Y arc was observed 5 min after the beginning of S phase (+5′) and indicated that initiation takes place within the fragment (see subsequently). At + 10 min (+10′), the RIs were essentially composed of Y-shaped molecules. This partial Y arc persisted up to + 60 min and was significantly detected until + 90 min, when about 75% of genomic DNA synthesis is completed. Quantifying hybridization signals evidenced the broad temporal window of php
locus replication. Indeed, replicative arcs represented ~20% of the total hybridization signal from + 5 min until + 60 min, decreased to ~8% at + 90 min and lowered to <1% as late as + 120 min.
Figure 1. Contrasted replication timings at php and proP loci. (A) Kinetic analysis of replication pattern at the php locus. 2D-gel analyses of DNA samples extracted at successive time points of S phase are shown. The 6.7 kb EcoRV (Ev) - EcoRI (E) fragment containing (more ...)
To rule out that this large temporal window of replication is due to a lack of synchrony of our plasmodia, we carried out flow cytometry analyses (B). Nuclei were isolated at specific time points of the cell cycle and DNA content of each population of nuclei was measured after nucleic acid staining with propidium iodide. B shows that each population exhibited homogeneous pattern of DNA content and that this latter increased synchronously from 2C to 4C as nuclei progressed in S phase. Therefore the synchrony of nuclei within a plasmodium is a property of the whole S phase.
Moreover, in previous studies we were able to pinpoint the replication timing of single copy DNA sequences within a 5–10 min period during S phase (26
). Therefore, as an internal control of DNA samples used for analysis of php
replication kinetics, we re-hybridized the same blots with a probe derived from proP
gene that replicates at the onset of S phase (26
). We detected in the 4.8 kb EcoRI fragment containing proP
gene RIs at +5 min, with a level of about 65% of the hybridization signal (C). This demonstrated that much more molecules containing proP
gene were engaged in replication than in the case of php
gene at this time point. In contrast, only a faint signal about (or “~”) (3%) was detectable at +10 min and no replicative signal could be detected later on, in agreement with reported results (26
). The size difference between proP
containing restriction fragments could not explain these contrasted replication patterns. Therefore, direct comparison of the two loci indicated radically different temporal windows of replication: proP
gene is replicated in less than 10 min whereas it takes ~90 min to replicate the php
gene-containing fragment. We also detected X-shaped molecule signals for both loci (see open arrowheads in A and C) after the forks have reached both ends of the fragment (i.e. from +25 min to +60 min for php
and from +10 min to +40 min for proP
). Such molecules correspond to transient post-replicative joint DNA molecules involving sister chromatids (33
). These X-shaped molecules had a maximum of intensity at +10 min for proP
and +60 min for php
. The delay in X-DNA apparition in the php
gene-containing fragment is consistent with a later period of replication.
We observed that, in contrast to proP
and other loci (26
), the timing of replication of php
locus is extended. This unexpected long period of replication of php
locus may be explained either by slow progression of replication forks or by different replication patterns among the millions of nuclei contained within a single plasmodium.
An early firing origin is located in the promoter region of php gene
To distinguish between these possibilities, we first wanted to map the replication origin of the php
gene-containing replicon (A) and to determine the fork position on the locus early in S phase. We thus performed a series of 2D-gels to analyze different restriction fragments of DNA extracted 5 min after the onset of S phase. After probing with php
probe, we compared RI patterns in overlapping fragments for deducing the localization of replication origin (A). We found a bubble arc in fragment a
, and a bubble to Y arc transition in fragments b
, indicating the firing of a bidirectional replication origin in these fragments. We located the origin at the middle of fragment a
, since it exhibited the most developed bubble arc (see the schematic extending bubble above the map in ). Consistently, the extent of the Y arc was more important in fragments b
in which the origin would be less centered. Only Y arcs were detected in fragments c2
, in agreement with an outside position of the origin. In the case of fragment e1
, due to the origin position close to its extremity and due to its size, no bubble arc could be detected and only a nascent Y arc was revealed. These results are consistent with an origin positioned at the 5′ side of the gene. The observation of a partial Y arc when analyzing the origin-containing fragment b
strongly suggests that the origin is efficiently fired. Otherwise, a complete Y arc would be observed in addition to the bubble arc, as a result of passive replication of the locus in some nuclei (34
Figure 2. Origin and replication forks mapping at php locus in early S phase. Overlapping restriction fragments were analyzed by 2D gels. a: 8.2 kb EcoRV fragment; b: 6.7 kb EcoRV-EcoRI fragment; c1: upstream 5.5 kb HindIII-EcoRV fragment; c2: downstream polymorphic (more ...)
Early replication of php gene by rightward moving fork and pausing of leftward moving fork
In order to follow the progression of replication forks, we compared the RI patterns shown in A with those obtained from similar analyses performed with DNA prepared at +10 min (B). A slow evolution of fork distribution was revealed by the different mean positions of RIs along the replicative arcs. Indeed, at +10 min, for fragment a, the maximal density of RIs was found at the end of the bubble arc. We also detected a faint terminal portion of a Y arc. For fragment b, the bubble arc was then essentially converted in a Y arc, as a result of fork movement within the fragment. Similarly the major position of RIs had moved along the Y arc for fragments c1 and e1, revealing the homogeneous displacement of replication forks. Knowing the origin position in a context of apparently smooth velocity for both forks, we could deduce which replication fork came out first of the restriction fragment. The downstream position of the origin in fragment b implied that the rightward moving fork reached first the end. Importantly, as at +10 min the bubble arc had almost disappeared in fragment b, and as fragments c2 and d were almost free of replication forks, we thus conclude that php gene is replicated in early S phase.
At +25 min, the fork movement was again evidenced by a change of RI mean position in the 2D-gels (B). Interestingly for fragment b, we also detected a spot (star) close to the intersection of the Y arc with the diagonal of linear molecules that corresponds to accumulation of RIs of a 2X size, like observed in the kinetics (A). This pattern differed from those obtained at +5 and +10 min where steady progression of RIs along the bubble and the Y arc could be detected. Such RI accumulation at +25 min indicated a stalling of the leftward moving fork close to the upstream EcoRV site (see the striped rectangle above the map). This stalling did not correspond to an arrest of the fork but rather to a slowing down. Indeed, the spot marked by the star for fragment b analysis spread on most of terminal portion of the Y arc for the shorter fragment c1. The accumulation of RIs at the apex of the Y arc for fragment e1 confirmed the fork stalling and allowed to map pausing at the middle of the fragment. We also noticed that replication forks go through the stalling region since RIs were found on the last part of the Y arc for fragment e1.
Thus, in agreement with kinetic analyses shown in A, we observed a low mobility of replication forks through 21 kb surrounding php gene (). The slow removal of replication forks from the restriction fragments is enhanced by fork stalling upstream the gene, while the coding region is rapidly replicated. Our results also indicated that, within a plasmodium, the collection of replication forks progresses concomitantly at php locus and argued against a randomly timed replication.
The php locus is replicated by a single origin
We have shown that a replication origin close to php
gene is fired at the onset of S phase; however, we still observed on kinetics a particularly prominent Y arc (~20% of the signal) at +25 min after the onset of S phase (A and B). The fact that this Y arc was never completed strongly suggested the conversion of bubble-containing fragments to single fork-containing fragments as a result of fork progression. To further confirm our assumptions and to rule out that these RIs originated from other origins activated in the vicinity of php
locus, we determined the direction of replication fork movement at php
locus in early S phase by using an adaptation of the 2D-gel method (29
In this approach, DNA contained in the agarose lane from the first dimension was digested again before it was submitted to the second electrophoresis. The resulting RI patterns depend on the polarity of replication forks in the shortened fragments (). We used a plasmodium at +20 min in S phase, when a strong intensity of RI signals was found. Following HindIII digestion, three restriction fragments were obtained, two of them (H2 and H2*) resulting from a restriction fragment length polymorphism. We observed Y arcs for the three fragments (see control experiment in ), with an accumulation of RIs at the apex of the Y arc for H1 fragment. This corresponds to the stalling of replication forks close to the EcoRV site at this stage of S phase (B). The second digestion in the gel was performed with EcoRV. For each resulting fragment, we found only one derived pattern (see fork polarity experiment and interpretative scheme in ). For the upstream fragment H1-RV, a faint bubble arc and the end of a Y arc were observed, which implied that forks moved leftward. The spot appearing on the left at a size of 2X corresponds to forks stalling close to the upstream EcoRV site, since digestion with EcoRV converted stalled forks into linear fragments. Rightward moving forks replicated the downstream polymorphic fragments H2. At this stage of S phase, part of the RIs has gone beyond EcoRV site so that the resulting fragments were linear. Shorter RIs formed the vertical end of the Y arc originating from H2-RV 1X spot.
Figure 3. Homogeneous replication fork direction at php locus. DNA preparation obtained from a +20 min plasmodium was restricted with HindIII and submitted to a first electrophoresis. The lane of interest was excised and DNA was digested in the gel with EcoRV before (more ...)
These results showed that fork directions are identical among the population of nuclei and also that replication forks have the same polarity for both alleles. Importantly, fork direction is not the same in the HindIII fragments: in the H1 upstream fragment we detected leftward moving forks, while in the H2-H2* downstream ones we detected rightward moving forks (see the arrowheads under the map in ). This fork divergence confirms the presence of a replication origin coinciding with the 5′ region of the gene. Therefore this analysis reinforces our previous data and rules out the possibility of a distant origin whose firing would produce forks reaching the EcoRI-EcoRV fragment 20 min after the onset of S phase. Since we have shown that the persistence of php RIs could not be the consequence of multiple initiation events among the nuclei, it was most likely due to a slow elongation rate of replication forks.
Slow elongation of php replicon in early S phase
To test this hypothesis, we measured the elongation rate of the php
replicon by analyzing the growing of nascent strands by alkaline gel electrophoresis (A). The natural synchrony of the plasmodium allows detection of the nascent strands of a single-copy replicon (24
). After probing with php
cDNA, short single stranded RI (stars) was observed from stages +7 to +40 min (A). At later stages, our electrophoresis procedure did not allow their separation from parental DNA. Although RIs were seen on 2D-gel at +5 min (A), they were not detected by alkaline gel electrophoresis at this stage due to lesser sensitivity of the latter method. We measured the mean size of php
RIs to calculate the mean rate of replication fork progression (C). Interestingly, the small size of php
RIs at earliest stages confirmed that the replication origin is close to the coding region. Furthermore, we measured a slow increase from 4 kb at stage +7 min to 17 kb at stage +40 min, that corresponds to an average rate of 0.4 kb/min/replicon. Comparison with the rate of elongation of proP
replicon was performed by re-hybridization of the blot with proP
gene (B). A stronger signal was obtained for nascent strands especially at earlier stages, suggesting a more acute firing of proP
nascent strands were detected slightly earlier, at +5 min, and had a size of 4 kb. The largest nascent strands that could be separated from parental DNA in these conditions were seen at +25 min with a size of 22 kb. Plotting the nascent strand lengths against time in S phase (C) revealed an average rate of 0.9 kb/min/replicon for proP
replicon. Although the rate of elongation of proP
replicon has been evaluated to be more than twice greater than the one of php
replicon, this value is in agreement with the canonical mean of 1.2 kb/min/replicon that has been calculated for Physarum
). We thus conclude that php
replicon is characterized by an unusually slow progression of replication forks.
Figure 4. Slow elongation of php replicon. (A) DNA fragments extracted at various time points of S phase were denatured and submitted to an alkaline agarose gel electrophoresis. Hybridization with php probe revealed the nascent strands containing the php gene (stars). (more ...)
We also compared these data with the evaluation of fork rates obtained from our 2D-gel analyses of php locus (). Indeed, the mean position of the signal on the arc of RIs indicated the mean location of forks within the fragment of interest. As indicated in D, alkaline gel and 2D-gel analyses gave consistent results. Both forks had covered each 2 kb as a mean after 5 min (i.e. at a speed of 0.4 kb/min/fork) and 3.0 kb after 10 min (i.e. 0.3 kb/min/fork). However, after 25 min the rightward moving fork had progressed over 6 kb (i.e. 0.24 kb/min/fork) whereas the leftward moving fork had progressed over less than 4 kb (i.e. 0.15 kb/min/fork) likely due to the stalling. Thus, the replication forks have an unequal rate. The accumulation persisted up to +60 min and implied that the replicon elongation is mostly unidirectional during this period.
php RIs have a long life span
In order to evaluate the life span of php RIs during the whole S phase, we studied php RI pattern in an asynchronous nuclei population prepared from microplasmodia where all replication events were represented. Our aim was to compare intensities of php and proP signals. We reasoned that, if the life span of php RIs was longer than the one of proP RIs, we would expect stronger signal intensity for php RIs since they were present during a longer period of S phase. On the opposite, a stochastic replication of php locus at a normal rate would not give any difference between signal intensities for proP and php loci in an asynchronous population.
shows a comparison of the replication pattern of php and proP loci obtained by 2D-gel analysis from DNA extracted from the same culture of exponentially growing microplasmodia. We could see on a single 2D-gel all the RIs appearing at any moment of S phase, in addition to the prominent 1X spot of non-replicating molecules. A similar transition from a bubble arc to a Y arc was detected for both genes. However, we obtained about 3–4-fold more RIs at php locus, as compared to proP locus, meaning that the php RIs life span is longer (the experiment was repeated four times). Clearly, this asynchronous population of nuclei, like plasmodium nuclei, exhibited a not fully expanded Y arc, demonstrating the efficient activation of the php-linked origin. The higher intensity found for php as compared to proP locus rules out the hypothesis of a random replication timing of the locus.
Figure 5. Longer life-span of php RIs as compared to proP RIs. DNA obtained from asynchronous liquid cultures of microplasmodia was restricted with EcoRI and EcoRV, submitted to 2D-gel, hybridized with either php or proP probe. Hybridization signals were quantified. (more ...)
Hydroxyurea blocks fork progression at different time points of S phase
To check that replication forks at php locus are bona fide moving forks, we inhibited DNA replication with HU, a drug that prevents replication fork elongation. Drug treatments were performed at successive periods of the S phase on half of each plasmodium and the other half was used as a control. We tested fork movement from the onset of S phase up to +25, 15–60, 60–90 and 120–150 min (). 2D-gel analyses revealed delays of RI patterns for treated plasmodia as compared to the control, except for the latest period of treatment (120–150 min). Therefore, fork progression was impeded by drug treatment. Note that a bubble arc was still observed after a 60–90 min treatment, indicating that, in a non-negligible part of nuclei, both replication forks were active within the EcoRI-EcoRV fragment at least at +60 min. These results underline the delayed activation of the origin in a small proportion of nuclei, which is consistent with the faint bubble arc seen until +60 min on . We conclude that, despite the long kinetics of elongation of php replicon, we detected on 2D-gels moving forks since they were sensitive to HU treatment. Therefore the slow replication of php locus is due to slow progression of replication forks rather than arrests randomly distributed along the replicon or fork collapsing.
The actively transcribed php gene is located in the vicinity of a developmentally regulated replication origin
gene has been previously described as developmentally regulated during the two alternative stages of growth, the diploid multinucleated plasmodium and the asynchronous haploid uninucleated amoebae (36
). We used northern blot analysis to compare the steady state level of php
mRNA in our strains of plasmodia and amoebae. We detected with php
probe an abundant 1070 nt mRNA in total RNA from plasmodium (Pl in A). In contrast, a weak signal could be detected in the amoebae sample only upon much longer exposure (Am’ in A). This indicated that php
gene is highly expressed in the plasmodium. On the opposite, expression of php
gene is almost extinguished in amoebae. Quantification and normalization against the constitutively expressed actinC gene mRNA (upper band in A, Pl and Am) indicates a 1 to 500 ratio of php
mRNA abundance between these two stages, confirming the developmental regulation of php
Figure 7. php origin is regulated during development. (A). Total RNA was extracted from either a G2 phase plasmodium or a liquid culture of amoebae. RNA samples of 10 μg each were analyzed by northern blot. Following hybridization with the actin probe (more ...)
Therefore, considering our previous results, showing a variation of origin usage in the case of developmentally regulated profilin genes proA
), we addressed the question of the php
origin usage during development. We compared the replication pattern of the gene in plasmodia and amoebae by 2D-gel analysis (B). In plasmodium, the 8.2 kb EcoRV fragment is replicated from an internal origin firing in early S phase and located at the center of the fragment, as deduced from the bubble to Y arc transition observed at +10 min (see above and B). In contrast, DNA prepared from amoebae exhibited only Y arcs when the same restriction fragment was analyzed (B). We previously showed that it is possible to detect a site-specific origin in this cell-type (15
). Therefore, these data demonstrated that the replication origin evidenced in the promoter region of php
gene in the plasmodium is inactive in the amoebae. We thus conclude that php
replicon is developmentally regulated and that usage of the origin upstream the coding region is correlated with transcriptional activity of the gene.