Molecular & Biochemical Parasitology 126 (2003) 9–14

Replication of thePlasmodium falciparum apicoplast DNAinitiates within the inverted repeat region

Divya Singh, Sushma Chaubey, Saman Habib∗Molecular and Structural Biology Division, Central Drug Research Institute, Post Box 173, Chattar Manzil, Mahatma Gandhi Marg, Lucknow 226001, India

Received 3 June 2002; received in revised form 26 September 2002; accepted 27 September 2002

Abstract

The 35 kb apicoplast genomes (plDNA) ofPlasmodium falciparum andToxoplasma gondii share close sequence similarity but differin their in vivo topologies. Although sequence analysis of tandem repeats ofT. gondii plDNA has suggested the presence of replicationinitiation sites within the inverted repeat region, the replication origins (ori) of theP. falciparum circular plDNA have not been identified.Using 5′ end-labelled nascent DNA as probe, we demonstrate that theori of P. falciparum plDNA is localised within the inverted repeatregion. Our results also indicate the presence of two initiation sites within each inverted repeat segment of the circular plDNA ofP.falciparum suggestive of a four D-loop/bi-directionalori mechanism of DNA replication.© 2002 Elsevier Science B.V. All rights reserved.

Keywords: Plasmodium falciparum; Apicoplast; Replication origin; Inverted repeat

1. Introduction

Parasites of the phylum Apicomplexa, includingPlasmo-dium andToxoplasma, possess a plastid-like organelle calledthe apicoplast[1,2]. The apicoplast has generated immenseinterest as a putative drug target for malaria[3,4] and isbelieved to be the site for type II fatty acid biosynthesis[5,6] and non-mevalonate pathway of isoprenoid biosynthe-sis within the parasite[7].

The apicoplast ofPlasmodium falciparum contains a35 kb circular genome (plDNA). Each sporozoan cell ofP. falciparum carries a single apicoplast with only∼1–3copies of the organelle genome per cell. Apicoplast genomesequence analysis has revealed the presence of an invertedrepeat (IR) that carries genes for large and small subunitrRNAs and several tRNAs. PlDNA also contains a minimalbut complete set of tRNA and ribosomal genes that are suffi-cient for translation of protein-encoding genes on the circle[1]. As none of the ORFs identified on the circle encodereplication proteins, plDNA replication must proceed by theutilisation of nuclear-encoded proteins as in plastids of al-gae and plants[8]. Ciprofloxacin (that targets DNA gyrase)

∗ Corresponding author. Tel.:+91-522-212411–212418x4282;fax: +91-522-223938/229504/223405.

E-mail address: samamit@lycos.com (S. Habib).

has been shown to specifically inhibit apicoplast genomereplication inP. falciparum [9]. Additionally, ciprofloxacinand clindamycin block replication of the 35 kb DNA of theToxoplasma gondii apicoplast and also inhibit parasite repli-cation in culture[10].

Although there is extensive sequence similarity betweenplDNA of T. gondii andP. falciparum, the former occurs invivo primarily as a precise oligomeric series of linear tandemarrays of the basic 35 kb genome while the in vivo topologyof the latter is genuinely circular[8,11]. This difference instructure has important implications for the mode of plDNAreplication. WhileT. gondii plDNA may replicate via therolling circle mode initiating at the centre of the invertedrepeat[11], plDNA of P. falciparum is likely to follow thebi-directional replication mode reported for most chloroplastgenomes[12–15].

The P. falciparum apicoplast exists as a complex,branched structure in the late trophozoite-early schizontstages and subsequently gives rise to multiple, discrete,slightly rod-shaped apicoplasts at cytokinesis[16]. The tim-ing of plDNA replication inP. falciparum seems to coincidewith that of total DNA synthesis in the parasite, starting inlate trophozoites a few hours before the onset of schizogony[8,17,18]. Electron microscopy of plDNA fromPlasmod-ium sp. has revealed the presence of fully double-strandedtheta-like structures suggesting a bi-directional replicationmode from a specific replication origin(s)[8,11,19]. While

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10 D. Singh et al. / Molecular & Biochemical Parasitology 126 (2003) 9–14

analysis of the tandem repeats ofT. gondii plDNA hassuggested that the replication origin of molecules replicatingby the rolling circle mechanism lies at the centre of the IR[11], the replication initiation site ofP. falciparum plDNAhas not been identified. Conventional two-dimensionalagarose gel electrophoresis carried out by Wilson’s group[8] and also by us (unpublished results) failed to identifya specific origin of replication ofP. falciparum plDNA.Using 5′ end-labelled nascent DNA strands of plDNA asprobe, we report the identification of replication initiationsites within the inverted repeat region of theP. falciparumapicoplast genome.

2. Materials and methods

2.1. Parasite culture

P. falciparum (strains 3D7 and NF-54) were cultured asdescribed[20]. The parasites were synchronised with sor-bitol [21] and harvested at the late trophozoite-early schizontstages. Total parasite DNA was isolated by phenol/chloro-form extraction[22].

Table 1Primers used for amplification of plDNA fragments

Fragment Primer sequence (restriction enzyme tags are underlined)

A650 U: 5′-CCGGAATTCGATGTGACGGGCGGTGTG-3′D: 5′-TGCTCTAGAAAAAGAATATCAAAGGCGGAAGC-3 ′

A1820 U: 5′-CCGGAATTCAGCCACATGTTCCACCACTTG-3′D: 5′-TGCTCTAGAGCTCGCCGCTACTATGAAAATC-3′

A1988 U: 5′-CCGGAATTCTTATAGAGAAAAGTACCGTGAGG-3′D: 5′-TGCTCTAGAGCTTTAATAGGCGAACAGACT-3′

A1288 U: 5′-CGCGGATCCATGTTTATCTATAATAATTTTAAAAAATATTCT-3 ′D: 5′-CGCGGATCCGTATTAATTACATTTTTATTATATTTCAT-3 ′

A1838 U: 5′-CCGGAATTCTAAAATAAATATGATAACTACAGG-3 ′D: 5′-CGCGGATCCTAAATAAATAAGAAGCTCCAAAAG-3 ′

A1967 U: 5′-CCGGAATTCTATGGTATTTAACTGGTCCTT-3′D: 5′-TGCTCTAGATTGTTAAATATCCTGAATCTGCT-3′

A1919 U: 5′-CCGGAATTCAGTGAAGCTATAAGTGAACCT-3′D: 5′-TGCTCTAGAATATCTATACCATCCATTACCAACA-3′

B483 U: 5′-TGCTCTAGATAAAGTTAATGCCTGAGTG-3′D: 5′-CCCAAGCTTTTCCCAAAATAGATATGTTACCA-3′

B1624 U: 5′-CCGGAATTCTTCAGAATATAGTGTAATGGTAAC-3′D: 5′-TGCTCTAGATATATCCTTTTGAATGTAATCC-3′

B2435 U: 5′-CCGGAATTCGATATGGGACAAAAAGTACATCC-3′D: 5′-TGCTCTAGACTCACAAAATATTATATTAGGTAAC-3 ′

B1308 U: 5′-CCGGAATTCGGTTGGATAGGATTAGGAGTTAG-3′D: 5′-TGCTCTAGACCTTCACCTGGTATATATGC-3′

B1426 U: 5′-CCGGAATTCTTGTATTAATTAAAGGAGGTAG-3′D: 5′-CGCGGATCCACTGTTGTTAAATTAGGAGATG-3′

B1880 U: 5′-CCGGAATTCGTATGATTTTAGCAACACCT-3′D: 5′-TGCTCTAGAATATTATCATTTACCCCTTCT-3′

B1639 U: 5′-CCGGAATTCAAACCTTTAATATTACCTACAACT-3 ′D: 5′-CGCGGATCCTAAAAATATAAAAGAGAAAATGG-3 ′

2.2. Cloning of plDNA fragments

Fourteen fragments (Fig. 1A) covering plDNA were am-plified by PCR from total parasite DNA using primers thatcarried restriction sites for cloning into pUC18. The primersused are described inTable 1. Two fragments, A1820 andA1838, were cloned in pGEMT-easy (Promega, USA). Theremaining fragments were cloned in pUC18 and their iden-tity confirmed by restriction digestion and DNA sequencing.

2.3. Preparation of P. falciparum plDNA

PlDNA-enriched DNA was purified according to themethod of Yap et al.[23] using the Qiagen plasmid minipreparation kit. Briefly, infected RBCs fromP. falciparumcultures were lysed with 0.05% saponin and washed exten-sively with PBS. The purified parasites were suspended inbuffer P1, lysed with buffer P2 and neutralised with bufferP3. After centrifugation, Proteinase K was added to the sup-ernatant at a final concentration of 0.75 mg ml−1 and incuba-ted for 2 h at 37◦C. The sample was passed through a Qiagentip-20 column previously equilibrated with QBT buffer.The column was washed with QC buffer and the extra-

D. Singh et al. / Molecular & Biochemical Parasitology 126 (2003) 9–14 11

Fig. 1. (A) Position of fragments covering the IRA and IRB sectors ofP. falciparum plDNA. The IRA sector includes the inverted repeat region.Nucleotides of the IRB sector are numbered after the end of the IRB half of the inverted repeat (shown as dotted line, not to scale). (B) Circular plDNAmap depicting selected restriction enzyme sites. Arrows indicate direction of transcription of LSU and SSU rRNAs on the IR.

chromosomal DNA was eluted with QF buffer preheated to65◦C. The DNA was precipitated with isopropanol, washedwith 70% ethanol, dried and suspended in TE.

2.4. The 5′ end-labelling of nascent strands andenrichment of replication intermediates

Labelling of nascent DNA strands followed by enrich-ment of replication intermediates was carried as described

by Kunnimalaiyaan and Nielsen[24] and Little and Schild-kraut [25]. Apicoplast DNA was dephosphorylated usingcalf intestinal alkaline phosphatase (CIAP). Proteinase K(50�g ml−1) was added to the sample and incubated at 37◦Cfor 1 h. The sample was subjected to phenol extraction andethanol precipitation. This was followed by labelling with[�-32P]-ATP (100�Ci) in the presence of 10 U of polynu-cleotide kinase at 37◦C for 1 h. The end-labelled DNA wasdigested with restriction enzymes (XbaI–ClaI or ClaI–ScaI).

12 D. Singh et al. / Molecular & Biochemical Parasitology 126 (2003) 9–14

The labelled DNA was loaded onto a benzoylated naphthoy-lated DEAE–cellulose (BND–cellulose) column that hadbeen pre-equilibrated with 0.3 M NET8 [0.3 M NaCl, 10 mMTris–HCl (pH 8), 1 mM EDTA]. The column was washedthrice with 0.3 M NET8 followed by three washes with 0.8 MNET8 [0.8 M NaCl, 10 mM Tris–HCl (pH 8), 1 mM EDTA].This fraction contains linear double-stranded DNA and istermed ‘salt wash’. DNA enriched in replication intermedi-ates was then eluted by washing the column three times with1 M NET8/1.8% (w/v) caffeine [1 M NaCl, 10 mM Tris–HCl(pH 8), 1 mM EDTA, 1.8% (w/v) caffeine]. The ‘salt’ and‘caffeine’ washes were precipitated with ethanol, washedwith 70% ethanol, dried and suspended in TE. DNA fromthe ‘salt wash’ and ‘caffeine wash’ were analysed for ra-dioactivity (Cerenkov counting). The latter was denaturedand used as probe in southern hybridisation.

2.5. Southern hybridisation

Plasmid constructs carrying plDNA sequences were di-gested with restriction enzymes to release their respectiveinserts, electrophoresed on a 0.8% agarose gel and blottedonto nylon membrane using standard procedures[26]. The5′ end-labelled nascent DNA (‘caffeine wash’) was used asprobe for hybridisation. The membranes were washed andexposed to X-ray film for autoradiography. Densitometricanalysis was carried out using ImageMaster 1D Elite soft-ware (Amersham-Pharmacia Biotech).

3. Results and discussion

3.1. The site of plDNA replication lies withinthe inverted repeat

Bi-directional DNA replication in plastid DNA oftenproceeds via the formation of D-loops of discrete sizesthat subsequently form bi-directional replication structures[12,27]. BND–cellulose, that binds single-stranded nucleicacids more strongly than double-stranded DNA has beenused to enrich for DNA molecules containing replicationintermediates from various organisms[25]. Single-strandedD-loop regions, that may contain up to∼2500 nt longsingle-stranded DNA, as well as expanding replication forkscontaining small transient single-stranded DNA regionsbind to BND–cellulose. If replication initiation occurs atspecific locations within a genome, it is possible to identifythese regions by using 5′ end-labelled nascent DNA (fromD-loops or bi-directionalori) as probe against the targetDNA [24,28].

Nascent strands ofP. falciparum plDNA isolated fromthe late trophozoite-early schizont stages were labelled atthe 5′ ends, digested with restriction enzymes, enrichedby BND–cellulose chromatography and used as probes forhybridisation with cloned inserts of plDNA (Fig. 2A andB). When labelled nascent DNA digested withXbaI andClaI

(Fig. 1B) was hybridised to fourteenP. falciparum plDNAfragments, a strong signal was obtained in three fragments(Fig. 2A, lanes 2, 7 and 13). All three fragments (A1988,A1820 and A650) are located within the IR region of plDNA(Fig. 1A). The two fainter signals observed for B1426and A1288 (Fig. 2A, lanes 10 and 12, respectively) disap-peared after more stringent washing of the membrane (datanot shown). Signal intensities were normalised for DNAamounts by comparing vector DNA levels (Fig. 2A, panel(i)). The normalised relative intensities of the three bandswere calculated to be 1:1:1.3 (A1988:A1820:A650) re-vealing near-equal representation of end-labelled fragmentscovering these regions in the nascent DNA preparation.Similar signals were obtained in two repeat experiments(data not shown). Our results indicate that replication ini-tiation sites ofP. falciparum plDNA are restricted to theIR region. Since sequences of the IRA and IRB sectors areinverted repeats of each other, replication origins of theP. falciparum plDNA must lie within each sector (IRA andIRB) of the inverted repeat.

3.2. Apicoplast DNA possibly contains two replicationorigins within each segment of the IR

As end-labelled nascent DNA digested withXbaI andClaIgave signals in both the 1820 and 1988 bp fragments, we ad-dressed the question as to whether the two fragments containindependent initiation sites. The nascent DNA preparationwas thus digested withClaI and ScaI (Fig. 1B). ScaI cutsplDNA at the edge of the 1820 bp fragment adjacent to the1988 bp fragment (Fig. 1A) and would digest 5′ end-labellednascent DNA molecules emanating from the 1988 bp frag-ment and entering the 1820 bp fragment or vice versa. Ifreplication initiates from a single initiation site (either in the1988 or 1820 bp region) within an IR segment, hybridisa-tion with the ClaI- and ScaI-digested nascent DNA probewould generate a signal only for the plDNA fragment con-taining the initiation site. However, Southern hybridisationof plDNA cloned inserts with theClaI- and ScaI-digestedprobe gave signals in both the 1820 and 1988 bp fragments(Fig. 2B, panel (ii)). An equivalent signal was also observedfor the 650 bp fragment. Normalisation for DNA amountswith vector DNA (Fig. 2B, panel (i)) gave the relative in-tensity ratio of 1.06:1:1.4 (A1988:A1820:A650). Our resultsindicate the existence of at least two initiation sites withineach IR segment ofP. falciparum plDNA. The signal seen inthe 650 bp fragment could be due to its 240 bp overlap withthe 1820 bp fragment (Fig. 1A) or a result of hybridisationwith nascent DNA emanating from the 1820 bp fragment.The presence of anori within the 650 bp region is, however,not ruled out.

Chloroplast DNA replication in algae (e.g.Chlamy-domonas andEuglena) and higher plants (e.g. pea, tobaccoand Oenothera) initiates from varying number of replica-tion origins at or near the inverted repeat region[12,28,29].There is no primary sequence similarity between the

D. Singh et al. / Molecular & Biochemical Parasitology 126 (2003) 9–14 13

Fig. 2. Hybridisation with end-labelled nascent DNA indicates that plDNA replication initiates within the IR. (A) Nascent DNA digested withXbaI andClaI was used as probe. Panel (i), ethidium bromide stained agarose gel containing vector-plDNA constructs digested to release the plDNA inserts; panel(ii), plDNA inserts hybridized with end-labelled nascent DNA. (B) End-labelled nascent DNA digested withClaI and ScaI was used as probe. Panel(i), plasmid vector hybridized with labelledamp gene fragment; panel (ii) plDNA inserts hybridized with end-labelled nascent DNA. Markers (M) areindicated in base pairs.

chloroplastori sequences of these species, although certainfeatures such as AT-rich stretches followed by GC-rich se-quences as well as hairpin loops at or nearori are sharedby these sequence elements. Theori sequences have beenfound to lie outside the conserved chloroplast 16S or 23SrRNA genes. Our results, however, indicate that aP. falci-parum apicoplastori may lie within the LSU rRNA gene(covered by the 1988 bp fragment). Although plDNA com-prises 86.9% AT, the LSU region does contain AT stretchesfollowed by GC-rich regions. Sequences capable of forminghairpin loops are found throughout the plDNA sequenceand cannot serve as indicators of putativeori elements.

The presence of at least two DNA replication initiationsites within each IR segment ofP. falciparum plDNA issuggestive ofori distribution similar toOenothera chloro-plast where two D-loops flanking the 16S rRNA genes ineach IR have been identified[29]. Unlike T. gondii plDNA,that has been proposed to follow the rolling circle mode of

replication[11], circular molecules ofP. falciparum plDNAare likely to follow the bi-directional replication mode uti-lizing initiation sites within each segment of the IR. Repli-cation may initiate at bi-directionaloris or via formationof D-loops (at least two bi-directionaloris/D-loops in eachsegment of the IR) followed by their fusion into Cairns-typeintermediates. It is also possible that the inverted repeatregion serves as a replication initiation zone containingmultiple sites of replication initiation that may be utiliseddifferentially by plDNA molecules. While this paper wasunder review, Williamson et al.[30] published their resultson P. falciparum plDNA replication. Using complementaryapproaches of two-dimensional gel analysis and electronmicroscopy they propose that plDNA replication proceedsvia two mechanisms. As also inferred by us, the first andpredominant mechanism follows the theta mode and in-volves initiation within the IR region. Williamson et al.additionally propose that a minor population of plDNA

14 D. Singh et al. / Molecular & Biochemical Parasitology 126 (2003) 9–14

molecules may follow the rolling circle mode utilising asyet unidentified initiation sites lying outside the IR.

Future studies to fine-map plDNA replication initiationsites within the IR, further confirmation of their in vivoactivity using other methods ofori analysis[25,31], anddetermination of DNA–protein interactions at or nearorisites would help elucidate the mechanism of replication oftheP. falciparum apicoplast genome.

Acknowledgements

We thank Dr. S.K. Puri for helpful discussions and J.P.Srivastava, Malavika Raman and S. Anuradha for technicalassistance. DS and SC are grateful to the Council for Scien-tific and Industrial Research for grant of Research Fellow-ships. This work was supported by a CSIR Young ScientistGrant to SH.

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