Molecular & Biochemical Parasitology 144 (2005) 133–141

Plasmodium falciparum DNA helicase 60 is a schizont stage specific,bipolar and dual helicase stimulated by PKC phosphorylation

Arun Pradhan, Virander S. Chauhan, Renu Tuteja∗

Malaria Group, International Centre for Genetic Engineering and Biotechnology, P.O. Box 10504, Aruna Asaf Ali Marg, New Delhi 110067, India

Received 5 April 2005; received in revised form 7 July 2005; accepted 8 August 2005Available online 31 August 2005

Abstract

The fundamental biology and the biochemical processes at different developmental stages of the malaria parasitePlasmodium falciparumhave not been explored in detail. As a step toward understanding the various mechanisms engaged in nucleic acid metabolism of this pathogen,particularly the essential enzymes involved in nucleic acid unwinding, recently, we have reported the isolation of the firstP. falciparum DEAD-box DNA helicase 60 (PfDH60), which contained striking homology with p68 protein [Pradhan A, Chauhan VS, Tuteja R. A novel ‘DEAD-box’

ovele schizontindsinase Cdependenttion and

metabolic

iza-es

cte-r all

leicro-

acidPin

nt-fily

DNA helicase fromPlasmodium falciparum is homologous to p68. Mol Biochem Parasitol 2005;140:55–60]. In this study, we show nimportant properties of PfDH60. Immunofluorescence assay studies revealed that the peak expression of PfDH60 is mainly in thstages of the development ofP. falciparum, where DNA replication is active. Interestingly, this is a bipolar DNA helicase, which unwdsDNA in both the directions. PfDH60 can also unwind RNA–DNA and RNA–RNA duplexes. PfDH60 is phosphorylated by protein kat the Ser and Thr residues. The helicase and ATPase activities of PfDH60 were stimulated after this phosphorylation. The cell-cycleexpression, bipolar translocation and dual nature collectively suggest that PfDH60 may be involved in the process of DNA replicadistinct cellular processes in the parasite and this study should make an important contribution in our better understanding of DNApathways such as repair, recombination and replication.© 2005 Elsevier B.V. All rights reserved.

Keywords: DEAD-box helicase; Malaria parasite; Schizont stage; Unwinding

1. Introduction

Malaria parasitePlasmodium falciparum is responsiblefor nearly all the malaria related deaths worldwide. Globalefforts to reduce the impact of malaria is intimately linked tothe study of complex biology of this parasite. In the humanhost during intra-erythrocyte development cycle ofP. falci-parum there are distinct stages such as ring stage, trophozoiteand schizont stage. A better understanding of the biologicalprocesses that the parasite uses during this developmentalcycle should lead to better therapeutics and amelioration ofthis devastating disease. In order to study one of the impor-tant biological processes such as nucleic acid metabolism, we

∗ Corresponding author. Tel.: +91 11 26189358; fax: +91 11 26162316.E-mail addresses: renu@icgeb.res.in, renututeja@hotmail.com

(R. Tuteja).

have started systematic study of isolation and charactertion of helicases from the parasite. DNA and RNA helicasare ubiquitously found in several species ranging from baria to mammals and have been shown to be important fothe cellular processes in prokaryotes and eukaryotes[1–3].These enzymes catalyze the unwinding of duplex nucacids and thereby play an essential role in virtually all pcesses involving DNA or RNA transactions[3,4]. A typicalhelicase reaction involves three sequential steps: nucleicbinding, NTP/dNTP binding and hydrolysis, and NTP/dNThydrolysis-dependent unwinding of duplex nucleic acideither 3′ to 5′ or 5′ to 3′ direction [1]. The energy forthe translocation is provided by intrinsic DNA-dependeNTPase activity of the helicase[1,3,4]. Because of the presence of sequence DEAD or DEAH or DEXH in motif II othe nine short conserved ‘helicase motifs’, the helicase famis also called DEAD-box protein family[1,3,5–7]. Multiple

0166-6851/$ – see front matter © 2005 Elsevier B.V. All rights reserved.doi:10.1016/j.molbiopara.2005.08.006

134 A. Pradhan et al. / Molecular & Biochemical Parasitology 144 (2005) 133–141

DNA helicases have been isolated from different organismsbut very little is known about the helicases from malariaparasites[4]. Studies regarding the function of helicases invarious systems excluding malaria parasite has indicated thatalthough some helicase functions may be redundant, otherhelicases are required for specific cellular processes[1,7,8].

In the recent past, we have cloned and characterizedin detail the first full-length DEAD-box DNA helicasefrom Plasmodium cynomolgi [9,10]. More recently, we havecloned the first full-length biochemically active DEAD-boxDNA helicase fromP. falciparum [11]. The ‘PlasmoDB’entry number for this gene is PFL1310c and its expressionprofile in ‘PlasmoDB’ shows that the maximum expressionis in the ‘early’ and ‘late’ trophozoite stages ofP. falciparum.ThisP. falciparum DNA helicase 60 (PfDH60) is homologousto p68 from various sources and contains ATP-dependentDNA helicase, DNA-dependent ATPase and ATP-bindingactivities [11]. In this paper, we report further characteri-zation of PfDH60. We show the stage-specific expression ofthis enzyme, dual nature and direction of unwinding and reg-ulation of helicase activity of PfDH60 by phosphorylationwith protein kinase C (PKC). The data suggest that PfDH60is a bipolar helicase and it can unwind a variety of nucleicacid hybrids. The phosphorylation of PfDH60 stimulates itshelicase and DNA-dependent ATPase activities. These datacollectively suggest that PfDH60 may be involved in DNAr

2

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pH 8.0) supplemented with 20 mM imidazole and proteaseinhibitor cocktail at 4◦C for 1 h (Sigma). The column wasextensively washed sequentially with binding buffer supple-mented with 50 and 100 mM imidazole and protease inhibitorcocktail. The recombinant His-tagged proteins were elutedwith 250 mM imidazole in protein buffer (20 mM Tris–HCl,pH 8.0, 250 mM NaCl, 10% glycerol and protease inhibitorcocktail). The protein was further dialyzed against the samebuffer without any imidazole or NaCl. The final purified pro-tein was checked for purity on 10% SDS-PAGE. This purifiedprotein was used for all the assays described in the followingsections. The concentration of the protein was determinedusing Bradford assay[13].

2.3. Preparation of substrate for DNA helicase assay

The various DNA helicase substrates used in the DNAunwinding assay were prepared in the same way asdescribed earlier[14]. The oligodeoxynucleotides were syn-thesized from Microsynth (Microsynth GmbH, Balgach,Switzerland). The regular substrate used in this study con-sisted of a32P-labelled 47-mer DNA oligodeoxynucleotide(5′-(T)15GTTTTCCCAGTCACGAC(T)15-3′) annealed toM13mp19 phage ssDNA to create a partial duplex. At both the5′- and 3′-ends, this oligodeoxynucleotide contains 15 base-pairs of non-complementary region. Ten nanograms of thiso -c MM d1 ).T with2 in2 la gi lyt tidew 4Bc pH7

2

thes ynth( nceso lows5T eo3T( esw boveb tide.F deo

eplication in the parasite.

. Materials and methods

.1. Materials

M13mp19 ssDNA was prepared using standard mets described[12]. NTPs and dNTPs were from Pharmia (Sweden) and [�-32P]ATP, and [�-32P]dCTP were purhased from Amersham Biosciences. Synthetic RNANA oligonucleotides were synthesized chemically. Pro-Sepharose was from Pharmacia.

.2. Expression and purification of protein

The complete open reading frame of PfDH60ubcloned into an expression vector pET-28a (Novaadison, WI, USA) atBamHI/HindIII sites. The expres

ion clones were transformed intoEscherichia coli BL21train (Novagen). Bacteria were grown in LB medium600 nm= 0.6. The expression of recombinant protein

nduced by 1 mM IPTG for 4 h. To purify the recombinrotein, the harvested bacterial cells were subjected to

reeze/thaw cycles at−70◦C. After suspension in lysis buff20 mM Tris–HCl, pH 8.0, 250 mM NaCl, 1% Triton a.5% Tween 20), the bacterial cells were further disrupteonication. After centrifugation at 4◦C, the soluble bacteriysates were allowed to bind to Ni-NTA (Qiagen, GmbH, G

any) in binding buffer (250 mM NaCl, 20 mM Tris–HC

ligodeoxynucleotide was labelled at 5′-end with T4 polynuleotide kinase (5U) in 50 mM Tris–HCl, pH 8.0, 10 mgCl2, 5 mM DTT, 0.1 mM EDTA, 0.1 mM spermidine an.85 MBq of [�-32P]ATP (specific activity 222 TBq/mmolhe labelled oligodeoxynucleotide was then annealed.5�g of single-stranded circular M13mp19 (+) DNA0 mM Tris–HCl (pH 7.5), 10 mM MgCl2, 100 mM NaCnd 1 mM DTT by heating at 95◦C for 1 min, transferrin

mmediately to 65◦C for 2 min and then cooling slowo room temperature. Non-hybridized oligodeoxynucleoas removed by gel filtration through a 1 ml Sepharoseolumn (Pharmacia, Sweden) with 10 mM Tris–HCl (.5), 1 mM EDTA and 100 mM NaCl.

.4. Preparation of other substrates

All the oligodeoxynucleotides used for makingubstrates in this study were synthesized from MicrosMicrosynth GmbH, Balgach, Switzerland). The sequef 17-mer and 32-mer oligodeoxynucleotides are as fol′-GTTTTCCCAGTCACGAC-3′ and 5′-TTCGAGCTCGG-ACCCGGGGATCCTCT AGAGT-3′. The sequence of thligodeoxynucleotides used for 5′-tail, 3′-tail and 5′- and′-tail substrates are as follows 5′-(T)15GTTTTCCCAG-CACGAC-3′, 5′-GTTTTCCCAGTCACGAC(T)15-3′, 5′-T)15GTTTTCCCAGTCACGAC(T)15-3′. These substratere also prepared in the same way as described ay using the respective labelled oligodeoxynucleoor DNA–RNA substrate, an RNA oligonucleotif the sequence 5′-GCAGGUCGACUCUA-3′ was

A. Pradhan et al. / Molecular & Biochemical Parasitology 144 (2005) 133–141 135

end-labelled as described above and annealed to a DNAoligodeoxynucleotide of 65 basepair of the sequence 5′-TTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGA-CCTGCAGGCATGCAAGC TTGGCGTAATCAT-3′. Afterpurification this substrate was used in the helicase reaction inthe presence of RNAase inhibitor (Sigma). For RNA–RNAsubstrate, the RNA helicase reaction was performed in thepresence of32P-labelled RNA duplex substrate and 1 unit ofRNAase inhibitor as described earlier[15].

2.5. Preparation of direction specific substrate

The partial duplex linear substrates were prepared byannealing the 5′-end labelled 65 basepair oligonucleotidementioned above with another 65 basepair oligonucleotide ofthe sequence 5′-CGGGTACCGAGCTCGAAGGGATCCT-CTAGAGTCGACCTGCAG GCATGCAAGCTTGGCGTA-ATCAT-3′. This gave the partial duplex structure shown inFig. 2A for measuring the 3′ to 5′ direction. The same labelledoligonucleotide was annealed with another 65 basepairoligonucleotide of the sequence 5′-TTCGAGCTCGGTA-CCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGC-ATGATTACGCCAAGCTT-3′. This gave the partial duplexshown inFig. 2B for measuring the 5′ to 3′ direction. Forconstructing another, 5′ to 3′ unwinding substrate, theoligodeoxynucleotide 32-mer (5′-TTCGAGCTCGGTA-C toMTDpia TP.Tt g oflu ro p19s wasd h1

2

l AmT1h tob r-w itiono ro-mt hores gel

was dried and exposed to hyper film with an intensifyingscreen for autoradiography. DNA unwinding was quantitatedas described previously[14].

2.7. DNA-dependent ATPase assay

The hydrolysis of ATP catalyzed by PfDH60 was assayedby measuring the formation of32P from [�-32P]ATP. Thereactions conditions were identical to those described forthe helicase reaction, except that the32P-labelled helicasesubstrate was replaced by a mixture of [�-32P]ATP (specificactivity 222 TBq/mmol) and cold ATP (1 mM). The reac-tion was performed for 2 h at 37◦C both in the presenceand absence of 100 ng of M13 mp19 ssDNA, followed bythin layer chromatography and the quantitation was done asdescribed earlier[16].

2.8. Protein phosphorylation

PfDH60 was phosphorylated by using PKC from Promegaunder optimal assay conditions. The reaction mixture con-tained 200 ng PfDH60, 1 ng PKC and PKC buffer (20 mMHEPES buffer, 2 mM CaCl2, 10 mM MgCl2, 1 mM ATP or5�Ci [�-32P]ATP (specific activity 222 TBq/mmol)). Thereaction was incubated at 30◦C for 30 min. After incubationthe phosphorylation reaction mixture was used for helicasea AGEg ning.F pho-r andh .5NHs rateda withs photy-r tog-r li wasd sedf

2

pro-d pro-t gGf

2

f dif-f ace-t omt 10%f midc BS

CCGGGGATCCTCTAGAGT-3′) was first annealed13mp19 ssDNA and then labelled at 3′-OH end in 40 mMris–HCl (pH 7.5), 10 mM MgCl2, 50 mM NaCl and 1 mMTT with 50�Curie [�-32P]dCTP and 5 units of DNAolymerase I (large fragment) at 23◦C for 20 min. The

ncubation was continued for an additional 20 min at 23◦Cfter increasing the dCTP to 50 mM using unlabelled dChis was digested withSmaI and purified by gel filtration

hrough 1 ml sepharose 4B. The substrates consistinong linear M13ssDNA with short duplex ends for 3′ to 5′nwinding was prepared by first 5′-end labeling of 32-meligodeoxynucleotide and then annealing with M13msDNA as described above. The annealed substrateigested withSmaI and purified by gel filtration througml of Sepharose 4B.

.6. DNA helicase assay

The helicase assay measures the unwinding of32P-abelled DNA fragment from a partially duplex DN

olecule. The reaction mixture (10�l) containing 20 mMris–HCl (pH 8.0), 8 mM DTT, 1.0 mM MgCl2, 1.0 mM ATP,0 mM KCl, 4% (w/v) sucrose, 80�g/ml BSA,32P-labelledelicase substrate (∼1000 cpm) and the helicase fractione assayed was incubated at 37◦C for 60 min (unless otheise indicated). The reaction was terminated by the addf 0.3% SDS, 10 mM EDTA, 5% glycerol and 0.03% bophenol blue. After further incubation at 37◦C for 5 min,

he substrate and products were separated by electropis on a 12% non-denaturing polyacrylamide gel. The

-

nd ATPase assays or SDS-PAGE analysis. The SDS-Pel was dried and exposed for autoradiography after staior phospho-amino acid analysis, the radioactive phosylated PfDH60 band was excised from the dried gelydrated in water. The eluted protein was hydrolyzed in 7Cl for 2 h in boiling water bath as described[17]. Theupernatant was collected after centrifugation, concentnd spotted on 3 MM Whatmann chromatography papertandards phosphoserine, phosphothreonine and phososine obtained from Sigma. The solvent used for chromaaphy was: propionic acid, 1 M NH4OH and isopropyl alcohon the ratio of 45:17.5:17.5, v/v. The chromatogramried, stained with ninhydrin solution (0.3%) and expo

or autoradiography.

.9. Antibody production

Recombinant and purified PfDH60 was used for theuction of polyclonal antibodies in mice using standard

ocols[13]. The polyclonal antibodies were purified as Iractions using protein A-Sepharose as described[13].

.10. Immunofluorescence assay

For this assay smears of parasitised red blood cells oerent developmental stages were prepared and fixed inone for 5 min followed by chilled methanol for 15 s at roemperature. For blocking, the slides were incubated inetal calf serum in phosphate buffered saline (PBS) in a huhamber at 37◦C for 2 h. The slides were washed with P

136 A. Pradhan et al. / Molecular & Biochemical Parasitology 144 (2005) 133–141

and incubated with purified IgG of anti-PfDH60 antibodiesat 1:200 dilutions in PBS containing 10% fetal calf serum for1 h at 37◦C. The slides were washed four times with PBSfor 15 min each and then incubated for 1 h at 37◦C with sec-ondary antibody (fluorescein isothiocyanate-conjugated anti-mouse IgG (Sigma) diluted 1:100 in PBS containing 10%fetal calf serum). After washing the slides were incubatedin 4′,6′-di-amidino-2-phenylindole dihydrochloride (DAPI)(2�g/ml in PBS) for nuclear staining. The slides were washedthrice with PBST (PBS, 0.5% Tween 20) for 10 min eachand twice with PBS for 10 min each and mounted with anti-fade reagent (Fluroguard, BioRad, USA) and viewed underoil immersion. Confocal images were collected using a Bio-Rad 2100 laser-scanning microscope attached to a NikonTE 2000U microscope and the figures were prepared usingAdobe Photoshop.

2.11. Immunoprecipitation and Western blotting

The parasites were released from infected erythrocytesby treatment with 0.1% (w/v) saponin. Cell-free proteinextracts from specific parasite stages or synchronous cul-tures[18] were prepared by sonicating the parasite pellets ina buffer containing 10 mM Tris–HCl, pH 7.5, 100 mM NaCl,5 mM EDTA, 1% Triton X-100 and protease inhibitor cock-tail (Sigma). The lysates were cleared by centrifugation at1 i-t zoiteo puri-fi s ofl to ibodyc eb eadsw timesa pro-t rans-f singt anew -t eni ma)c opedu fac-t uri-fi asp suree

3

3a

lizet

(Fig. 1A–C) as described in Section2. It is interesting tonote that there was no expression of PfDH60 in the ringand trophozoite stages and expression was mainly in theschizont stages of the parasite. The results indicate thatPfDH60 is expressed in a cell-cycle dependent manner withpeak expression in the schizont stages. The Western blottingresults with protein extracts from various erythrocyticdevelopmental stages also show expression of PfDH60mainly in the schizont stage (Fig. 1D (i), lane 3). Anidentical blot was probed with anti-HRPII antibodies, whichconfirmed that equal amount of protein, was loaded for eachtime point (Fig. 1D (ii)). The ‘PlasmoDB’ entry number forPfDH60 is PFL1310c and the transcriptome data indicatethat its transcript peaks in late trophozoites. Whereas in‘PlasmoDB’ proteome data the mass spectrometry basedexpression evidence indicates that it is present in trophozoitestages.

3.2. PfDH60 unwinds DNA bi-directionally from 3′ to 5′and from 5′- to 3′-ends

The standard strand-displacement helicase assay mea-sures the unwinding of32P-labelled DNA fragment frompartially duplex nucleic acid. The unwinding activity ofhomogenously pure PfDH60 was tested by using this assaywith various direction-specific substrates. All helicases pref-e ov-i ofu thee irec-t es,t -ea ction2 td uslypp ub-sa t in3 arf urs c-t tha ounto onec est-i itha n ofu

3

Au l then

3,000× g for 20 min at 4◦C. PfDH60 was immunoprecipated from the synchronized parasite lysate (ring, trophor schizont stage) and uninfected RBC lysate usinged IgG of anti-PfDH60 antibodies. Hundred microgramysate was incubated with the purified IgG at 4◦C overnighn an end-to-end shaker. Subsequently, antigen–antomplexes were incubated with 50�l of protein A-sepharoseads (Amersham) for 2 h with end-to-end shaking. Bere washed with phosphate-buffered saline severalnd were finally suspended in PBS. Immunoprecipitated

eins were resolved by SDS-PAGE and subjected, after ter onto nitrocellulose membrane, to western blotting uhe purified IgG of anti-PfDH60 antibodies. The membras first incubated overnight at 4◦C with a 1:1000 dilu

ion of the purified IgG of anti-PfDH60 antibodies and thncubated with the appropriate secondary antibody (Sigoupled to alkaline phosphatase. The blot was develsing BCIP and NBT (Sigma) according to the manu

urer’s instructions. An identical blot, which contained ped HRPII (histidine rich protein II) instead of PfDH60 wrepared and probed with anti-HRPII antibodies to enqual loading of parasite proteins for each time point.

. Results

.1. Localization of PfDH60 by immunofluorescencessay

The purified antibodies to PfDH60 were used to locahis protein in various developmental stages ofP. falciparum

rentially unwind nucleic acids in a polar fashion by mng unidirectionally on the bound strand. The directionnwinding by helicase is defined by the strand to whichnzyme binds and moves. For the determination of d

ion of unwinding by PfDH60, four different substratwo specific for 3′ to 5′ direction (Fig. 2A and C top panls) and other two specific for 5′ to 3′ direction (Fig. 2Bnd D, top panels) were prepared as described in Se. The DNA unwinding activity, using∼1 ng of differenirection-specific substrates with 100 ng of homogenourified PfDH60 enzyme is shown inFig. 2A–D (bottomanels). The release of radiolabelled DNA from the strates ofFig. 2A and C and from the substrates ofFig. 2Bnd D by PfDH60 enzyme will indicate the movemen′ to 5′ and 5′ to 3′ directions, respectively. It is clerom the results that PfDH60 could unwind all the foubstrates (Fig. 2A–D) indicating that it contains bidireional DNA unwinding activity. The helicase activity will these substrates was directly proportional to the amf PfDH60 used in the reaction but the data for onlyoncentration of PfDH60 have been shown. It is interng to note that PfDH60 showed almost same activity wll the substrates used for the determination of directionwinding.

.3. PfDH60 helicase activity on various substrates

In order to determine the specificity of PfDH60, its DNnwinding activity was tested with various substrates. Alon-forked or forked substrates used in this study (Fig. 3A–D)

A. Pradhan et al. / Molecular & Biochemical Parasitology 144 (2005) 133–141 137

Fig. 1. Localization of PfDH60 in different intra-erythrocytic stages ofP. falciparum by immunofluorescence staining and confocal microscopy. The cellswere fixed and immunostained with purified primary antibodies against PfDH60 followed by fluoroisothiocyanate-labelled secondary antibody and thencounterstained with DAPI. In each panel, single confocal image of each stage is shown. (A) Ring stage, (B) trophozoite stage and (C) schizont stage. Ineachpanel (i) phase contrast image; (ii) image of cell stained with DAPI (blue); (iii) immunofluorescently stained cell (green); (iv) super-imposed image. Thesedata are representative of three separate experiments. Control normal mouse sera produced no fluorescence (data not shown). (D) Western blotting (i)the totalprotein lysate from different developmental stages was immunoprecipitated with purified IgG of anti-PfDH60 antibodies and the resulting immunoprecipitateswere separated by SDS-PAGE and probed with the same anti-sera after blotting on to nitrocellulose as described in Section2. Lane 1, protein from ring stage;lane 2, protein from trophozoite stage; lane 3, protein from schizont stage; lane 4, protein from uninfected RBC; lane 5, purified PfDH60. (ii) An identicalblot, which contained purified HRPII in lane 5 (instead of purified PfDH60), was incubated with anti-P. falciparum HRPII antibodies to ensure equal loadingof parasite proteins for each time point.

had identical sequence and same duplex length (17 basepair)but differed only in the presence of non-complementary tailsof 15 nucleotides. The assay was performed by using about100 ng of homogenously purified PfDH60 and∼1 ng of eachlabelled substrate in the presence of 1.0 mM MgCl2, 1.0 mMATP and 10 mM KCl. PfDH60 showed almost same activitywith substrates with no tail (Fig. 3A, lane 2) or with sub-strates having a tail at 3′-end, 5′-end or at both ends (lane 2of Fig. 3B–D). All of these reactions were dependent on theconcentration of PfDH60 used (data not shown). It was inter-esting to note that PfDH60 was unable to unwind the longerduplex region of 32 basepair in the no-tail substrate (Fig. 3E,lane 2) or in the substrate with tails at both the ends (Fig. 3F,lane 2).

3.4. RNA–DNA, RNA–RNA and DNA–RNA helicaseactivities of PfDH60

RNA–DNA unwinding activity was measured by usingRNA–DNA substrate (consisting of radiolabelled 14-merRNA oligonucleotide annealed to 65-mer oligodeoxynu-cleotide) with 100 and 200 ng of purified PfDH60 enzyme.The results reveal that PfDH60 shows RNA–DNA helicaseactivity in a concentration dependent manner (Fig. 3G, lanes2 and 3). The RNA unwinding activity of PfDH60 wasmeasured by using partial duplex of RNA–RNA (radiola-belled 17-mer RNA oligonucleotide annealed to 1 kb RNA)with 100 and 200 ng of purified PfDH60 enzyme. Withthis substrate also PfDH60 protein shows RNA unwinding

138 A. Pradhan et al. / Molecular & Biochemical Parasitology 144 (2005) 133–141

Fig. 2. (A–D) Direction of unwinding by PfDH60. The structures of thelinear substrates for 3′ to 5′ direction (A and C) and for 5′ to 3′ direction(B and D) are given at the top of each panel. The release of radiolabelled16-base DNA from the substrate of panel C and of 17-base DNA from thesubstrate of panel D by the PfDH60 indicates the movement in the 3′ to 5′or in the 5′ to 3′ direction, respectively. In each gel, lane 1 is the reactionwithout enzyme (C); lane 2 is the reaction with 100 ng of pure PfDH60 (E)and lane 3 is the boiled substrate (B). Asterisk denotes the32P-labelled end.S denotes the substrate and UD is unwound DNA.

activity in a concentration dependent manner (Fig. 3H, lanes2 and 3). The DNA–RNA unwinding was measured by usinga partial duplex substrate (consisting of radiolabelled 17-meroligodeoxynucleotide annealed to the same 1 kb RNA) with100 and 200 ng of purified PfDH60 enzyme. The resultswith this substrate show that the activity is dependent on theenzyme concentration (Fig. 3I, lanes 2 and 3). All of theseactivities were dependent on the presence of Mg2+ and ATPsince the enzyme did not show unwinding activity withoutMg2+ and ATP (data not shown).

3.5. Phosphorylation of PfDH60 by protein kinase C

Computer analysis (NetPhos, Expasy) of amino acidssequence of PfDH60 revealed that it contains a few potentialSer and Thr residues for PKC phosphorylation. In order tocheck the effect of phosphorylation on enzyme activity ofPfDH60, its phosphorylation was performed by incubatingthe protein (PfDH60) with [�-32P]ATP in the presence ofPKC (from animal sources). In this experiment, myelinbasic protein (MBP) was used as a positive control andbovine serum albumin (BSA) as a negative control forPKC. The phosphorylation of proteins was examined

Fig. 3. (A–F) Unwinding activity of PfDH60 with various forked and non-forked substrates. The helicase reaction was performed under standard assayconditions as described in Section2. Each panel shows the structure of thesubstrate used and the autoradiogram of the gel. Asterisks denote the32P-labelled ends. In each panel, lanes 1 and 3 are the reaction without enzyme (C)and with boiled substrate (B), respectively. In each panel, lane 2 is reactionwith 100 ng of pure PfDH60 (E). (G–I) Unwinding activity of PfDH60 withRNA–DNA (G), RNA–RNA (H) and DNA–RNA (I) substrates. The helicasereaction was performed under standard assay conditions as described inSection2. The structure of the substrate used is shown on the top of theautoradiogram of the gel in each panel. Asterisks denote the32P-labelledends. In each panel, lanes 1 and 4 are the reaction without enzyme (C)and with boiled substrate (B), respectively. In each panel, lanes 2 and 3 arereaction with 80 and 100 ng of pure PfDH60 (E).

by SDS-polyacrylamide gel electrophoresis followed byautoradiography. The results clearly indicate that 60 kDapolypeptide of PfDH60 (Fig. 4A, lane 3) and 32 kDapolypeptide of MBP (Fig. 4A, lane 2) were phosphorylated

A. Pradhan et al. / Molecular & Biochemical Parasitology 144 (2005) 133–141 139

Fig. 4. (A–E) In vitro phosphorylation and stimulation of DNA helicase and ATPase activities of PfDH60 by PKC. (A) The kinase reaction was carried out understandard conditions in the presence of Mg2+ and [�-32P] ATP followed by SDS-PAGE and autoradiography as described in Section2. The phosphorylationreaction was performed with 100 ng of PfDH60 protein. Autoradiogram shows 60 kDa band of PfDH60 phosphorylated with PKC (lane 3). Lane 2 is thereaction with myelin basic protein (MBP) protein (as positive control) and lane 1 is reaction with BSA (as negative control). (B and C) Phosphoamino acidanalysis of PfDH60 phosphorylated by PKC. The phosphorylated32P-labelled 60 kDa protein of PfDH60 was hydrolyzed in 6N HCl and finally analysed bypaper chromatography as described in Section2. The positions of standard phosphoamino acids visualised after ninhydrin staining are shown in the panelB. (D) DNA helicase activity of PfDH60 after phosphorylation with PKC. Lane 1, control without PfDH60; lane 2, heat-denatured substrate; lanes 3 and 4,PfDH60 sample used for phosphorylation (80 and 100 ng, respectively); lanes 5 and 6, PfDH60 treated under phosphorylation conditions (in the absenceofPKC; 80 and 100 ng, respectively) lanes 7 and 8 PfDH60 after phosphorylation with PKC (80 and 100 ng, respectively). The structure of the substrate usedisshown on extreme left. Asterisk denotes the32P-labelled end. Percent unwinding is written at the bottom of each lane. (E) ssDNA-dependent ATPase activityof PfDH60 after phosphorylation with PKC. Lane 1, prephosphorylated PfDH60 (80 ng) without PKC (PKC sample); lane 2, phosphorylated PfDH60 (80 ng).The position of Pi and ATP are marked on the right side. Percent Pi released is written at the bottom of each lane.

by PKC, while the negative control (66 kDa BSA) did notshow any phosphorylation (Fig. 4A, lane 1). The phospho-rylation was dependent on the time of incubation and theamount of PfDH60 (data not shown).

3.6. PfDH60 is phosphorylated at Ser and Thr residues

The phosphorylated 60 kDa band of PfDH60 was elutedfrom the gel and subjected to phospho-amino acid analysisfollowed by paper chromatography as described in Section2. The positions of standard Ser and Thr after staining areshown inFig. 4B, lanes 1 and 2. The autoradiogram afterchromatography revealed that PKC phosphorylates on Serand Thr residues of PfDH60 (Fig. 4C, lane 1).

3.7. Stimulation of DNA helicase and ATPase activitiesof PfDH60 by phosphorylation

The effect of PKC phosphorylation on DNA helicaseand ssDNA-dependent ATPase activities of PfDH60 wastested. DNA helicase activity of the homogenously puri-fied PfDH60 used for the phosphorylation experiment isshown in Fig. 4D, lanes 3 and 4. The results reveal thatboth the ssDNA-dependent ATPase (Fig. 4E, lane 2) andthe DNA helicase (Fig. 4D, lanes 7 and 8) activities ofPfDH60 were stimulated after phosphorylation of PfDH60with PKC as compared to without PKC phosphoryla-tion (Fig. 4E, lane 1 andFig. 4D, lanes 5 and 6, res-pectively).

140 A. Pradhan et al. / Molecular & Biochemical Parasitology 144 (2005) 133–141

4. Discussion

The investigation of components of the machineryinvolved in DNA–RNA metabolism ofP. falciparum is animportant step towards understanding this complex process.It has been shown that DNA replication occurs at distinctdevelopmental stages in the parasite. We have made an effortto study one of the important component namely helicase ofthis machinery. In this paper, we show for the first time theimportant properties of the first helicase PfDH60 (homologueof p68) and report that it is expressed in a cell-cycle depen-dent manner. p68 is one of the best characterized member ofthe ‘DEAD-box’ protein family of helicases and it has wellconserved orthologues from yeasts to humans[19–22]. Ourdata demonstrate that PfDH60 is a dual and bipolar DNAhelicase and its phosphorylation by PKC up-regulates thehelicase and DNA-dependent ATPase activities. The bipolardirection, dual nature and expression data collectively sug-gest that PfDH60 may be involved in the process of DNAreplication in the parasite.

Interestingly, PfDH60 translocates in both 3′ to 5′ and 5′to 3′ directions along the bound strand, suggesting that it is abipolar DNA helicase. Generally, bipolar helicase activitiesare not very common among the DEAD-box family of heli-cases. But bi-directional RNA unwinding has been reportedfor human p68[23]. In addition to this, the bipolar DNA heli-c fromBD sb ro-c ofP cidst kedw H60c ndl gerd ). ThelRMa

P-d Au tudyta s ini asa rk d sof on-t nlya andR

tinge d isk ction

and regulation of activities of many proteins[32–34].Recently, a kinase PfPKB, which has about 65% homologywith the catalytic domain of PKC has been reported fromP.falciparum [35]. Our results reveal that PfDH60 is a substrateof PKC from animal sources and its enzyme activities arestimulated after this phosphorylation. On the other hand, ithas been reported that the RNA helicase and ATPase activitiesof human p68 protein are inhibited by PKC phosphorylation[32,34]. Furthermore, phospho-amino acid analysis showedthat both Ser and Thr residues are phosphorylated with PKC.It has been reported previously that cdc2 and CK2 phos-phorylation up-regulate the helicase activity of PDH65 andPKC phosphorylation up-regulates the pea topoisomeraseI activity [33,36]. Overall, the results of phosphorylationstudy suggest that PKC might contribute to the physiologicalregulation of DNA helicase and ATPase activities in the cell.

In this study, we have reported numerous important activ-ities of PfDH60. We have shown that it is a dual and bipolarhelicase, which suggests that it may be involved in multiplesteps in nucleic acid metabolism in the parasite. The PKCphosphorylation may contribute to the physiological regu-lation of the activities of PfDH60 and overall nucleic acidmetabolism in the parasite. We have shown that PfDH60accumulates to peak levels in the schizont stages of the devel-opment ofP. falciparum, which is in agreement with theexpression of otherP. falciparum DNA replication genes sucha asa es ofp ndP e fort ani plea ro-v dingo

A

evel-o cro-s anI omeT t ofB wl-e

R

NA

ing

ind

ase activities have also been reported for PcrA helicaseacillus anthracis andStaphylococcus aureus [24] and HerANA helicase from thermophilic archea[25]. Since p68 haeen shown to be involved in a number of biological pesses[26] it may be possible that this bipolar propertyfDH60 helps it to work on a wide spectra of nucleic a

argets in the parasite. The activity of PfDH60 was checith various substrates and results demonstrated that PfDontains limited unwinding activity and it could not unwionger duplexes in vitro, perhaps in order to unwind lonuplexes it requires some additional accessory protein(s

imited unwinding activity has also been reported forE. coliep protein helicase[27], E. coli DnaB helicase[28] humanCM4, -6 and -7 protein complex[29], PDH45/eIF4A[30]nd PcDDH45[9].

In this study, we show that PfDH60 contains ATependent RNA–RNA, RNA–DNA as well as DNA–RNnwinding activities. We have reported in the previous s

hat PfDH60 contains RNA-dependent ATPase activity[11]nd this RNA-dependent ATPase activity of PfDH60 help

ts RNA unwinding activity. The RNA helicase activity hlso been shown in human p68[23,31]but to the best of ounowledge its DNA helicase activity has not been reportear. Most probably PfDH60 is the first p68 homologue caining both DNA and RNA helicase activities. There are ofew proteins known which are endowed with both DNANA helicase activity[4].The phosphorylation plays important role in regula

nzymatic activities. PKC is a Ser and Thr kinase annown to be involved in gene expression, signal transdu

s topoisomerase I[37]. Interestingly, the kinase PfPKB wlso reported to be present mainly in the schizont stagarasite[35]. The concomitant expression of PfDH60 afPKB suggest that PfDH60 may be an in vivo substrat

his kinase inP. falciparum. The characterization of suchmportant dual and bipolar helicase with intrinsic multictivities and possible role in DNA replication may pide a significant contribution to our better understanf DNA–RNA metabolism in parasite.

cknowledgements

This work was supported by Defence Research and Dpment Organization grant (to R.T.). The confocal micope facility at ICGEB, New Delhi, is funded bynternational Senior Research Fellowship of the Wellcrust (UK). Infrastructural support from the Departmeniotechnology, Government of India is gratefully acknodged.

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