A novel ‘DEAD-box’ DNA helicase from Plasmodium falciparum is homologous to p68

Molecular & Biochemical Parasitology 140 (2005) 55–60

A novel ‘DEAD-box’ DNA helicase from Plasmodium falciparum is homologous to p68夽 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 27 October 2004; received in revised form 7 December 2004; accepted 8 December 2004 Available online 13 January 2005 Keywords: ATPase activity; DNA helicase; Malaria; p68; Plasmodium falciparum; Unwinding activity

Duplex DNA unwinding is catalyzed by a group of enzymes called DNA helicases, which act in an ATP-dependent fashion to produce the ssDNA template [1]. By catalyzing the unwinding of duplex DNA, helicases play an essential role in many cellular processes such as DNA replication, repair, recombination and transcription [2–5]. Mostly helicases from the variety of organisms contain about nine short conserved amino-acid sequence fingerprints (designated Q, I, Ia, II, III, IV, V and VI), called ‘helicase motifs’ [6–9]. Because of the presence of the sequence DEAD or DEAH or DEXH in motif II the helicase family is also called ‘DEAD-box’ protein family. In this family of proteins, the core, which harbors the conserved motifs, is usually flanked by specific aminoand carboxy-terminal extensions that vary widely in length and sequence [5]. It has been suggested that ‘DEAD-box’ proteins usually perform very specific roles in vivo [5]. Although a number of DNA helicases have been characterized from a variety of sources such as bacteriophage, bacteria, fungal, viral, plant and animal systems [4,5] but not much work has been done on helicases from malaria parasites. It is interesting to note that the genomic sequence of Plasmodium falciparum shows the presence of multiple putative ‘DEADbox’ helicase genes. The pathogenicity of P. falciparum mainly results from its rapid rate of asexual reproduction in the host and its ability to sequester in small blood vessels. There are a limited number of drugs in widespread use for the treatment of P. falciparum 夽 The sequence data reported in this paper have been submitted to the

GenBank and the accession number is AY700082. ∗ Corresponding author. Tel.: +91 11 26189358; fax: +91 11 26162316. E-mail addresses: [email protected], renu [email protected] (R. Tuteja). 0166-6851/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.molbiopara.2004.12.004

malaria and this parasite slowly has developed resistance to nearly all available anti-malarial drugs [10,11]. The search for novel effective, safe and affordable anti-malarial drugs for P. falciparum malaria is one of the most important tasks to pursue. In the case of P. falciparum, based on differential gene expression study in the presence of chloroquine, it has been predicted that RNA helicase like proteins may be involved in anti-malarial action of the drug [12]. In order to find alternate ways to control malaria through inhibitors specific for DNA/RNA helicases, we have initiated a systematic study of helicases from malaria parasites. Recently we have purified and characterized an eIF-4A homologue, which is the prototype of the DEAD-box family of helicases, from Plasmodium cynomolgi [13,14]. In the present study we report the cloning of the first full-length helicase gene from P. falciparum and its sequence analysis. We also report the expression in Escherichia coli, purification and detailed characterization of the encoded functionally active helicase protein. Our studies show that this enzyme contains DNA helicase, ssDNAdependent ATPase and ATP-binding activities. It is highly homologous to p68, a member of the ‘DEAD-box’ protein family, which has well conserved orthologues from yeasts to humans [15–18]. The nuclear protein p68 was identified because of its immunological relations to the SV40 large tumor antigen and it has been shown to contain RNA helicase activity [18,19]. The ‘DEAD-box’ family of proteins has been reported from a variety of sources from bacteria to mammals [5]. In order to clone the first ‘DEAD-box’ helicase from P. falciparum, the genome of P. falciparum was searched using ‘DEAD-box’ motif as query. Out of a number of positive hits found, some were selected and analyzed further. The sequence with complete reading frame upstream and

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downstream of ‘DEAD-box’ motif, which also contained all the other helicase motifs including the ‘Q’ motif was chosen for further studies. Accordingly the primers PfH3F (5 GGGATCCATGGAAAGGCAAAATCTA-3 ) and PfH3R (5 -CCAAGCTTTCATCTTGAATAAGCTAT-3 ) were synthesized and used for PCR using P. falciparum genomic DNA as template. A single band of ∼1.5 kb was obtained which agreed with the size of the gene. To further confirm the validity of the gene, the primer pair PfH3M (5 GTTATTGATGAAGCTGATCGT-3 ) and PfH3R was used with the product of the first PCR as template. A product of expected size of ∼700 bp was obtained. The product of the first PCR was cloned in pGEMT vector and positive clones were sequenced. The sequence analysis showed a complete genomic clone of 1551 bp with a methionine at the start and termination codon at the end. The deduced amino-acid sequence of PfDH60 revealed a protein consisting of 516 amino acid residues with a predicted molecular mass of approximately 59.8 kDa. It is a basic protein with a calculated isoelectric point of ∼8.5. The sequence analysis also confirmed the presence of all the conserved domains of the DEAD-box protein family including the ‘Q’ motif [7,9] and therefore it belongs to the ‘DEAD-box’ protein family. Hence this protein is designated as Plasmodium falciparum DEAD-box helicase 60 kDa in size (PfDH60). The blast analysis of PfDH60 against ‘PlasmoDB’ database revealed that this gene is located on chromosome 12 of P. falciparum and it contains no introns. The ‘PlasmoDB’ entry number for this gene is PFL1310c and the expression profile of this protein in ‘PlasmoDB’ shows that the maximum expression is in the ‘early’ and ‘late’ trophozoite stages of P. falciparum. Southern blotting analysis of the genomic DNA of P. falciparum digested with BamHI showed a single hybridizing band at ∼9.0 kb suggesting the existence of a single copy of PfDH60 gene in P. falciparum (data not shown). A multiple alignment of amino-acid sequence homology search using NCBI database revealed that PfDH60 aligned contiguously and showed highest homology with a putative ‘DEAD-box’ helicase from Plasmodium yoelii (∼95%) and ∼68–70% similarity with p68 from various sources (Fig. 1) [20]. The blast analysis also indicated that this gene is more homologous to p68 from plants and is evolutionary closest to a protein from Arabidopsis thaliana. PfDH60 has ∼67% homology with Homo sapiens p68 and this protein has been shown to contain the helicase activity [19]. The distantly related protein is the well-characterized p68 protein from Saccharomyces cerevisiae [16]. Many of these proteins also contain glycine rich regions with RGG boxes but it is interesting to note that PfDH60 contains no obvious RGG boxes in its sequence. PfDH60 also contains the conserved phenylalanine 15 amino acid upstream of the ‘Q’ motif (Fig. 2A). The alignment results show that it contains a similar core region encompassing the conserved domains but it has different N- and C-terminal extensions which endow the protein with specialized function [5]. In contrast to all the other members of the

‘DEAD-box’ protein family, PfDH60 contains PTRELC in motif Ia in place of PTRELA (Fig. 2A). The substitution of Ala-Cys is a result of G–T transversion and C–G transition in the nucleotide sequence. In domain II, PfDH60 contains VIDEAD in place of VLDEAD. The substitution of Leu-Ile is due to the C–A transversion in the nucleotide sequence. There is also one amino-acid change in domain III: in place of commonly present sequence SAT, PfDH60 contains TAT (Fig. 2A). The Ser-Thr substitution is a result of G–C transition in the nucleotide sequence. The sequence TAT is the same as the consensus motif located at the corresponding position in the DEA/H family of proteins [21]. The sequence RGLD is present in motif V of PfDH60 (Fig. 2A). It has been speculated that this motif may function as an RNA-binding site in p68 family of proteins [19]. Only a few of the ‘DEAD-box’ proteins have been biochemically characterized. Therefore for biochemical characterization of PfDH60, the gene was cloned in the bacterial expression vector pET28a and the protein was produced after induction with IPTG. The SDS-PAGE analysis showed an additional polypeptide of ∼60 kDa that is IPTG-induced in E. coli transformed with pET-PfDH60 (Fig. 2B-i, lane 2) as compared to un-induced (Fig. 2B-i, lane 1). Although PfDH60 after expression is localized in both the soluble (cytosolic) fraction and inclusion bodies (Fig. 2B-i, lanes 3 and 4), it was further purified using Ni2+ -NTA-agarose matrix and the soluble fraction only. The final purification step yielded a purified PfDH60 enzyme, which showed a 59.8 kDa band on the SDS-PAGE (Fig. 2B-i, lane 5). The purity of this preparation was further confirmed by western blotting using anti-his antibodies, which detected a single band of 59.8 kDa (data not shown). This purified protein was used for all the assays described in the following sections. The results of western blotting with anti-pea p68 show that PfDH60 cross reacts with the polyclonal antibodies to pea p68 (data not shown). The DNA unwinding activity of PfDH60 was determined and characterized in detail by using the standard stranddisplacement assay as described [4]. The substrate for all the studies contained non-complementary tails of 15 nucleotides on both the 5 and 3 ends. Purified PfDH60 was allowed to react separately with penta-his antibodies, and IgG purified from the pre-immune sera and from the sera of the rabbit immunized with pea p68. The results showed that both the DNA helicase (Fig. 2B-ii, lanes 4 and 5) and ATPase (Fig. 2B-iii, lanes 3 and 4) activities were depleted with both the anti-pea p68 and penta-his antibodies. On the other hand there was no reduction of DNA helicase (Fig. 2B-i, lane 2) and ATPase activities (Fig. 2B-iii, lane 1) in pre-immune IgG treated samples. These data confirm that the DNA helicase and ATPase activities are due to the PfDH60 protein and not due to the contamination of E. coli helicases in the purified preparation. Further characterization of the helicase activity of PfDH60 showed that the enzyme was inactivated upon heating at 55 ◦ C for 5 min or after prolonged storage at 4 ◦ C (data not shown). The enzyme activity was inhibited with EDTA (5 mM), M13 ssDNA (10 ␮g/ml), Poly [A], Poly [U] (30 ␮M as P) and

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Fig. 1. Comparison of amino acid sequence of P. falciparum DEAD-box helicase (PfDH60) protein with other DEAD-box helicases (p68 homologues) from Plasmodium yoelii, Arabidopsis thaliana, Aspergillus nidulans, Homo sapiens and Saccharomyces cerevisiae. Multiple alignments were done using CLUSTAL W program, identical amino acids of each protein are black-boxed and similar ones are in gray box. The accession numbers of the aligned sequences are Plasmodium yoelii EAA15859, Arabidopsis thaliana NP974985, Aspergillus nidulans EAA57794, Homo sapiens NP006377 and Saccharomyces cerevisiae CAA36874.

synthetic RNA (1 ␮g/ml) but M13 RF1DNA, Poly [C], Poly [C/G] (30 ␮M as P) had no effect on the helicase activity (data not shown). For optimum activity PfDH60 required 30 mM KCl and at higher concentration (200 mM) the activity was completely inhibited (data not shown). It was very interesting to note that PfDH60 was able to unwind the partially duplex DNA substrate over a wide pH range from 5.0 to 10.0 (data not shown). But further characterization of the helicase activity was carried out at pH 8.0 only. In a recent study it has been shown that DNA unwinding by HCV NS3 helicase is less sensitive to pH ranging from 6.25 to 7.7 [22].

DNA helicase activity of PfDH60 was totally dependent on the hydrolysis of ATP, with an optimum concentration requirement of 1.0 mM and at 8.0 mM ATP concentration the helicase activity was completely inhibited (data not shown). Although dATP showed ∼95% of the activity, no other NTPs or dNTPs could be utilized as a cofactor (data not shown). This property is similar to a number of other reported helicases such as HDH I, calf thymus helicase I, PDH 65, PcDDH45 and eukaryotic eIF-4A [4,13,23]. The unwinding activity of PfDH60 required the hydrolysis of ATP since the poorly hydrolysable analog ATP␥S was inactive as a cofac-

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Fig. 2. (A) Schematic diagram showing the various conserved motifs of PfDH60. Open boxes represent the conserved helicase motifs and the amino acid sequence of each motif is written by the single letter code inside the box. Labels below the open boxes (Q, I, Ia, etc.) are the names assigned to the motifs. The number between the motif and above the arrow is the number of amino acids separating the various motifs. (B) (i) The induction and purification of over-expressed PfDH60 in E. coli. The proteins were separated by SDS-PAGE and visualized by Coomassie blue staining. Lane M is the molecular weight marker. Lane 1, un-induced bacterial cell lysate; lane 2, IPTG-induced bacterial cell lysate; lane 3, cytosolic fraction of cell lysate; lane 4, pellet of cell lysate; lane 5, purified PfDH60 after Ni2+ -NTA agarose column. (ii) Immunodepletion of DNA helicase activity. The structure of the substrate used is shown on the left side of the autoradiogram. Asterisks denote the 32 P-labeled end. Lane 1, heat denatured substrate; lane 2, PfDH60 pretreated with pre-immune IgG; lane 3, control without enzyme; lane 4, PfDH60 pretreated with anti-pea p68 IgG; lane 5, PfDH60 pre-treated with anti-his antibody. (iii) Immunodepletion of ATPase activity. Lane 1, PfDH60 pretreated with pre-immune IgG; lane 2, control without enzyme; lane 3, PfDH60 pretreated with anti-pea p68 IgG; lane 4, PfDH60 pre-treated with anti-his antibody. The positions of the Pi and ATP spots are indicated by arrows. (C) Kinetics and concentration dependence of helicase activity of PfDH60. The quantitative enzyme activity data from the autoradiogram are shown on the right side. The structure of the substrate used is shown on the extreme left. Asterisks denote the 32 P-labeled end. (i) The standard helicase reaction was carried out with 90 ng of pure PfDH60 for the times indicated on the top of each lane. (ii) An increasing amount of PfDH60 was used in the standard helicase assay. The concentrations used are written at the top of each lane. Lanes marked ‘Control’ and ‘Boiled’ are the reactions without the enzyme and with heat-denatured substrates, respectively. The activity is shown as percent unwinding.

tor (data not shown). The enzyme showed a requirement for divalent cation Mg2+ for its activity. The optimum concentration of MgCl2 was 1 mM and at 8 mM the unwinding activity was completely inhibited (data not shown). In the presence of Mn2+ the enzyme had almost the same activity as in the presence of Mg2+ but other divalent cations such as Ag2+ , Ca2+ , Co2+ , Cu2+ , Ni2+ , and Zn2+ were unable to support any unwinding activity (data not shown). The kinetics of the unwinding reaction of PfDH60 at optimum assay conditions (1.0 mM ATP, 1.0 mM MgCl2 , 30 mM KCl) using 90 ng purified PfDH60 showed a linear rate up to 60 min (Fig. 2C-i, lane 8) and deviated from linearity with longer incubation. These results are similar to PDH65 [4] and this behavior suggests

co-operation in the enzyme reaction, which is most likely due to the interactions between enzyme molecules. Titration of the unwinding activity of PfDH60 under optimal assay conditions with increasing amount of purified protein showed linearity up to 105 ng of protein (Fig. 2C-ii, lane 8). ATPase activity has been reported to be the intrinsic property of all the helicases and the energy released is also required for the translocation of the helicase protein on the DNA [4]. The release of radioactive phosphate (Pi) from [␥32 P] ATP by PfDH60 was measured as described earlier [24] and it was observed that Mg2+ , ssDNA and synthetic RNA were able to stimulate ATP hydrolysis several folds (data not shown). Maximum ATPase activity was observed with 2 h

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of incubation at 37 ◦ C and with 90 ng of PfDH60 (data not shown). It is evident that ATP hydrolysis is required for the DNA unwinding activity of PfDH60 as the non-hydrolysable analogue of ATP (ATP␥S) could not support any unwinding activity (data not shown). The results of ATP-binding activity of PfDH60 showed that a polypeptide of 60 kDa is radiolabeled after the reaction (data not shown). Most of the members of the ‘DEAD-box’ protein family have been identified as ‘putative computer predicted helicases’. Only a few members have been shown to contain DNA or RNA unwinding activity. The main examples of DNA helicases from this family are the E. coli RecQ gene product, a 74 kDa protein, yeast Rad3, a product of excision repair gene ERCC3, a 72 kDa protein encoded by the human REQL gene, the 172 kDa protein encoded by DNA2 gene in yeast, PDH45 from pea and eIF-4A from P. cynomolgi [4,13]. On the other hand some other members of ‘DEAD-box’ protein family such as Arabidopsis thaliana DRH1, Drosophila VASA, pea PDH45, mouse eIF-4A, Xenopus-an3 and Xp54, human p68, E. coli CsdA, RhlE and SrmB and hepatitis C virus NS3 helicase [17,19,22,25] have been shown to contain RNA unwinding activity. Although a number of helicases have been characterized biochemically, till to date the biological role of only a few selected helicases have been determined [5]. In this study we have cloned, expressed, purified and characterized a novel functionally active ‘DEAD-box’ DNA helicase named PfDH60 from P. falciparum and have shown that it is a homologue of p68 from various sources and it contains DNA helicase, ssDNA-dependent ATPase and ATP-binding activities. To the best of our knowledge this is the first report of cloning and characterization of a novel member of ‘DEAD-box’ protein family from P. falciparum and also the first report which shows that a homologue of p68 from P. falciparum encompasses the DNA helicase activity. Previously p68 has been reported as RNA helicase but its DNA helicase activity has not been reported so far. The importance of p68 has been well documented by various studies [18]. A gene closely related to p68 has been found both in Saccharomyces cereviseae (DBP2) and in Scizosaccharomyces pombe (dbp2) and it has been shown that the deletion of DBP2 gene renders yeast cells cold-sensitive for growth [26]. PfDH60 could be a novel homologue of p68 from malaria parasite with DNA helicase activity. Our findings demonstrate that p68 might also have a role in DNA metabolism. For testing the RNA helicase activity of PfDH60 in future we are designing the required RNA substrates. The isolation and characterization of the first DNA helicase from the malaria parasite is the first step towards elucidating the DNA transaction mechanism in the parasite.

Acknowledgements The authors are grateful to Dr. Narendra Tuteja (ICGEB, New Delhi) for the gift of polyclonal antibodies to pea p68. This work was supported by Defence Research and Devel-

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