Characterization of replication fork and

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May 16, 2008 - Characterization of replication fork and phosphorylation stimulated Plasmodium falciparum helicase 45. Arun Pradhan a,b, Ejaz M. Hussain b, ...

Gene 420 (2008) 66–75

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Gene j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / g e n e

Characterization of replication fork and phosphorylation stimulated Plasmodium falciparum helicase 45 Arun Pradhan a,b, Ejaz M. Hussain b, Renu Tuteja a,⁎ a b

Malaria Group, International Centre for Genetic Engineering and Biotechnology, P. O. Box 10504, Aruna Asaf Ali Marg, New Delhi-110067, India Department of Biosciences, Jamia Millia Islamia, New Delhi-110025, India

A R T I C L E

I N F O

Article history: Received 28 February 2008 Received in revised form 29 April 2008 Accepted 1 May 2008 Available online 16 May 2008 Received by A.J. van Wijnen Keywords: DEAD-box protein ssDNA-dependent ATPase Plasmodium falciparum Translation initiation factor Unwinding enzyme

A B S T R A C T Helicases are essential enzymes, which play important role in the metabolism of nucleic acids. In the present study we report further characterization of PfH45 (Plasmodium falciparum helicase 45), which is an essential enzyme for parasite survival. The results show that the helicase activity of PfH45 is significantly stimulated by replication fork like structure. The studies using truncated derivatives of PfH45 show that its nucleic acid dependent ATPase activity resides in the N-terminal one third of the protein and its RNA and DNA-binding activity predominantly resides in the C-terminal two third of the protein. The phosphorylation of PfH45 by protein kinase C at Ser and Thr residues stimulated its DNA and RNA helicase and ssDNA and RNA-dependent ATPase activities. DNA-interacting compounds actinomycin, DAPI, daunorubicin, ethidium bromide, netropsin and nogalamycin were able to inhibit the helicase and ssDNA-dependent ATPase activity with apparent IC50 values ranging from 0.5 to 5.0 μM respectively. These compounds distinctively inhibit the helicase activity probably by forming complex with DNA and obstructing enzyme movement. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Helicases are ubiquitous motor proteins that can transiently catalyze the unwinding of energetically stable duplex nucleic acids by using nucleoside triphosphate (NTP) hydrolysis as the source of energy (Matson et al., 1994; Tuteja and Tuteja, 2006). They are important enzymatic tools of cells and can be considered as a “screw driver” of the cellular machinery. All the helicases share at least three general biochemical properties (i) nucleic acid binding, (ii) NTP/dNTP binding and hydrolysis and (iii) NTP/dNTP hydrolysis-dependent unwinding of duplex nucleic acids (Tuteja and Tuteja, 2004a; Tuteja and Tuteja, 2004b). The DNA and RNA helicases play many essential roles in most aspects of nucleic acid metabolism. RNA helicases catalyze the ATPdependent unwinding of local RNA secondary structures and play a major role in remodeling RNA structures (Gorbalenya et al., 1989; Linder et al., 1989; Luking et al., 1998; Pause and Sonenberg 1992). Most of the helicases contain nine short conserved amino acid sequence motifs known as ‘helicase motifs’ (Tuteja and Tuteja, 2004b). Because of the presence of sequence DEAD in motif II this helicase family is also termed as ‘DEAD-box’ protein family.

Abbreviations: ATPase, adenosine triphosphatase; DAPI, diamidino phenyindole; DEAD, single letter code for amino acids Asp–Glu–Ala–Asp; eIF4A, eukaryotic initiation factor 4A; NTP, nucleoside triphosphate; PKC, protein kinase C; ssDNA, single stranded DNA. ⁎ Corresponding author. Tel.: +91 11 26741358; fax: +91 11 26742316. E-mail addresses: [email protected], [email protected] (R. Tuteja). 0378-1119/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2008.05.005

The proteins of the DEAD-box family are widely distributed in nature and required for virtually most aspects of nucleic acid metabolism (Rocak and Linder, 2004). eIF4A is a bonafide member of DEADbox protein family of helicases and is involved in almost all aspects of nucleic acid metabolism (Hall and Matson, 1999; Rogers et al., 2001; Hernandez and Vazquez-Pianzola, 2005; Linder, 2006). It is a multifunctional protein and is known to facilitate translation of mRNA by unwinding inhibitory secondary structures in the 5′-untranslated region of the mRNA, which facilitates binding of the mRNA to the 40S ribosomal subunit (Hernandez and Vazquez-Pianzola, 2005; Rogers et al., 2001). The pea DNA helicase 45 (PDH45) and the hepatitis C virus non-structural protein 3 (NS3) helicase both are eIF4A homologues, which belong to DEAD-box protein family and have been shown to contain both RNA and DNA unwinding activities (Du et al., 2002; Pham et al., 2000). Malaria is caused by the members of the genus Plasmodium and Plasmodium falciparum causes the most virulent form of this disease (Tuteja, 2007a). Anti-malarial drugs are the mainstays of control of malaria but in some cases the current treatments are insufficient because of increasing drug resistance (Hyde, 2007). The genomes of most of the cellular pathogens and several viruses code for helicases and there has been enormous interest in developing helicases as potential drug targets (Frick and Lam, 2006; Tuteja, 2007b). The genome sequence of P. falciparum has been completed and annotation is in progress (Gardner et al., 2002; Bahl et al., 2003). A number of putative helicases were identified in the genome of P. falciparum and the detailed bioinformatics analysis demonstrated that the genome

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contains at least 22 full-length putative DEAD-box helicase genes (Gardner et al., 2002; Bahl et al., 2003; Tuteja and Pradhan, 2006). In order to understand the basic biology of malaria parasite, we have initiated a systematic study of helicases and recently we reported the isolation and cloning of PfH45 (P. falciparum helicase 45 kDa) (Pradhan and Tuteja, 2007). Here we present further characterization of PfH45 and show that its DNA helicase activity is modulated by replication fork like structure. The studies with truncated derivatives of PfH45 indicate that its nucleic acid dependent ATPase activity resides in the N-terminal one third of the protein. PfH45 is a homologue of eIF4A and we have previously shown that it contains RNA helicase activity also (Pradhan and Tuteja, 2007). In the present study we report that PfH45 contains nucleic acid binding activity and this activity predominantly resides in the C-terminal two third of the protein. Phosphorylation is the most common post-translational modification and previous studies have shown that it plays a role in regulating the enzymatic activities of a number of enzymes such as p68, P. falciparum DNA helicase 60 (PfDH60) and pea DNA helicase 47 (PDH47) (Buelt et al., 1994; Pradhan et al., 2005; Vashisht et al., 2005). Our results show that all the enzymatic activities of PfH45 such as DNA and RNA helicase along with DNA and RNA-dependent ATPase activity are modulated by phosphorylation with protein kinase C. We also report that the DNA-interacting compounds inhibit the DNA unwinding and ssDNA-dependent ATPase activities of this enzyme. These studies will make important contribution in better understanding of the mechanism of nucleic acid transaction in the parasite.

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replication fork like structure. The other substrate used consisted of the partial duplex containing the 32P-labeled oligodeoxynucleotide of 17 bases with the sequence 5′-GTTTTCCCAGTCACGAC-3′ annealed to M13mp19 ssDNA. The oligodeoxynucleotide used were synthesized from Microsynth (Microsynth GmbH, Balgach, Switzerland). For studying the effect of DNA-interacting compounds on helicase activity, different compounds were added to the helicase reaction mixture prior to the addition of the helicase enzyme. The RNA helicase substrate was prepared by using the same method as described earlier (Pradhan and Tuteja, 2007) and the following RNA oligonucleotides synthesized from Primm srl (Milan, Italy): 13 mer 5′-AUAGCCUCAACCG-3′ and 39 mer 5′-GGGAGAAAUCACUCGGUUGAGGCUAUCCGUAAAGCACGC-3′. The RNA helicase assay was performed by using the same method as described earlier (Pradhan and Tuteja, 2007). 2.4. ATPase assays

2. Materials and methods

The hydrolysis of ATP catalyzed by PfH45 was assayed by measuring the formation of 32P from [γ-32P]ATP and the reaction was performed for 2 h at 37 °C in the presence of the enzyme and 100 ng of M13 mp19 ssDNA. This was followed by TLC and the quantitation was done as described earlier (Pradhan and Tuteja, 2007). For studying the effect of DNA-interacting compounds on ssDNA-dependent ATPase activity, different compounds were added to the reaction mixture prior to the addition of the enzyme. The RNA-dependent ATPase assay was performed using a protocol similar to that described above for ssDNA-dependent ATPase assay, except that the ssDNA was replaced with 39-mer RNA oligonucleotide.

2.1. Materials

2.5. Preparation of truncated derivatives of PfH45

M13mp19 ssDNA was purchased from Invitrogen (Carlsbad, CA, USA). Nucleoside tri-phosphates and deoxy-nucleoside tri-phosphates were from Pharmacia (Sweden) and [γ-32P]ATP was purchased from Perkin Elmer (Boston, MA, USA). Synthetic DNA oligonucleotides were synthesized chemically. The DNA-interacting compounds camptothecin, daunorubicin, ellipticine and VP-16 were purchased from Topogene Inc. (Columbus, Ohio, USA). Actinomycin was from Boehringer Mannheim (Indianapolis, IN, USA), ethidium bromide was from BDH (E. Merck, Mumbai, India) and aphidicolin, cyclophosphamide, DAPI, distamycin, genistein, mitoxantrone, nalidixic acid, netropsin, nogalamycin and novobiocin were from Sigma Chemical Co. (St. Louis, MO, USA). All of these compounds were dissolved in dimethyl sulfoxide and stored at 4 °C in dark.

In order to check the activity contributed by various domains, the full-length gene was divided into two fragments: N-terminal and C-terminal. The N-terminal fragment was amplified by using the primers PfH45F (5′-GGGATCCATGAGTACTAAAGAAGA-3′) and PfR1 (5′-CCTCGAGCTCTTATCAATCATATCA-3′) and the C-terminal fragment was amplified by using the primers PfF1 (5′-GGGATCCGATATGATTGATAAGAGA-3′) and PfH45R (5′-CCTCGAGTTATAAATAGTCAGCAA-3′). The target DNA was denatured for 5 min initially and then 35 cycles of PCR were performed. PCR conditions for amplification are as follows: 95 °C for 1 min, 54 °C for 1 min, 72 °C for 1 min followed by 72 °C for 10 min. The resulting clones were verified by sequencing and the fragments were sub-cloned in the protein expression vector pET-28a obtained from Novagen (Madison, WI, USA). The truncated proteins were purified by the same procedure as described for full-length protein (Pradhan and Tuteja, 2007).

2.2. Purification of PfH45 The his-tagged protein was expressed and purified using the same method as described earlier (Pradhan and Tuteja, 2007). In brief the expression clone transformed into E. coli strain BL21(DE3)pLysS was used for the production and purification of the recombinant protein. The expressed protein was purified using standard methods with NiNTA (Qiagen, GmbH, Germany) affinity chromatography and was checked for purity by SDS-PAGE and coomassie blue staining using the standard protocol (Sambrook et al., 1989). This purified protein was used for all of the assays described below. 2.3. Preparation of helicase substrates and helicase assays The helicase assay was carried out using the same substrate and the method as described previously (Pradhan and Tuteja, 2007). The substrate consisted of the partial duplex containing the 32Plabeled oligodeoxynucleotide of 47 bases with the sequence 5′-(T15)GTTTTCCCAGTCACGAC-(T15)-3′ annealed to M13mp19 ssDNA. This oligodeoxynucleotide contains 15 nucleotides of non-complementary region at both the 5′ and 3′ ends to give a substrate resembling a

2.6. In vitro RNA binding assay The RNA binding assay was done by using the same method as described previously with slight modifications (Cheng et al., 2005). For these equal amounts (1 µg) of BSA, PfH45 (full length) and truncated derivatives of PfH45 (PfH45-N and PfH45-C) were dot-blotted on precharged PVDF membrane. This membrane was blocked for 1 h at room temperature in blocking buffer (25 mM NaCl, 10 mM MgCl2, 10 mM HEPES, 0.1 mM EDTA, 1 mM DTT, 3% BSA). The 13 mer RNA oligonucleotide used for the preparation of RNA helicase substrate was labeled at the 5′-end with 1.85 MBq of [γ32P] ATP (specific activity 222 TBq/mmol) using T4 polynucleotide kinase (NEB, England) and purified using Sepharose 4B (Pharmacia, Sweden) column chromatography. After blocking the membrane was incubated for 2 h in binding buffer (50 mM NaCl, 10 mM MgCl2, 10 mM HEPES, 0.1 mM EDTA, 1 mM DTT, 1.5% BSA) containing 10 pmol of 32P-labeled RNA substrate. After binding, the membrane was washed thrice with binding buffer and exposed for autoradiography. The spots obtained were quantitated by densitometry. To check for equal loading of proteins, equal amounts

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(1 µg) of PfH45, PfH45-N and PfH45-C were dot-blotted on another precharged PVDF membrane. This membrane was blocked with blocking buffer (1% BSA in Tris buffered saline) for 1 h at room temperature and probed for a further 1 h with alkaline phosphatase conjugated anti-his antibody (Sigma Chemical Co. (St. Louis, MO, USA) in same buffer. The blot was washed and developed using standard protocol. 2.7. In vitro DNA-binding assay The DNA-binding assay was performed by using the same method as described for RNA binding but end-labeled DNA oligodeoxynucleotide of 17 bases used for preparation of DNA duplex substrate described under Section 2.3 was used. The rest of the procedure was similar as described for RNA binding. 2.8. Protein phosphorylation P. falciparum lysate was prepared from asynchronous culture containing asexual stages using RIPA buffer (1% Triton X100, 0.5% sodium deoxycholate and 1 mM PMSF). This lysate was used as source of kinase to phosphorylate PfH45 protein. A fraction of the same lysate was used to immunoprecipitate endogenous PfH45 by using purified IgG against PfH45. 100 µg of total parasite protein was mixed with purified PfH45 IgG (5 µg) and the mixture was incubated at 4 °C for 2 h. The immunoprecipitate was collected with protein A-Sepharose

beads and the beads were extensively washed before use. About 10 µl of these beads containing endogenous PfH45 were phosphorylated by increasing concentration of lysate (1.0 and 5.0 µg) in 20 µl reaction volume in 20 mM HEPES buffer, 2 mM CaCl2, 10 mM MgCl2, 5 μCi [γ-32P] ATP (specific activity 222 TBq/mmol). A control reaction was also set up using the higher concentration of lysate (5.0 µg) and no protein. To check if the endogenous PfH45 is a substrate for protein kinase C (PKC), in a separate tube 10 µl of the beads containing endogenous PfH45 were phosphorylated in same reaction conditions using 6 ng of PKC from Promega (Madison, WI, USA). After incubation for 30 min at 30 °C, the mixture was resolved on 10% SDS-PAGE, the gel was dried and autoradiographed. Recombinant purified PfH45 was phosphorylated under optimal assay conditions by using PKC. The reaction mixture contained 500 nM PfH45, 6 ng PKC and PKC buffer (20 mM HEPES buffer, 2 mM CaCl2, 10 mM MgCl2, 1 mM ATP or 5 μCi [γ-32P] ATP (specific activity 222 TBq/mmol)). The reaction was incubated at 30 °C for 30 min. After incubation the phosphorylation reaction mixture was used for helicase and ATPase assays or SDS-PAGE analysis. The SDSPAGE gel was dried and exposed for autoradiography after staining. For phosphoamino acid analysis, the radioactive phosphorylated PfH45 band was excised from the dried gel and hydrated in water. The eluted protein was hydrolyzed in 7.5 N HCl for 2 h in boiling water bath as described (Hunter and Sefton, 1980). The supernatant was collected after centrifugation, concentrated and spotted on 3 MM

Fig. 1. (A) The proteins were separated by SDS-PAGE and visualized by coomassie blue staining. Lane M, molecular weight marker and lane 1, purified PfH45. (B) and (C) DNA unwinding activity of PfH45 with forked and non-forked substrates. The helicase reaction was performed under standard assay conditions. Two different concentration of purified PfH45 (25 and 50 nM, lanes 1 and 2 respectively) were used for the reaction. Lane C in both panels is reaction without enzyme and lane B is heat-denatured substrate. The structure of the substrate is shown on the left side of the autoradiogram. The quantitative data are shown as a histogram above the autoradiograms. (D) Preference of nucleotide triphosphate (NTP) for the helicase activity of PfH45. Lane 1 is reaction without enzyme, lane 2 is reaction in the presence of ATPγS and lanes 3–10 are reactions in the presence of one of the NTPs or dNTPs.

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Fig. 2. (A) Motif structure: Schematic diagram showing the various conserved motifs of PfH45. Open boxes represent the conserved motifs and the name of the motif is written inside the box. (B) The motifs present in the N-terminal fragment of PfH45. (C) The motifs present in the C-terminal fragment of PfH45. In (B) and (C) the size of the fragment is written in bracket. (D) Western blot analysis of purified full-length PfH45 (lane 1), PfH45-N (lane 2) and PfH45-C (lane 3). (E) DNA helicase activity of full-length and truncated derivatives of PfH45. Lane 1 is heat-denatured substrate and lane 2 is control without enzyme. (F) RNA helicase activity of full-length and truncated derivatives of PfH45. Lane 1 is control without enzyme and lane 2 is heat-denatured substrate. (G) ssDNA-dependent ATPase activity of full-length and truncated derivatives of PfH45. Lane 1 is control without enzyme, lane 2 is reaction of PfH45 in the absence of DNA, lane 3 is reaction of PfH45 in the presence of DNA, lane 4 is reaction of PfH45-N, lane 5 is reaction of PfH45-C and lane 6 is reaction of PfH45-N and PfH45-C. The positions of ATP and released Pi are marked on the left side of autoradiogram. (H) RNA-dependent ATPase activity of full-length and truncated derivatives of PfH45. Lane 1 is control without enzyme, lane 2 is reaction of PfH45 in the absence of RNA, lane 3 is reaction of PfH45 in the presence of RNA, lane 4 is reaction of PfH45-C, lane 5 is reaction of PfH45-N and lane 6 is reaction of PfH45-N and PfH45-C. The positions of ATP and released Pi are marked on the left side of autoradiogram.

Whatmann chromatography paper with standards phosphoserine and phosphothreonine, which were obtained from Sigma (St. Louis, MO, USA). The solvent used for chromatography was: propionic acid, 1 M

NH4OH and isopropyl alcohol in the ratio of 45:17.5:17.5 v/v. The chromatogram was dried, stained with ninhydrin solution (0.3%) and exposed for autoradiography.

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3. Results 3.1. Characterization of PfH45 DNA helicase activity PfH45 was purified using the procedure described previously (Pradhan and Tuteja, 2007) and the SDS-PAGE and coomassie blue staining of the purified protein showed that the preparation is homogeneous (Fig. 1A, lane 1). This purified homogeneous preparation of PfH45 was used for all the subsequent assays described in the following sections. The DNA unwinding activity of PfH45 was ascertained and characterized in detail by using the commonly used strand-displacement assay. Both the forked and non-forked substrate used for the assay contained the same duplex length (17 base pair) with identical sequence but differed only in the presence of tails of 15 non-complementary nucleotides at both ends. The assay was performed by using two different concentrations (25 and 50 nM) of purified PfH45. PfH45 showed significant unwinding activity when a forked substrate with non-complementary tails at both ends was used and this activity increased with an increase in the amount of purified PfH45 protein (Fig. 1B, lanes 1 and 2). There was no difference in the stimulation of the unwinding activity of PfH45, if the substrate contained a single tail at 5′ or 3′ end (data not shown). But PfH45

showed negligible activity with the substrate containing no tail and this activity increased only slightly with the increase in the concentration of PfH45 (Fig. 1C, lanes 1 and 2). These results suggest that a replication fork like structure is essential and it stimulates the activity of PfH45. Therefore further characterization of PfH45 was performed using the forked substrate. The hydrolysis of ATP was necessary for the unwinding activity of PfH45 because the poorly hydrolysable analog ATPγS was unable to support the unwinding activity (Fig. 1D, lane 2). Besides ATP, only dATP, dCTP or UTP were able to support the DNA helicase activity of PfH45 to some extent (Fig. 1D, lanes 3, 4, 6 and 9 respectively). But no other NTPs or dNTPs could be utilized as a cofactor (Fig. 1D, lanes 5, 7, 8 and 10 respectively). 3.2. Characterization of truncated derivatives of PfH45 For characterizing the contribution of various motifs in enzyme activities of PfH45, the protein was divided into two fragments: PfH45-N (~ 17 kDa) contained the motifs Q, I, Ia and Ib and PfH45-C (~ 29 kDa), which contained the motifs II to VI (Fig. 2A–C). Both of these truncated derivatives were purified and checked by western blot analysis with anti-his antibodies (Fig. 2D). Same concentrations (75 nM) of the full length, PfH45-N and PfH45-C were checked for

Fig. 3. RNA binding activity of PfH45. (A) Color coded scale of RNA binding. Models of PfH45-N (B) and PfH45-C (C) showing the residues responsible for RNA binding according to the color coding scheme shown in (A). (D) RNA binding activity. This experiment was repeated three times and the representative blot for one experiment is shown. (E) A graphical representation of the data of panel (D). (F) Western blot probed with anti-his antibody.

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DNA and RNA helicase and ssDNA and RNA-dependent ATPase activities. The results clearly indicate that the full-length PfH45 showed both the DNA and RNA helicase activity (lane 6 of Fig. 2E and F respectively) while PfH45-N showed no helicase activity (lane 3 of Fig. 2E and F respectively) and PfH45-C showed insignificant helicase activity (lane 4 of Fig. 2E and F respectively). The physical mixing of PfH45-N and PfH45-C did not result in restoration of any unwinding activity (lane 5 of Fig. 2E and F respectively). It was observed that full-length PfH45 showed ATPase activity only in the presence of ssDNA or RNA (lane 3 of Fig. 2G and H respectively) and activity is negligible in the absence of nucleic acid cofactor (lane 2 of Fig. 2G and H respectively). Furthermore it was interesting to note that only PfH45-N contained the ssDNA (Fig. 2G, lane 4) or RNAdependent ATPase activity (Fig. 2H, lane 5). But PfH45-C had no ssDNA (Fig. 2G, lane 5) or RNA-dependent ATPase activity (Fig. 2H, lane 4) and the physical mixing of PfH45-N and PfH45-C did not result in any increase in the ssDNA or RNA-dependent ATPase activity (lane 6 of Fig. 2G and H respectively). 3.3. RNA binding activity of PfH45 and truncated derivatives Our analysis has shown that PfH45 is homologous to eIF4A and we have previously reported that it also contains RNA helicase activity (Pradhan and Tuteja, 2007). Therefore in order to check the efficiency of PfH45 to bind RNA, the RNA binding propensity analysis was performed. The bioinformatics analysis using the program RNA interface residue prediction from protein 3D structure (http://yayoi. kansai.jaea.go.jp/qbg/kyg/index.php) with structure obtained from 3D-JIGSAW (www.expasy.org) showed that the amino acids with maximum propensity to bind RNA are located at the C-terminal region of PfH45 protein (Supplemental data 1) (Kim et al., 2006). This RNA interface residue prediction program labels the amino acids highly likely to present at interface in red and the buried amino acids (not considered as an interface residue) in deep blue (Fig. 3A). As shown in Fig. 3C the amino acids responsible for RNA binding are mostly located on the C-terminal fragment (PfH45-C) of the protein as compared to the N-terminal fragment (PfH45-N) (Fig. 3B). In order to find the RNA binding efficiency of PfH45, PfH45-N and PfH45-C the RNA binding assay was performed using the method described. This experiment was repeated three times and the results were reproducible and a representative autoradiogram is shown in Fig. 3D. The results of RNA binding show that PfH45 showed maximum efficiency to bind RNA (Fig. 3D, lane 1) while PfH45-C has higher propensity (Fig. 3D, lane 3) than PfH45-N (Fig. 3D, lane 2) to bind RNA and bovine serum albumin (BSA) used as a control showed no RNA binding (Fig. 3D, lane C). These results were expressed as percentage RNA binding with PfH45 considered to provide 100% RNA binding efficiency. The histogram shows that PfH45-N and PfH45-C bind RNA with 20% and 75% efficiency respectively as compared to the full-length PfH45 (Fig. 3E). These results are in agreement with the bioinformatics analysis, which revealed that the prominent RNA binding residues are located predominantly in the C-terminal (PfH45-C) region of the PfH45 protein (Fig. 3C). An identical blot of PfH45-full length, PfH45-N and PfH45-C was probed with anti-his antibody, which confirmed that equal amount of protein was loaded for this assay (Fig. 3F). 3.4. DNA-binding activity of PfH45 and truncated derivatives In order to check if same domains of PfH45 are involved in binding to DNA, the DNA-binding assay was performed using the same procedure as described for RNA binding but the labeled RNA was replaced with labeled DNA oligodeoxynucleotide. The results of DNAbinding assay revealed that PfH45 showed maximum efficiency to bind DNA (Fig. 4A, lane 1) while PfH45-C has higher propensity (Fig. 4A, lane 3) than PfH45-N (Fig. 4A, lane 2) to bind DNA and BSA used as a control showed no DNA binding (Fig. 4A, lane C). These

Fig. 4. DNA-binding activity (A) DNA-binding activity of PfH45 and its derivatives. (B) A graphical representation of the data of panel (A). (C) Western blot probed with anti-his antibody.

results were expressed as percentage DNA binding and the histogram showed that PfH45-N and PfH45-C bind DNA with 12% and 78% efficiency respectively as compared to the full-length PfH45 (Fig. 4B). These results suggest that most probably the same domains of PfH45 are involved in DNA and RNA binding. An identical blot of PfH45-full length, PfH45-N and PfH45-C was probed with anti-his antibody, which confirmed that equal amount of protein was loaded for this DNA-binding assay (Fig. 4C). 3.5. Phosphorylation of PfH45 by protein kinase C Bioinformatics-based analysis of amino acid sequence of PfH45 using NetPhosK (www.expasy.org) program revealed that it contains multiple potential phosphorylation sites (Blom et al., 2004) (Supplemental data 2). Therefore the phosphorylation of endogenous PfH45 protein was checked using P. falciparum total lysate (as a source of kinase) and protein kinase C (PKC). An asynchronous P. falciparum culture was used for the preparation of total parasite lysate and from an aliquot of this preparation the endogenous PfH45 protein was recovered by immunoprecipitation. The phosphorylation of endogenous PfH45 protein was checked using two different concentrations of P. falciparum total lysate as a source of kinase. It was observed that endogenous PfH45 was phosphorylated by endogenous P. falciparum kinases in a concentration dependent manner (Fig. 5A, lanes 3 and 2 respectively). But only PfH45 or only lysate reactions had no corresponding band (Fig. 5A, lanes 1 and 4 respectively). Furthermore it was noteworthy that the endogenous PfH45 was phosphorylated with PKC also (Fig. 5A, lane 5). The phosphorylation map of PfH45 also suggested that it contains the maximum number of putative PKC phosphorylation

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Fig. 5. Phosphorylation of PfH45 (A) Phosphorylation of endogenous PfH45 with P. falciparum lysate. Lane 1 is only endogenous PfH45 with all the components of the reaction but no lysate, lane 2 is endogenous PfH45 phosphorylated with 5.0 μg of lysate, lane 3 is endogenous PfH45 phosphorylated with 1.0 μg of lysate, lane 4 is phosphorylation with 5.0 μg of the lysate only without any endogenous PfH45 and lane 5 is endogenous PfH45 phosphorylated with PKC (B) Phosphorylation of PfH45 with PKC. Lane 1 is recombinant PfH45, lane 2 is PfDH60 and lane 3 is BSA. (C) Phosphoamino acid analysis of PfH45 phosphorylated with PKC. The positions of standard phosphoamino acids visualized after ninhydrin staining are shown. (D) PfH45 DNA helicase activity after phosphorylation. Lane 1, control without enzyme, lane 2, heat-denatured substrate. The percent unwinding in lanes 3 and 4 is shown at the bottom of the autoradiogram. (E) ssDNA-dependent ATPase activity after phosphorylation. Lane 1, control without enzyme. The positions of the inorganic phosphate (Pi) and ATP are marked on the left-hand side of the autoradiogram. The percentage of inorganic phosphate (Pi) released in lanes 2 and 3 is shown at the bottom of the autoradiogram. (F) PfH45 RNA helicase activity after phosphorylation. Lane 1, control without enzyme, lane 4, heat-denatured substrate. The percent unwinding in lanes 2 and 3 is shown at the bottom of the autoradiogram. (G) RNA-dependent ATPase activity after phosphorylation. Lane 1, control without enzyme. The positions of the inorganic phosphate (Pi) and ATP are marked on the left-hand side of the autoradiogram. The percentage of inorganic phosphate (Pi) released in lanes 2 and 3 is shown at the bottom of the autoradiogram. All the experiments were performed at least three times and the results were reproducible.

sites (Supplemental data 2). Therefore in order to check the effect of phosphorylation on enzymatic activity of PfH45, its phosphorylation was performed by incubating the protein (PfH45) with [γ32P]

ATP in the presence of PKC. The phosphorylation of proteins was examined by SDS-PAGE followed by autoradiography. In this experiment, PfDH60 was used as a positive control (Pradhan et al.,

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2005) (Fig. 5B, lane 2) and BSA (Fig. 5B, lane 3) as a negative control. The results clearly indicate that 45 kDa polypeptide of PfH45 (Fig. 5B, lane 1) was phosphorylated by PKC and the phosphorylation was dependent on the time of incubation and the amount of PfH45 (data not shown). 3.6. PfH45 is phosphorylated at serine and threonine residues The phosphorylated band of PfH45 was eluted from the gel and subjected to phosphoamino acid analysis followed by paper chromatography. The positions of standard phosphoserine and phosphothreonine after staining are also shown in Fig. 5C. The autoradiogram after chromatography revealed that PKC phosphorylates on serine and threonine residues of PfH45 (Fig. 5C). 3.7. Stimulation of helicase and ATPase activities of PfH45 after phosphorylation The effect of PKC phosphorylation on helicase and ATPase activities of PfH45 was tested. The results revealed that both the DNA helicase (Fig. 5D, lane 3) and the ssDNA-dependent ATPase (Fig. 5E, lane 2) activities of PfH45 were stimulated about two to three-fold after phosphorylation of PfH45 with PKC as compared to without PKC phosphorylation (Fig. 5D, lane 4 and Fig. 5E, lane 3 respectively). Furthermore it was interesting to note that the RNA helicase (Fig. 5F, lane 2) and the RNA-dependent ATPase (Fig. 5G, lane 3) activities were also stimulated to almost same extent after phosphorylation of PfH45 with PKC as compared to without PKC phosphorylation (Fig. 5F, lane 3 and Fig. 5G, lane 2 respectively).

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3.8. Effect of DNA-interacting compounds on DNA helicase and ssDNA-dependent ATPase activities of PfH45 and kinetics of inhibition A variety of DNA-interacting agents have been reported to contain anti-helicase activities (Bachur et al., 1992; George et al., 1992), therefore, the effects of various kinds of these agents on DNA unwinding and ssDNA-dependent ATPase activities of PfH45 were studied. The compounds used in this study belong to the following categories of DNA-binding ligands: (a) DNA polymerase alpha inhibitor, such as aphidicolin, non-intercalating topoisomerase inhibitors such as camptothecin, nalidixic acid, novobiocin, and VP-16 (b) minor groove binders, such as netropsin; and (c) DNA-intercalating compounds, which included actinomycin, cyclophosphamide, daunorubicin, ellipticine, ethidium bromide, genistein and nogalamycin. In the present study, the effect of these compounds on the DNA unwinding activity and ssDNA-dependent ATPase activities of PfH45 were initially tested by including 50 μM of each compound separately in the standard helicase and ATPase assays. The results indicated that actinomycin, DAPI, daunorubicin, ethidium bromide, netropsin and nogalamycin inhibited the DNA unwinding and ssDNA-dependent ATPase activities of PfH45 effectively (Fig. 6A and B, lanes 3, 7, 8, 11, 15 and 16 respectively). However, at 50 μM concentration, aphidicolin, camptothecin, cyclophosphamide, distamycin, ellipticine, genistein, mitoxantrone, nalidixic acid and novobiocin were unable to inhibit the DNA unwinding as well as ssDNA-dependent ATPase activities of PfH45 significantly (Fig. 6A and B, lanes 4, 5, 6, 9, 10, 12, 13, 14 and 17 respectively). These experiments were repeated at least three times and the results were reproducible. The compounds, which inhibited the helicase and ATPase activities, were investigated further for the

Fig. 6. Effect of various DNA-interacting compounds on DNA helicase (A) and ssDNA-dependent ATPase activities of PfH45. In (A) the structure of the DNA helicase substrate is shown on the left side of the autoradiogram. Asterisks denote the 32P-labeled end. Lane 1 is control without enzyme and lane 18 is heat-denatured substrate. In (B) the position of ATP and released Pi is shown on the left side of the autoradiogram. In both the panels, lane 2 is the reaction with enzyme and without any compound but in the presence of 1 μl of dimethylsulfoxide. Lanes 3–17 are the reactions with enzyme in the presence of different compounds. The compounds used are written above the autoradiogram.

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A. Pradhan et al. / Gene 420 (2008) 66–75

kinetics of inhibition. For this, each inhibitor was included in the helicase and ATPase reactions at final concentrations ranging from 0.1 to 10 μM. The apparent IC50 value for each inhibitor was determined from these data (Supplemental data 3). For DNA helicase activity of PfH45, the most effective inhibitors were nogalamycin and netropsin with apparent IC50 values of 0.5 μM, followed by DAPI, ethidium bromide, daunorubicin, and actinomycin with apparent IC50 values of 1.0, 1.0, 1.5 and 1.8 μM, respectively (Supplemental data 3). For ssDNAdependent ATPase activity of PfH45, the results show that the most effective inhibitor was nogalamycin with an apparent IC50 value of 0.8 μM, followed by netropsin, ethidium bromide, actinomycin, DAPI and daunorubicin with apparent IC50 values of 1.5, 1.5, 4.0, 4.25 and 5.0 μM respectively (Supplemental data 3). 4. Discussion The DEAD-box family of proteins is one of the largest families of helicases but only few of its members have been biochemically characterized from various systems (Linder, 2006). In the present study we report detailed characterization of PfH45 and our results show that its ssDNA and RNA-dependent ATPase activity mainly resides in the N-terminal one third of the protein and the DNA and RNA binding activity predominantly resides in the C-terminal two third of the protein. We further demonstrate that the enzymatic activities of PfH45 are modulated after phosphorylation with PKC and inhibited by some DNA-interacting compounds. We have previously reported that PfH45 encodes a biochemically active DEAD-box protein, which is highly homologous to eIF4A (Pradhan and Tuteja, 2007). Some of the other eIF4A homologues such as pea DNA helicase 45 (PDH45), pea DNA helicase 47 (PDH47), hepatitis C virus (non-structural protein 3, NS3) and Plasmodium cynomolgi DEAD-box DNA helicase 45 (PcDDH45) have also been reported as DNA helicases (Du et al., 2002; Pham et al., 2000; Tuteja et al., 2003; Vashisht and Tuteja, 2005). The DNA unwinding activity of PfH45 is significantly stimulated by a replication fork like substrate and the stimulation of DNA helicase activity by a similar structure of the substrate has also been reported for PcDDH45 and human DNA helicase (Tuteja et al., 2003; Seo and Hurwitz, 1993). PfH45 prefers to use mainly ATP or dATP as a cofactor similar to the previously reported eIF4A homologue from P. cynomolgi (PcDDH45) (Tuteja et al., 2003). The studies with truncated derivatives of PfH45 indicate that the ssDNA and RNA-dependent ATPase activity resides in the N-terminal fragment of PfH45 but neither PfH45-N nor PfH45-C alone was able to show any unwinding activity. This observation further strengthens the notion that the energy released after ATP hydrolysis is essentially required for the unwinding activity because PfH45-C is unable to perform the unwinding without any associated ATPase activity. Furthermore these activities are concomitant and physical mixing of PfH45-N and PfH45-C did not result in any unwinding. This observation further suggests that all the helicase motifs on a single polypeptide are required for the ATPase and unwinding activities of a protein. There are few reports in the literature, which show that without the presence of Walker A and Walker B motifs, some proteins still show the helicase and ATPase activities (Tuteja et al., 1994; Tuteja et al., 1995; Nasirudin et al., 2005). PfH45 is an eIF4A homologue and contains both the DNA and RNA unwinding and ATPase activities. The nucleic acid binding activity analysis of PfH45 indicated that this activity mainly resides in the C-terminal fragment, which contains motifs II to VI. In a previous study with yeast Dhh1p (a homologue of eIF4A) it has been reported that the motif V and VI contribute to its RNA binding activity (Cheng et al., 2005). Phosphorylation is one of the common mechanisms for posttranslational modification of proteins and a number of kinases have been identified and classified in P. falciparum genome, which shows that it contains a variety of kinases homologous to the wellcharacterized kinases from other sources (Ward et al., 2004). PKC is

a serine/threonine kinase and is known to be involved in a variety of processes such as gene expression, signal transduction and regulation of the activities of numerous proteins including helicases (Buelt et al., 1994; Pradhan et al., 2005; Vashisht et al. 2005; Yang et al., 2004). This study shows that both the endogenous and recombinant PfH45 are substrate of PKC and the activities of recombinant PfH45 are stimulated after this phosphorylation at serine and threonine residues. But which serine and threonine residues of PfH45 are getting phosphorylated is a subject for further study. It is interesting to note that some enzyme activities have been reported to be upregulated and others inhibited after phosphorylation with a variety of kinases (Buelt et al., 1994; Pradhan et al., 2005; Vashisht et al. 2005; Yang et al., 2004; Tuteja et al., 2003; Tuteja et al., 2001). A kinase PfPKB has been reported from P. falciparum but no homologue of PKC can be identified in the genome of the parasite (Kumar et al., 2004; Ward et al., 2004). Therefore it is most likely that another, possibly unrelated kinase is responsible for the in vivo regulation of the activity of PfH45 via phosphorylation. Many DNA-binding agents have been shown to modulate DNA and RNA metabolism by binding to the nucleic acid and disrupting the enzymatic machinery that interacts with it (Bachur et al., 1992; George et al., 1992). Therefore, it is of interest to determine the effect of these agents on helicases, which are likely to be the first component of the “protein machines”. If the DNA-binding agents completely inhibit unwinding of duplex DNA, any effect they might have on further DNA transition processes would be deleterious (Pham and Tuteja, 2002; Tuteja, 2007b). A comparison of apparent IC50 values of some of these inhibitors for various eIF4A homologues shows that the apparent IC50 value for almost all the inhibitors is lowest for PfH45 as compared to some of the other helicases (Table 1). Actinomycin, DAPI, daunorubicin, ethidium bromide, netropsin and nogalamycin inhibited the activities of PfH45. Actinomycin is an antibiotic that binds specifically to the double-helical DNA and is a universal inhibitor for most of the helicases such as pea chloroplast DNA helicase I, HDHII (human DNA helicase II), PDH45 (pea DNA helicase 45), PDH47 (pea DNA helicase 47) and PcDDH45 (P. cynomolgi DEAD-box DNA helicase 45) (Tuteja and Phan, 1998; Pham and Tuteja, 2002; Tuteja et al., 2003; Vashisht and Tuteja, 2005). Ethidium bromide, a phenathridium compound has been reported to inhibit a few other helicases, such as PDH120 (pea DNA helicase 120) and PfDH60 (P. falciparum DNA helicase 60) (Phan et al., 2003; Pradhan and Tuteja, 2006). An oligopyrrolamidine, netropsin binds to the minor groove of DNA and inhibits the WRN and BLM helicases (Brosh et al., 2000) but it did not inhibit E. coli UvrD helicase activity (George et al., 1992). The inhibition by nogalamycin depends on the source of the enzyme and is highly variable because the IC50 value for this compound ranged from 0.1 to N650 μM for various viral helicases (Borowski et al., 2002). The anthracyclines are also universal inhibitors of most of the helicases tested so far, such as PcDDH45, HDHII, SV40 large T antigen and the viral helicases of the Flaviviridae family (Tuteja et al., 1997; Bachur et al., 1998; Pham and Tuteja, 2002; Tuteja et al., 2003). The compounds used in the present Table 1 Comparison of inhibitory potential of DNA-interacting compounds Compounds

PfH45a

PcDDH45b

PDH45c

PDH47d

HCVe

Actinomycin Daunorubicin Ethidium Bromide Mitoxantrone Netropsin Nogalamycin

1.8 1.5 1.0

N 50 7.5 N 50

N50 4.0 6.0

2.0 2.0 8.0

nd nd nd

nd 0.5 0.5

nd nd 1.7

nd nd 2.0

6.0 2.0 0.5

6.7 Nd 0.1

a b c d e

Plasmodium falciparum helicase 45 (this study). Plasmodium cynomolgi DEAD-box DNA helicase 45 (Tuteja et al., 2003). Pea DNA helicase 45 (Pham and Tuteja, 2002). Pea DNA helicase 47 (Vashisht and Tuteja, 2005). Helicase from hepatitis C virus (HCV) (Borowski et al., 2002).

A. Pradhan et al. / Gene 420 (2008) 66–75

study most probably inhibit the unwinding reaction of PfH45 by intercalation into the different grooves of the duplex DNA strand, which causes a physical block to the progression of the helicase. On the other hand, the interaction of these compounds with ssDNA might also block the helicase from translocating along the ssDNA. The detailed characterization of PfH45 suggests that it is a multifunctional protein and these studies might provide a significant contribution to our better understanding of nucleic acid metabolism in the malaria parasite. Furthermore the inhibitor study will be useful in understanding the mechanism of nucleic acid unwinding in the parasite. Acknowledgements The authors sincerely thank four anonymous reviewers for the helpful and constructive comments. The authors sincerely thank Dr. Narendra Tuteja, ICGEB, New Delhi for the critical reading of the manuscript and Mr. Mamadou Modibo Tekete from University of Bamako, Mali for his help in preparation of mutant proteins. The work on helicases in R. T's laboratory is partially supported by Department of Science and Technology grant. We thank Indian Council of Medical Research, New Delhi for fellowship to Arun Pradhan. Infrastructural support from the Department of Biotechnology, Government of India is gratefully acknowledged. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.gene.2008.05.005. References Bachur, N.R., et al., 1998. Anthracycline antibiotic blockade of SV40 T antigen helicase action. Biochem. Pharmacol. 55, 1025–1034. Bachur, N.R., Yu, F., Johnson, R., Hickey, R., Wu, Y., Malkas, L., 1992. Helicase inhibition by anthracycline anticancer agents. Mol. Pharmacol. 41, 993–998. Bahl, A., et al., 2003. PlasmoDB: the Plasmodium genome resource. A database integrating experimental and computational data. Nucleic Acids Res. 31, 212–215. Blom, N., Sicheritz-Ponten, T., Gupta, R., Gammeltoft, S., Brunak, S., 2004. Prediction of post-translational glycosylation and phosphorylation of proteins from the amino acid sequence. Proteomics 4, 1633–1649. Borowski, P., et al., 2002. NTPase/helicase of Flaviviridae: inhibitors and inhibition of the enzyme. Acta Biochimica Polonica 49, 597–614. Brosh Jr., R.M., Karow, J.K., White, E.J., Shaw, N.D., Hickson, I.D., Bohr, V.A., 2000. Potent inhibition of Werner and Bloom helicases by DNA minor groove binding drugs. Nucleic Acids Res. 28, 2420–2430. Buelt, M.K., Glidden, B.J., Storm, D.R., 1994. Regulation of p68 RNA helicase by calmodulin and protein kinase C. J. Biol. Chem. 269, 29367–29370. Cheng, Z., Coller, J., Parker, R., Song, H., 2005. Crystal structure and functional analysis of DEAD-box protein Dhh1p. RNA 11, 1258–1270. Du, M.X., Johnson, R.B., Sun, X.L., Staschke, K.A., Colacino, J., Wang, Q.M., 2002. Comparative characterization of two DEAD-box RNA helicases in superfamily II: human translation-initiation factor 4A and hepatitis C virus non-structural protein 3 (NS3) helicase. Biochem. J. 363, 147–155. Frick, D.N., Lam, A.M., 2006. Understanding helicases as a means of virus control. Curr. Pharm. Des. 12, 1315–1338. Gardner, M.J., et al., 2002. Genome sequence of the human malaria parasite Plasmodium falciparum. Nature 419, 498–511. George, J.W., Ghate, S., Matson, S.W., Besterma, J.M., 1992. Inhibition of DNA helicase II unwinding and ATPase activities by DNA-interacting ligands. Kinetics and specificity. J. Biol. Chem. 267, 10683–10689. Gorbalenya, A.E., Koonin, E.V., Donchenko, A.P., Blinov, V.M., 1989. Two related superfamilies of putative helicases involved in replication, recombination, repair and expression of DNA and RNA genomes. Nucleic Acids Res. 17, 4713–4730. Hall, M.C., Matson, S.W., 1999. Helicase motifs: the engine that powers DNA unwinding. Mol. Microbiol. 34, 867–877. Hernandez, G., Vazquez-Pianzola, P., 2005. Functional diversity of the eukaryotic translation initiation factors belonging to eIF4 families. Mech. Dev. 122, 865–876. Hunter, T., Sefton, B.M., 1980. Transforming gene product of Rous sarcoma virus phosphorylates tyrosine. Proc. Natl. Acad. Sci. USA 77, 1311–1315. Hyde, J.E., 2007. Drug-resistant malaria: an insight. FEBS J. 274, 4688–4698. Kim, O.T.P., Yura, K., Go, N., 2006. Amino acid residue doublet propensity in the proteinRNA interface and its application to RNA interface prediction. Nucleic Acids Res. 34, 6450–6460.

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