Pre-replication complex organization in the atypical DNA replication cycle of Plasmodium falciparum:...

Molecular & Biochemical Parasitology 145 (2006) 50–59

Pre-replication complex organization in the atypical DNA replication cycleof Plasmodium falciparum: Characterization of the mini-chromosome

maintenance (MCM) complex formation

Shelley Pattersona, Claudia Roberta, Christina Whittlea,Ratna Chakrabartia, Christian Doerigb, Debopam Chakrabartia,∗

a Department of Molecular Biology and Microbiology, University of Central Florida, Orlando, FL 32826, USAb INSERM U609, Wellcome Centre for Molecular Parasitology, Anderson College, Glasgow G11 6NU, Scotland, UK

Received 8 June 2005; received in revised form 9 September 2005; accepted 15 September 2005Available online 3 October 2005

Abstract

The overall organization of cell division inPlasmodium is unique compared to that observed in model organisms because DNA replicates moret f manyk replicationc in thet ssionl elopmentc r to form ad 7 subunitsa earl 2, 6 and7 sphorylationv©

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han once per cell cycle at several points of its life cycle. The sequencing of thePlasmodium genome has also revealed the apparent absence oey components (e.g. Cdt1, DDK and Cdc45) of the eukaryotic cell cycle machinery that are responsible for the formation of the pre-omplex (pre-RC). We have characterized thePlasmodium falciparum minichromosome maintenance complex (MCM) that plays a key roleransition of pre-RC to the RC. Similar to other eukaryotes, thePlasmodium genome encodes six MCM subunits. Here, we show that expreevels of at least three of the PfMCM subunits, the homologues of MCM2, MCM6 and MCM7, change during the intraerythrocytic devycle, peaking in schizont and decreasing in the ring and trophozoite stages. PfMCM2, 6 and 7 subunits interact with each otheevelopmentally regulated complex: these interactions are detectable in rings and schizonts, but not in trophozoites. PfMCM2, 6 andre localized in both cytosolic and nucleosolic fractions during all intraerythrocytic stages ofP. falciparum development, with increased nucl

ocalization in schizonts. Only PfMCM6 is associated with the chromatin fraction at all stages of growth. No phosphorylation of PfMCMwas detected, but two as yet unidentified threonine-phosphosphorylated proteins were present in the complex, whose pattern of pho

aried during parasite development.2005 Elsevier B.V. All rights reserved.

eywords: Plasmodium falciparum; MCM; Pre-replication complex; Cell cycle

. Introduction

In spite of spectacular advances in molecular medicine,alaria continues to be a major health problem in much of

he developing world, affecting the lives of over 500 millioneople[1]. A clear understanding of the molecular basis of thealaria parasite cell growth and differentiation is essential torovide a basis for the development of novel chemotherapeu-

ic agents. The malaria parasite has a complex developmentalycle in its mosquito and human hosts and there are severaloints in the life cycle where DNA replication occur[2,3].

Abbreviations: MCM, mini chromosome maintenance; ORC, origin recog-ition complex; CDK, cyclin-dependent kinase∗ Corresponding author. Tel.: +1 407 882 2256.

E-mail address: [email protected] (D. Chakrabarti).

Sporozoites, which are in cell cycle arrest, are injectedthe mosquito salivary glands into blood stream of the huhost during a blood meal. Upon entering hepatocytes, szoites undergo exoerythrocytic schizogony, a process requmultiple rounds of DNA replication. Merozoites resulting frthe exoerythrocytic schizogony invade erythrocytes to inithe erythrocytic life cycle stages. During the intraerythrocgrowth, the parasite matures through the ring, trophozoiteschizont stages and undergoes multiple rounds of nuclearsion. Chromosomes do not condense, and the nuclear memremains intact throughout the nuclear divisions (endomitowhich results in the formation of a syncytium containing ab16 nuclei. An apparent asynchrony of nuclear division hasobserved in individual schizonts[4]. Daughter merozoites aformed through the movement of individual nuclei into mezoite buds[3]. Some of the merozoites undergo cell cycle ar

166-6851/$ – see front matter © 2005 Elsevier B.V. All rights reserved.oi:10.1016/j.molbiopara.2005.09.006

S. Patterson et al. / Molecular & Biochemical Parasitology 145 (2006) 50–59 51

to differentiate into male and female gametocytes. The molec-ular basis for sexual differentiation remains to be elucidated.Ingestion of gametocytes by mosquito initiates further devel-opment of gametocytes into gametes, a process that implies,for male gametocytes, rapid cell division to form eight gametes.Zygotes are formed, the only diploid life cycle stage, when maleand female gametes are fertilized. In the subsequent ookinetestage meiotic reduction occurs. The ookinete develops into anoocyst and undergoes many rounds of cell division to formsporozoites. Hence, there are five points in the life cycle thatinvolve cell division. In an effort to understand the molecularmechanism of the onset of DNA replication, we have initiallyfocused on the characterization of the pre-replication complexin the intraerythrocytic stage ofPlasmodium falciparum, whichis the causative agent for the most virulent form of the disease.

The basic mechanism of the pre-replication complex for-mation and the subsequent onset of DNA synthesis are wellconserved in eukaryotes[5]. In yeast an origin recognition com-plex composed of high molecular weight proteins ORC1 through6 is bound to the origin of replication throughout the cell cycle.It is believed that the ORC acts as a landing pad to nucleate thestep-wise assembly of other components, e.g., Cdc6, Cdt1, andMCM (for mini-chromosomemaintenance) complex. MCM is aheterohexameric complex composed of MCM2–7 that is thoughtto function as the replicative helicase[6]. Although the MCMcomplex is heterohexameric, subcomplexes of various configu-r tot osisa bcel cle[ ringG to thc tightc hata intso cu-l inaryc orte[ M2,6

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ns (plasm werd wae3 tem( (dT)p Kit( werec uickP edP into

pET30 Ek/LIC vector (Novagen) utilizing the LIC sequence,and transformed into NovaBlueE. coli cells (Novagen). Cloneswith the correct size insert, determined by restriction digestion,were further verified by sequencing using a Beckman CoulterCEQ 2000 XL sequencer. Following sequence verification, theexpression plasmid constructs were electroporated intoE. coliBL21(DE3) cells. The cultures were induced at 0.6A600 nmwith0.5 mM isopropyl-beta-d-galactoside for 6 h at 22◦C.

2.2. Purification of recombinant PfMCM proteins andantibody production

Of all the constructs made to express recombinant proteins,only the N-terminus fragment of PfMCM2 (residues 276–466)and the C-terminus fragments of PfMCM6 (residues 658–803)and PfPMC7 (residues 635–814) yielded proteins in the solubleform. The bacterial pellet was resuspended in 1 ml talon extrac-tion/wash buffer (50 mM sodium phosphate, pH 7.0, 300 mMsodium chloride, 10% glycerol) plus mini-complete EDTA-free protease inhibitors (Roche) per 25 ml culture. The cellsuspension was lysed using a French Press (three times at900 psi) followed by brief sonication. The suspension was clar-ified by centrifugation at 20,000× g for 20 min at 4◦C. Thesupernatant was added to 1 ml bed volume of Talon resin andHis6-tagged recombinant protein was purified following themanufacturer’s batch/gravity-flow column purification proto-c d in1 ates6 y twow ns-f the1 ith1 sedt pro-t tionb hlo-r ounto withG l tor ent toC tionu ectedt iesf ingr nes[

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ations are also detectable[7]. The MCM complex is recruitedhe pre-replication complex (pre-RC) after the exit from mitnd the onset of DNA synthesis. The abundance and su

ular localization of MCM proteins change with the cell cy5,6]. In budding yeast MCM proteins are in the nucleus du1 and S phase whereas in G2 and M they are exported

ytosol[8]. Although it is expected that the parasite has aontrol mechanism for cell proliferation and differentiation tllows precise duplication of the genome during various pof its complex life cycle, very little is known about the mole

ar machinery that regulates this process. Only the prelimharacterization of MCM4 and ORC1 subunits has been rep9,10]. In this paper we report the characterization of PfMC, and 7 subunits.

. Materials and methods

.1. Molecular cloning and expression in bacteria

Using the Saccharomyces cerevisiae and human proteiequence for the MCM subunits, the PlasmoDB databaseodb.org) was screened for matches. The putative ORFsetermined using EditSeq (LaserGene) software. Total RNAxtracted from asynchronous intraerythrocyticP. falciparumD7 parasites using the RNAgents Total RNA Isolation SysPromega). First-strand cDNA was synthesized using oligo-rimer from 200 ng total RNA using the ProStar RT-PCRStatagene). Either full-length or fragments of the genesloned. The PCR products were purified using the QIAqurification Kit (Qiagen). Approximately 0.2 pmol of purifiCR product was treated with T4 DNA Polymerase, cloned

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ol (Clontech). Briefly, the talon resin was pre-equilibrate× extraction/wash buffer before adding the clarified lysupernatant. Binding to the resin was performed at 4◦C for0 min on a shaker. The unbound sample was removed bashes with the 1× extraction/wash buffer before being tra

erred to the column. The two stringent washes, one with× extraction/wash buffer with 5 mM imidazole, the other w× extraction/wash buffer with 20 mM imidazole, were pashrough the column to remove any non-specific bindingeins. The bound proteins were then eluted with 5 ml eluuffer (50 mM sodium phosphate, pH 7.0, 300 mM sodium cide, 150 mM imidazole, 10% glycerol). The necessary amf purified protein was resolved on SDS-PAGE, stainedelCode® Blue Reagent (Pierce) and excised from the ge

emove contaminating bands. The gel slices were then socalico Laboratories (Pennsylvania, USA) for immunizasing complete Freund’s adjuvant. The animals were subj

o four injections before obtaining the final bleed. Antibodrom the final bleed were affinity-purified on the correspondecombinant proteins immobilized to nitrocellulose membra11].

.3. Parasite extracts

P. falciparum 3D7 maintained in a modified erythrocyte cure [12] were synchronized by sorbitol treatment[13]. Thenfected erythrocytes were treated with 0.1% saponin to rehe parasites, which were then washed with phosphate-bualine. Cell-free extracts were prepared by resuspendinevelopment stage-specific parasite pellet in M-PER (Pieagent containing protease inhibitor cocktail (Roche), 5

52 S. Patterson et al. / Molecular & Biochemical Parasitology 145 (2006) 50–59

sodium fluoride, and 5 mM�-glycerophosphate. The suspen-sion was incubated at 4◦C with agitation for 20 min. The celllysate was clarified by centrifugation at 20,800× g for 15 minat 4◦C.

2.4. Western blotting and immunoprecipitation

Western blot analysis was performed using the SuperSig-nal Chemiluminescent Reagent (Pierce). For immunoprecipi-tation, the antibody was incubated with protein A Dynabeads®

(Dynal) for 10 min with constant agitation at room tempera-ture for binding. The antibody-protein A Dynabeads® complexwas next equilibrated with 0.2 M triethanolamine, pH 8.2, cross-linked with 20 mM dimethyl pimelimidate (DMP) for 30 min,quenched by adding 50 mM Tris–HCl, pH 7.5 for 15 min, fol-lowed by three washes with PBS containing 0.1% BSA. Twohundred micrograms of stage-specific extract was incubatedwith the cross-linked antibody bead complex for 1 h at 4◦C.The beads were then washed three times with Buffer B (10 mMsodium phosphate, pH 7.2, 1 mM EDTA, 0.5% Triton X-100,1 M sodium chloride). Next the beads were washed with bufferC (10 mM sodium phosphate, pH 7.2, 1 mM EDTA, 0.5% Tri-ton X-100, 1 M sodium chloride, 0.1% SDS) followed by two10 min washes with Buffer A (10 mM sodium phosphate, pH7.2, 1 mM EDTA, 0.5% Triton X-100) to remove unbound pro-teins. The proteins were eluted by two washes with 30�l 0.1 Ms utrai edo

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with PBS, and a second time with PBS plus EDTA-free proteaseinhibitors (Roche). The collected pellets were immediatelyresuspended in 100�l of Buffer A (10 mM Hepes, pH 7.9,10 mM KCl, 1.5 mM MgCl2, 0.34 M sucrose, 10% glycerol,1 mM DTT, and EDTA-free protease inhibitors) plus NP-40(final concentration 0.65%). The suspension was incubatedon ice for 10 min, then centrifuged at 20,800× g for 10 minat 4◦C. The supernatant (S1), containing the cytoplasmicfraction, was separated from the pellet (P1) and centrifuged at20,800× g for 5 min at 4◦C to clarify. The supernatant (S2)was transferred to a new microfuge tube and any pellet wasdiscarded. P1 was washed once with Buffer A, before beingresuspended in 50�l of Buffer B (10 mM Hepes, pH 7.9, 3 mMEDTA, 0.2 mM EGTA, 1 mM DTT, and EDTA-free proteaseinhibitors). The pellet was incubated on ice for 30 min, and thencentrifuged at 20,800× g for 10 min at 4◦C. The pellet (P1) wasseparated from the supernatant (S3), containing the nucleosolicfraction, and washed once with Buffer B. P1 was resuspendedin 49�l Buffer C (10 mM Hepes, pH 7.5, 1 mM EDTA, 0.5 MNaCl, 10 mM MgCl2, 5 mM CaCl2, and EDTA-free proteaseinhibitors) plus 1�l DNase I (10 mg/ml), then incubated for3 h at 37◦C. The suspension was collected by centrifugation at20,800× g for 10 min at 4◦C. The supernatant (S4) containedthe once chromatin bound proteins after being released due tothe DNase I treatment. Samples from different fractions wereresolved on a 10% SDS-PAGE, transferred to nitrocellulose,t

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odium citrate, pH 2.07. The eluates were pooled and nezed with 20�l 1 M Tris, pH 10.4. The samples were resolvn SDS-PAGE for Western blot analysis.

.5. Indirect immunofluorescence microscopy

An asynchronous culture ofP. falciparum 3D7 was washewice with serum-free RPMI. The culture was resuspendeerum-free RPMI to a final concentration of 5× 107 RBC/ml.hree hundred microliters of the culture suspension was pn a poly-l-lysine coated coverslip and incubated in a CO2 incu-ator at 37◦C for 30 min. The coverslip was washed with PBxed with 4% paraformaldehyde, and washed again with Phe coverslip was permeabilized and blocked for 20 minBS containing 5% BSA and 0.05% saponin. Primary antiboere incubated on the coverslip for an hour followed by sevashes with PBS containing 5% BSA. Secondary antiboere incubated on the coverslip in the dark for 30 min folloy several washes with PBS containing 5% BSA. Chromostaining was done with 1�M 4′,6-diamidino-2-phenylindolDAPI) for 5 min. The coverslips were mounted with gel/moBiomeda) on slides. The slides were then viewed usieltaVision image restoration microscope system (Appliedision) equipped with a Nikon TE200 microscope and a Pometrix cooled CCD camera. DeltaVision software (SoftWoas used for image restoration to eliminate out of focus bl

.6. Nuclear and chromatin fractions

The parasite pellet (from 100 ml culture at a 10% pitemia) obtained following saponin treatment was washed

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hen immunoblotted with specific antibodies.

. Results

.1. Identification of P. falciparum MCM (PfMCM)ubunits

To identify MCM subunits ofP. falciparum, we performedLASTP searches of the PlasmoDBPlasmodium genomennotated database (http://www.PlasmoDb.org) using MCM asquery for “text search”. This allowed us to identify

lasmodium MCM subunits, all of which are large polypeides (>100 kDa) containing the MCM signature seque(IVT)(LVAC) 2(IVT)D(DE)(FL)(DNST)KM. Comparison o

he PfMCM amino-acid sequences shows a stretch of ar00 amino acids in the central region that is conserved in allnits (Fig. 1). In addition, a zinc-finger motif is located towa

he N-terminus of MCM2, 4, 6 and 7, as would be expectehese MCM proteins[14]. Another conserved sequence foun MCMs, the Walker A type ATP-binding motif, is presen all six Plasmodium MCM proteins. The last notable traithe MCM family of proteins at the C-terminal region thatotential to form� helical structure, can also be found inix putative malarial MCM proteins. It is interesting to notelthough the PfMCM subunits are large proteins (∼100 kDa)one of the sequences have introns.

.2. Dynamics ofPfMCM2, 6 and 7 expression duringntraerythrocytic development

To analyze the steady-state expression of PfMCM2, 6 auring the maturation of the parasite through intraerythro

http://www.plasmodb.org/


Fig. 1. Sequence alignment of Pf MCMs. Sequence alignment was performed by Clustal method using DNAStar Megalign program. Box I indicates the zinc-fingermotif found in MCM2, MCM4, MCM6, and MCM7. The box II indicates a conserved ATP/GTP-binding site motif A (P-loop). The MCM signature sequence is boxIII. Box IV is a degenerate heptad-repeat sequence similar to that found in MCMs.

cell cycle, we used affinity purified polyclonal antibodiesgenerated against partial recombinant proteins for Western blotanalysis with synchronized stage-specific parasite extract. Theseantibodies are specific and did not exhibit any cross-reactivitywith non-cognate PfMCM proteins. As can be seen fromFig. 2, PfMCM2, 6 and 7 show similar expression profiles. Theproteins are present throughout the intraerythrocytic life cycle,

although their expression levels fluctuate. PfMCM2 expressionappears to peak in late schizont/segmented schizonts stage, thendrop quickly in rings. Interestingly, the expression of PfMCM6also appears to peak during the late schizont/segmentedschizonts stage, however the reduction in the protein level isnot apparent until early trophozoite stage and there is a secondpeak in the late trophozoite stage. PfMCM7 expression appears

F cle. A sitesh 0.1% lq ana PfMCM7.L tage; zoite s

ig. 2. Expression of MCM2, 6, and 7 duringP. falciparum intraerythrocytic cyarvested every 6 h starting 12 h post-synchronization by saponin lysis (uantities (20�g) of the protein extracts were resolved by SDS-PAGE andanes 1 and 2 correspond to trophozoite stage; lanes 3 and 4 schizont s

P. falciparium 3D7 culture was synchronized by sorbitol treatment and para). Cell-free extracts were prepared from parasite pellet by M-Per lysisreagent. Equalyzed by Western blots using antibodies against PfMCM2, PfMCM6, andlane 5 segmented schizonts; lanes 6 and 7 ring stage; and lane 8 trophotage.


Table 1Summary of MCM quantification

MCM2 MCM3 MCM4 MCM5 MCM6 MCM7

MW (kDa)Calculated 104 109 117 86 106 94Observed 132 – – – 113 92

Abundance (fmol/�g total protein extract)Ring 0.79 – – – 0.69 0.60Trophozoite 0.74 – – – 0.59 0.25Schizont 1.65 – – – 1.83 2.10

to peak in early schizont and continues at a high level throughschizont/segmented schizonts. The PfMCM7 protein levelsslowly drop during ring and early trophozoite stages, with thelowest amount of protein found in the mid trophozoite stage.Our Western blot analysis correlates well with the microarraydata in PlasmoDB (plasmodb.org) where the level of transcriptsof PfMCM2, 6 and 7 are the lowest in rings (at 12 h) and peaksin schizonts (36 h). To obtain accurate profiles of PfMCM2,6, and 7 expression, the signals from each antibody werequantified. To achieve this, known amounts of the recombinantproteins, alongside with 20�g of stage-specific extracts, weresubjected to Western blot analysis, and the intensity of thesignals was measured by densitometry. The signal of thestandard recombinant purified proteins was linear in relationwith protein quantity, so that the stage-specific native PfMCMsubunit signals plotted on the graph allowed the calculation ofthe specific quantity of the polypeptides in the parasite extract(Table 1). As mentioned above, all three proteins peak at theschizont stage. In addition, during ring and schizont stages theproteins appear to be in equimolar amounts. However, in tropho-zoites, while PfMCM2 and PfMCM6 appear to be in equimolaramounts, the molar ratio of PfMCM7 is significantly lower.

3.3. Subcellular localization and chromatin association ofPfMCM subunits

dur-i tiono icm licft fr ed asm His-t tage,i il-a utedi ery-t whatd -p orea onlyP nceW e thai ge.

Fig. 3. Localization of PfMCM2, 6, and 7 in cytosolic or nuclear fractions.Parasites isolated from synchronized stage-specific cultures were subjected todetergent lysis (0.65% Nonidet P-40) to isolate cytosolic and nuclear fractions[15]. Twenty micrograms of cytosolic and nuclear fractions were resolved on a10% SDS-PAGE and subjected to Western blot analysis using specific antibodies.C, cytosolic; N, nuclear; R, ring (16 h); T, trophozoite (26 h), S, schizont (36 h).PfRNR2,P. falciparum ribonucleotide reductase small subunits.

Compared to the Western blot data (Fig. 2) levels pf PfMCM2and 6 appears to be enhanced at the ring stage. This may be dueto the fact that Western blot analysis was done with tightly syn-chronized samples that were harvested at 6 h intervals whereasthe subcellular localization experiment was performed with sam-ples that are predominantly at the ring (16 h), trophozoite (26 h)or schizont (36 h) stages of intraerythrocytic growth and somecross-contamination cannot be ruled out.

The subcellular localization PfMCM subunits was furtherassessed by indirect immunofluorescence assay (IFA). PfMCM2and PfMCM6 colocalizes with the DNA stain in the schizontstage (Fig. 4A, upper parasite). In ruptured merozoites of asegmented schizont (Fig. 4B), some association of PfMCM2and PfMCM6 was evident. However, a significant pool ofPfMCM2 does not interact with PfMCM6 as evident from theirdistinct localization. Because of small size of merozoites, itis difficult to ascertain exact subcellular localization of theseproteins. As expected from the Western blot experiments,the intensity of PfMCM2 and PfMCM6 fluorescence wasconsiderably reduced in trophozoites (Fig. 4A, lower parasite).Some co-localization of PfMCM2 and PfMCM6 was evidentfrom yellow colored spots. The PfMCM7 antibodies were notsuitable for immunofluorescence.

Because chromatin association of MCM complex isstringently regulated during cell cycle progression[16], weinvestigated a possible interaction of PfMCM subunits withc on ofP actsi und( atedw qual

Because the subcellular distribution of PfMCM changesng cell cycle in model organisms, we analyzed the localizaf PfMCM2, PfMCM6, and PfMCM7 during intraerythrocytaturation ofP. falciparum. We prepared nuclear and cytoso

ractions following a method used forP. falciparum Replica-ion Protein A analysis[15]. P. falciparum small subunit oibonucleotide reductase (PfRNR2) and histone were usarkers of the cytosolic and nuclear fractions, respectively.

one expression was significantly higher in the schizont sn agreement with thePlasmodium transcriptome data avable on PlasmoDB. PfMCM2, 6, and 7 appear to be distrib

nto both nuclear and cytoplasmic fractions throughout thehrocytic developmental cycle. There appears to be someifferential distribution among the subunits (Fig. 3). For examle, compared to other PfMCM subunits, PfMCM2 is mbundant in the cytosol during schizogony. Interestingly,fMCM7 contains a clear bipartite nuclear targeting sequee have also detected a lower molecular mass polypeptid

s recognized by the PfMCM2 antibody only in the ring sta

.t

hromatin. To analyze the dynamics of chromatin associatifMCM2, 6 and 7, we fractionated stage-specific cell extr

nto cytoplasmic (S2), nucleosolic (S3), and chromatin boS4) preparations. The pellet from the S3 fraction was treith DNase I to release chromatin bound proteins. An e


Fig. 4. Subcellular localization of PfMCM2 and PfMCM6. Parasites on coverslips were fixed with paraformaldehyde and permeabilized with 0.05% saponin. Thecells were probed with PfMCM2 and PfMCM6 antibodies followed by Alexafluor 555 labeled antimouse (green, PfMCM2) and Alexafluor 647 labeled antirabbit(red, PfMCM6) secondary antibodies. DNA was stained with DAPI (blue). Images were analyzed by DeltaVision deconvolution microscopy. The bar indicates 5�M.

amount of proteins from the three fractions of intraerythrocyticdevelopmental stages were subjected to Western blot analysiswith anti-PfMCM2, PfMCM6, and PfMCM7 antibodies (Fig. 5).To obtain detectable signals from the ring stage fractions, the sig-nals from the schizont fractions were overexposed. Our resultsshow that of the three PfMCM subunits under study, PfMCM6is the only one that is tightly associated to the chromatin (Fig. 5,S4 fraction). The results suggest that PfMCM6 is bound to thechromatin throughout the intraerythrocytic life cycle, as well asbeing present in both the cytoplasmic and nucleosolic fractions.Interestingly, PfMCM2 does not appear to be bound to thechromatin during any stage of the intraerythrocytic life cycle,and is found predominantly in the cytoplasmic fraction until theschizont stage, where there appears to be an equal distributionbetween cytoplasmic and nucleosolic. PfMCM7 is found in thecytoplasmic and nucleosolic fractions throughout the intraery-throcytic life cycle, increasing in both fractions from ring toschizont.

3.4. Interaction between PfMCM subunits

We next investigated whether PfMCM subunits interact witheach other to form a heterohexamer or at least a subcomplex. As

evident fromFig. 6, the antibodies against PfMCM2 were ableto immunoprecipitate both PfMCM6 and 7 (the latter to a lesserextent). The antiserum against PfMCM6 was able to precipi-tate both PfMCM2 and PfMCM7 in rings and schizonts, but introphozoites the co-precipitation was less efficient for PfMCM2,and did not yield detectable levels of PfMCM7. On the otherhand, the antibodies against PfMCM7 were able to pull-downboth PfMCM2 and PfMCM6 in all three stages. The inability ofthe antibodies against PfMCM6 to pull-down PfMCM7 in thetrophozoite could be a result of the antibodies against PfMCM7not being able to detect a small quantity of MCM7. The co-precipitation results suggest that PfMCM2, 6, and 7 interact andcan be found in a complex. Interestingly, as seen in Western blots(Fig. 3) lower molecular mass PfMCM2, possibly a truncatedform of PfMCM2, was also detected in ring stage parasites afterimmunoprecipitations.

3.5. Phosphorylation status of PfMCM subunits

It is established that MCM subunits are phosphorylatedand the phosphorylation status influences the subcellularlocalization (and hence the activation) of the MCM complex[17,18]. Therefore, we analyzed whether the phosphorylation

F onate tsc d nuc 0%S t PfM

ig. 5. Chromatin association PfMCM subunits. Parasite pellet was fractiytoplasmic fractions; S3, nucleosolic fraction, and S4, chromation bounDS-PAGE. Western blot analysis was performed with antibodies agains

d into cytosolic and nuclear fractions as described in Section2. Fraction S2 represenlear fraction. Equal amounts (20�g protein) of each fraction were resolved on 1CM2, 6, and 7 antibodies.


Fig. 6. Co-immunoprecipitation of the PfMCM Subunits. Stage-specificP. falciparum 3D7 protein extracts (200�g) were immunoprecipitated with the specificPfMCM 2, 6 and 7 antibodies that were cross-linked to protein A Dynabeads by dimethyl pimelimidate. The immunoprecipitates were washed extensively,eluted,and resolved on 8% SDS-PAGE. Western blot analysis was performed with antibodies against PfMCM2, 6, and 7. R, ring; T, trophozoite; S, schizont.

Fig. 7. Phosphorylation of PfMCM subunits. Stage-specificP. falciparum 3D7 extract (700�g) was immunoprecipitated with the indicated antibody. The immuno-precipitates were subjected to Western blot analysis with phosphoserine or phosphothreonine antibodies.

status of PfMCM2, 6 and 7 changes during the intraerythrocyticmaturation of the parasite. We used well-established commer-cial anti-phosphoserine and anti-phosphothreonine antibodiesto detect phosphorylation of subunits by Western blots of thecomplex immunoprecipitated by the anti-MCM antibodies. Ascan be seen inFig. 7, phosphoserine and phosphothreoninespecific antibodies did not detect phosphorylation of PfMCM2,6 or 7. However, all three immunoprecipitates yielded similarpositive signals with anti-phosphothreonine antibody in allthree stages of intraerythrocytic development. The same twophosphothreonine-containing bands of approximately 85 and95 kDa were observed in all three immunoprecipitates, buttheir relative intensity varied dramatically during parasitedevelopment: in the ring stage the 95 kDa phosphorylatedpolypeptide is more abundant than the 85 kDa protein, whereasthe reverse situation is observed in trophozoites. It is evidentfrom the Western blot using PfMCM2, 6 and 7 antibodies (tothe right ofFig. 7) that these two bands do not correspond to anyof these three PfMCM proteins. The fact that the 85 and 95 kDaphosphorylated proteins are immunoprecipitated with highreproducibility by three distinct anti-PfMCM antibodies (i) isconsistent with the existence of a single complex encompassingPfMCM2, 6 and 7, as suggested by the co-immunoprecipitationexperiments described in the previous section, and (ii) demon-

strates that the complex does include other polypeptides inaddition to these three PfMCM proteins.

4. Discussion

The cell cycle in the developmental stages ofPlasmodiumdeviates from the canonical eukaryotic model. In the intraery-throcytic cell cycle and at several other points in the develop-mental cycle, multiple rounds of DNA replication take place,followed by nuclear division without cytokinesis and forma-tion of syncytial cells. Although thePlasmodium cell cycleis unique, some resemblance to theDrosophila early embry-onic cycle can be seen. In the first 13 cycles of the Drosophilaembryonic cycles only S and M phases are observed withoutintervening gap phases[19]. In these cycles, the nuclei dividein a shared cytoplasm, thus forming a syncytium. The exit frommitosis in the syncytial division requires degradation of cyclins[20]. Although some progress has been made in identifyingCDK-related kinases and cyclins inPlasmodium, the precisemolecular mechanism of the cell cycle regulation remain unclear[21,22]. Studies on pre-replication formation and onset of repli-cation in Plasmodium will provide insight into the molecularmechanism of multiple rounds of DNA replication. Only twoproteins of the pre-RC, ORC1 and MCM4, have been identified


in Plasmodium, and both have been reported to be expressedonly in gametocytes[9,10]. This is in contrast to our unpub-lished observations, and to data from transcriptome analysis bymicroarray[23,24], which show presence of mRNA for PfORC1and PfMCM4 in asexual parasites. Our analysis (this paper, andunpublished observations) demonstrates the presence of all sixsubunits of the MCM complex in theP. falciparum genome.PfMCM2, 4, 6, and 7 contain a putative C4-type zinc fingerdomain at the N-terminus region as observed in MCMs fromother eukaryotes. The zinc finger domain may play a role in thebinding of PfMCMs to chromatin because these domains areknown to be involved in protein-DNA interactions[25]. S. cere-visiae zinc finger MCM mutants were non viable, suggesting theimportance of this domain for the function of the protein[26].

The presence of six subunits of PfMCM raises the possibil-ity that heterohexameric MCM complexes form in the malariaparasite as they do in other eukaryotes. Our study shows that theexpression of PfMCM2, 6, and 7 polypeptides are observed pre-dominantly in late trophozoites and during schizont maturation.This expression profile coincides with DNA replication inPlas-modium [27]. The quantification results suggest that PfMCM2,6, and 7 are found in approximately stoichiometric amounts dur-ing ring and schizont stages. In a “typical” eukaryotic cell, theMCMs form a stable heterohexamer complex (containing allsix subunits) in G1, and this complex is present until the initi-ation of DNA synthesis has occurred[28,29]. At the beginningo lex ist culee tiveh cq ro-t of als ringt o bes et-e -RCs for af 7m lexea wn-r celc

thet atione odiea , asd atesT ormat , ith hee it oat lsod ge,t M7a ayi uld

coincide with the time frame after the activation of DNA syn-thesis.

Our observation of PfMCM complex formation in the sch-izont and ring stages suggests of a model where in late schizonts,following DNA replication, the cell cycle is reset in G1 and thepre-RC is being formed. Whereas in the trophozoite stage theparasites are in the S phase and the pre-RC complex has beendisrupted and hence the interaction between the subunits is notobserved. It is interesting to note that only PfMCM6 shows tightchromatin association. It is possible that the PfMCM6 subunitmakes direct contact with chromatin whereas other subunits areassociated through protein–protein interactions. Weak associa-tion of PfMCM6 with other subunits may have led to the dis-ruption of the complex during the fractionation procedure. Phos-phorylation of MCM2, MCM3, MCM4, MCM6, and MCM7 hasbeen observed in vivo and in vitro in different eukaryotic cells[14,17,34–37]. The MCM proteins also appear to be differen-tially phosphorylated during the cell cycle[36,37]. Althoughwe have not detected any phosphorylation of PfMCMs, we haveidentified phosphorylation of two as yet unidentified proteins (85and 95 kDa), which are part of the complex. In model eukary-otes, phosphorylation of MCM2 by Cdc7 is thought to play arole in the removal of the MCM complex from the chromatinand be a trigger for DNA synthesis[35,38].

Interestingly, no obvious Dbf4-Cdc7 (DDK) has been identi-fied in thePlasmodium genome, which may explain our inabilityt

f ocia-t NAr in theP siteu ppar-e rk Ms.Am sonw niz-a teado theO ainw dc6,o mm RC2at onge lysp NAr beenfT es oft delo andp reg-u rton what

f DNA synthesis, the status of the heterohexamer comphought to shift to a heterohexamer composed of two moleach of MCM4/6/7, which is believed to function as a replicaelicase at the replication forks[30,31]. With the stoichiometriuantities of MCM2, 6, and 7, one could infer that the MCM p

eins are found in the heterohexamer complex consistingix MCMs during the ring and schizont stages. However, duhe trophozoite stage, the quantity of PfMCM7 was found tignificantly lower than that of PfMCM2 and PfMCM6. The hrohexamer complex is essential for the formation of the preince the binding of six subunit heterohexamer is requiredunctional Pre-RC[32]. Thus, the reduced quantity of PfMCMay decrease the number of MCM heterohexamer comp

vailable for the production of the Pre-RC, and hence doegulate DNA synthesis initiation at the proper phase of theycle.

The decrease in heterohexamer MCM complexes inrophozoite stage is supported by the co-immunoprecipitxperiments. In the ring and schizont stages, the antibgainst PfMCM2, 6 and 7 pull-down the other two proteinsetermined by Western blot analysis of the immunoprecipithese results support the six-subunit heterohexamer conf

ion or a subcomplex of PfMCM2/6/7 formation. Recentlyas been shown that in humans MCM subunits exist as arohexameric complex consisting of one of each subuns a homodimer of MCM4/6/7 heterotrimers[33]. In addition,

etramers of MCM2/4/6/7 and dimers of MCM3/5 were aetected[33]. It is to be noted that during the trophozoite sta

he antibody against PfMCM6 does not pull-down PfMCnd pulls down PfMCM2 very inefficiently. These results m

ndicate disassociation of the PfMCM complex, which wo

s

l

s

l

s

.-

t-r

o detect any phosphorylation of PfMCMs.Our study demonstrates thatP. falciparum MCM proteins

orm a multi-protein complex whose expression and assion with the chromatin coincides with the preparation for Deplication. In the absence of Cdc6 and Cdt1 homologueslasmodium genome, it is uncertain which protein(s) the parases as a MCM chromatin-loading factor. Because of the ant absence of DDK in thePlasmodium genome, CDKs or otheinases may play a dominant role in phosphorylating PfMCnalysis of thePlasmodium genome has revealed thatPlas-odium may have a minimal pre-RC complex in compariith that of higher eukaryotes: there are only three recogble ORC subunits, PfORC1, PfORC2, and PfORC5, insf six subunits usually found in eukaryotic cells. BecauseRC1 shows significant similarity with Cdc6, it is uncerthether PfORC1 functions as a component of the ORC or Cr fulfils both functions. The identity of PfORC5 to ORC5 froodel organisms is rather low. This leaves only the PfOs a likely candidate protein that binds toPlasmodium replica-

ion origins. This also raises a tantalizing possibility that amukaryotesPlasmodium ORC is one of the simplest and onlightly more complex than archaea (unless otherPlasmodiumroteins are able to function as ORCs). The initiation of Deplication in archaea, once thought to be a bacterium, hasound to be a simplified version of the eukaryotic event[39,40].he chromosomal replication has many of the basic featur

he eukaryotic replication.Fig. 8, describes a comparative mof pre-replication complex formation in eukarya, archaea,lasmodia. Clearly, studies of the pre-RC formation andlation in Plasmodium have the potential to elucidate hitheovel mechanisms and unresolved questions with regard to


Fig. 8. Comparison of models of pre-replication complex formation in eukaryotes, archaea, andPlasmodium. (A) In eukaryotes, the pre-RC complex formationinvolves an ordered assembly of a number of components: origin recognition complex (ORC) composed of six subunits, Cdc6, Cdt1, and Mcm2–7. Two proteinkinases, CDK and DDK (Dbf4-dependentkinase) are required for the firing of licensed origins for initiation. Activation by these protein kinases leads to changes inthe pre-RC causing the binding of Cdc45 to the MCM complex. This results in an unwinding of replication origin and the recruitment of replication proteins. (B)The chromosomal replication in archaea has many of the basic features of the eukaryotic replication. A Cdc6/ORC1 and MCM homologues have been identified.Instead of a heterohexameric structure in eukaryotes, the archaeal homologue forms a double homohexameric structure. (C)P. falciparum has only three subunits ofORC complex instead of usual six subunits found in all eukaryotes. As discussed in Section4, possibility exists that ORC2 homologue could be the only true ORCcomplex component in the malaria genome. Although its physiological function is unclear, PfPK5 is a homologue of p34cdc2cyclin-dependent kinase that is requiredfor both entry into S-phase and mitosis in fission yeast cells. Other key regulators of pre-replication complex formation, Cdt1, DDK, and Cdc45 appearto be absentin Plasmodium. This suggests that in comparison to the eukaryotic model, the malaria parasite may have a minimal pre-RC for the onset of S phase slightly morecomplex than archaea. ThePlasmodium model is based on the eukarya and archaea models developed by Kelman and Hurwitz[40].

is the minimum structure of a functional pre-RC in eukaryotes.The present study on PfMCM complex provides the first step inunderstanding the molecular mechanism of pre-RC formation inPlasmodium.

Acknowledgements

This work is supported by a grant from National Insti-tutes of Health (AI48036) to DC Work in CD’s laboratory issupported by INSERM, the Wellcome Trust, the French Min-istry of Defence (DGA) and the UNDP/WorldBank/WHO spe-cial programme for research and training in Tropical Diseases(TDR).

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