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J. Biol. Chem., Vol. 279, Issue 20, 20692-20698, May 14, 2004
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From the
Department of Biological Science and Technology, Faculty of Industrial Science and Technology, Tokyo University of Science, Noda, Chiba 278-8510, Japan and the
Department of Ecological Engineering, Toyohashi University of Technology, Toyohashi, Aichi 441-8580, Japan
Received for publication, January 9, 2004 , and in revised form, March 1, 2004.
| ABSTRACT |
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| INTRODUCTION |
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The DEAD box proteins have seven to eight distinctive motifs. The DEAD is derived from the amino acid sequence of motif II, the Walker B motif (2, 3). In vitro analyses of DEAD box proteins such as the translation initiation factor eIF-4A and the human nuclear protein p68 have demonstrated that these proteins possess RNA-dependent ATPase activity and are capable of melting short RNA duplex structures in an ATP-dependent manner (35). For example, eIF-4A, an archetypical member of the DEAD box protein family, is capable of unwinding partial duplex RNA in a bidirectional manner and acting on RNA or DNA-RNA, but not on the DNA duplex (4, 6). Extensive mutational analyses of the conserved regions of DEAD box proteins have demonstrated that these regions are important to ATP binding, ATP hydrolysis, RNA binding, RNA unwinding, and coupling of these different activities. In addition to these typical DEAD box RNA helicases, some DEAD box proteins have recently been shown to have peculiar characteristics. Hepatitis C virus NS3 drives the unwinding activity with all ribo- and deoxyribo-NTPs (6). Moreover, CsdA, an Escherichia coli DEAD box protein, unwinds double-stranded RNA in the absence of NTP (7). These reports suggest that the DEAD box family includes proteins with a wide variety of biochemical activities.
The yeast Saccharomyces cerevisiae contains over 20 different DEAD box proteins, many of which are essential to viability. Combined with biochemical analyses, yeast genetical analyses have revealed the functions of many DEAD box proteins. For example, the DEAD box proteins Prp5p and Prp28p are involved in pre-messenger RNA splicing (810), whereas Ded1p, which has ATP-dependent RNA helicase activity, is necessary for translation initiation (1113). Tif1p and Tif2p also participate in translation initiation (14). However, it is ribosome biogenesis that DEAD box proteins are most often associated with, because 13 DEAD box proteins have been implicated in ribosome biogenesis (1). Ribosomes are one of the largest and most complex macromolecular assemblies (reviewed in Ref. 15). DBP9 encodes an essential nucleolar DEAD box protein. Genetic depletion of Dbp9p results in a deficit of 60 S ribosomal subunits and in the appearance of half-mer polysomes. In vivo depletion of Dbp9p results in reduced synthesis of the 27 S precursor to the mature 25 S and 5.8 S ribosomal RNA, which terminally leads to a net deficit in 60 S ribosomal subunits (16). These results strongly suggest that Dbp9p participates in ribosome biogenesis.
Dbp9p is classified as a DEAD box protein based on sequence homology (17). It has never, however, been shown experimentally to unwind RNA. In this study, therefore, we purified and characterized Dbp9p, which was found to display several specific characteristics: it unwinds RNA in the absence of NTP, its ATPase activity is inhibited by RNA but stimulated by DNA, and it exhibits DNA-DNA and DNA-RNA helicase activity. Our work suggests that the DEAD box family proteins vary widely with respect to substrate specificity.
| MATERIALS AND METHODS |
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Expression and Purification of Recombinant Dbp9 Plasmids containing E. coli were grown in 2xYT medium to A600 = 0.4 at 30 °C. The expression of recombinant protein was induced by 1 mM isopropyl-1-thio-
-D-galactopyranoside at 30 °C for 2.5 h. The cells were harvested by centrifugation, washed with sonication buffer (20 mM Tris-HCl, pH 8.0, 2 mM EDTA, 0.5 M NaCl), and sonicated in the same buffer. The lysate was clarified by centrifugation at 12,000 rpm for 20 min at 4 °C. The lysate was dialyzed against buffer containing 20 mM potassium phosphate, pH 7.5, 0.5 M KCl, and 0.5 mM MgCl2 and then loaded onto a 9-ml Probond nickel-chelating resin (Invitrogen). The lysate was washed with 10-column volumes of the loading buffer (50 mM imidazole, 20 mM phosphate buffer, pH 7.5, 0.5 M KCl, 0.5 mM MgCl2). Step elutions were carried out in 3-column volumes by increasing the imidazole to 100 and then 500 mM. Dbp9p was mostly eluted in the 500-mM elution, as verified by SDS-PAGE. The elution was loaded on a 150-ml Sephacryl S 400 (Amersham Biosciences) gel-filtration column equilibrated in the same buffer. The proteins were eluted in 200 ml of the same buffer. Forty 5-ml fractions were collected and analyzed. Two fractions mostly containing Dbp9p were pooled and dialyzed against 20 mM potassium phosphate, pH 7.5, 20 mM KCl, and 10% glycerol. The protein was then loaded on a phosphocellulose cation exchange column equilibrated in the same buffer and eluted with a gradient of 0.021.0 M KCl. Fractions were analyzed for ATPase activity. Peak ATPase activity was found to correlate with the peak of Dbp9p.
ATPase AssayThe measurement of hydrolysis of [
-32P]ATP was carried out in a reaction mixture containing 50 µM ATP, 20 mM Hepes-KOH (pH 7.6), 2 mM DTT,1 2% PEG8000, 3 mM MgCl2, 0.5 µCi (18.5 kBq) of [
-32P]ATP, and various amounts of purified Dbp9p samples. The mixture was incubated at 37 °C for 1 h. Aliquots of each reaction were spotted onto a thin layer chromatography plate. ATP and free phosphate were separated by thin-layer chromatography in 0.5 M LiCl, 1 M formic acid. The sheets were dried and exposed on x-ray film. When needed, Pi on the TLC plate was quantitated by counting in a liquid scintillation counter.
RNA Unwinding AssayPreparation of substrate S1:S1 substrate for the RNA-unfolding and RNA binding assay was prepared following a previously described method (18). S1 was 81 bp T7 RNA polymerase transcript from plasmid Bluescript II KS (Stratagene) digested with HindIII. These transcripts were synthesized in the presence of [
-32P]GTP as described previously (19). We next purified S1 using a QIA quick nucleotide removal kit (Qiagen, Valencia, CA) and a Micro-Spin G25 column (Amersham Biosciences).
RNA Unwinding and Binding AssaysRNA substrate mixtures (20 µl) containing 20 mM Hepes-KOH (pH 7.6), 3 mM MgCl2, 100 mM KCl, 2 mM DTT, and 25, 50, or 100 ng of Dbp9p or 100 ng of mutated Dbp9p (R414I, R414T, R414K) were incubated at 37 °C for 30 min. Reactions were stopped by the addition of 5 µl of a mixture containing 0.1 M Tris-HCl, pH 7.4, 20 mM EDTA, 0.5% SDS, 0.1% Nonidet P-40, 0.1% xylene cyanol, 50% glycerol, and 0.2 mg/ml proteinase K. As controls, we prepared a sample with an RNA substrate that was heat-denatured by incubating for 5 min at 99 °C and without the addition of Dbp9p. Aliquots of each sample were loaded onto an 8% SDS-polyacrylamide gel and electrophoresed at 100 V. The gel was packed into a hybridization bag and exposed on an imaging plate (Fuji).
The RNA binding assay was carried out by the same method except that the RNA unwinding buffer lacked proteinase K. The mixture was loaded onto 4% nondenaturing polyacrylamide gel and separated by electrophoresis.
Cofactors for ATPasePoly(A), poly(U), and poly(C) were from Amersham Pharmacia Biotech. Heat-denatured yeast total DNA refers to yeast total DNA that was heated at 100 °C for 10 min. DNaseI-treated yeast total DNA was prepared by digestion with 1260 units of DNaseI at 25 °C for 1 h. pUC19 linear DNA was prepared by EcoRI (TAKARA) digestion. We added 10-, 100-, or 1000-ng nucleotides to ATPase reaction mixtures containing 50 ng of Dbp9p and then stirred. Measurement of ATP hydrolysis was carried out as described in ATPase assay.
DNA Helicase, DNA-RNA Helicase AssayThis assay is based on Tuteja et al. (20). The DNA substrate used in the helicase assay consists of 32P-labeled complementary oligonucleotides annealed to M13mp19 phage ssDNA to create a partial duplex. The DNA probe sequence is 5'-TTT TTT TTT TTT TT GTT TTC CCA GTC ACG AC TTT TTT TTT TTT T. The T-3' RNA probe sequence is 5'-UUU UUU UUU UUU UU GTT TTC CCA GTC ACG AC UUU UUU UUU UUU UU-3'. Twenty-five nanograms of these DNAs or RNAs was 5'-end-labeled by T4 polynucleotide and 0.925 MBq [
-32P]ATP in a T4 polynucleotide kinase buffer (50 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 5 mM DTT). Free [
-32P]ATP was removed using a MicroSpin G-25 column (Amersham Pharmacia Biotech). This labeled oligomer was annealed to M13mp19 ssDNA (1 µg) in 40 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 50 mM NaCl, and 1 mM DTT.
Forty-nine microliters of 32P-labeled oligomer mixture and 5 µlof0.2 µg/µl DNA were mixed in a solution containing 40 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 50 mM NaCl, and 1 mM DTT. The mixture was heated at 95 °C for 3 min and allowed to anneal at 65 °C for 20 min followed by slow cooling to room temperature for 40 min. The substrates were purified using a MicroSpin S-400 HR column (Amersham Biosciences). The standard reaction mixture (15 µl) consisted of 20 mM Tris-HCl (pH 8.5), 8 mM DTT, 1 mM MgCl2, 1 mM ATP, 30 mM KCl, 4% sucrose 80 µg/µl bovine serum albumin (Sigma), 5 µl of 32P-labeled helicase substrate and Dbp9p. The reaction mixture was incubated at 37 °C for 30 min and stopped by the addition of 10 µl of a helicase reaction stop buffer (0.3% SDS, 10 mM EDTA, 5% glycerol, and 0.03% bromphenol blue). After further incubation at 37 °C for 5 min, the substrate and product were separated by electrophoresis on a 12% native polyacrylamide gel (mini gel size, 8 x 10 cm) in TBE buffer (89 mM Tris borate (pH 8.2) and 2 mM EDTA (pH 8.0)). After electrophoresis, the gel was exposed to an imaging plate for 2 h and the bands were detected by a BAS2000 bioimaging analyzer (Fuji). The helicase activity using the DNA-RNA substrate was also determined as described above but in the presence of RNasein.
| RESULTS |
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-D-galactopyranoside at 30 °C for 2.5 h. Large-scale preparations of the recombinant protein were purified with anti-His tag affinity chromatography (Probond) and gel-filtration column chromatography (Sephacryl S400). This partially purified Dbp9p was purified further through phosphocellulose cation exchange column chromatography (Fig. 1A). During the purification, fractions were simultaneously assayed for ATPase activity. In the course of phosphocellulose column chromatography, the elution profile was monitored by an ATPase assay. The peak fraction of the ATPase consisted of a homogeneous preparation of a protein with an apparent molecular mass of 80 kDa (Fig. 1B), which is in good agreement with the predicted molecular mass of Dbp9p with all the tags described above. Identification of the purified protein was also verified by an anti-FLAG tag antibody (data not shown).
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| DISCUSSION |
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Some DEAD box proteins need the interaction with other proteins to exert their ATPase or helicase activity (4). Here we show that Dbp9p possesses an unwinding activity on S1 ssRNA without ATP. This result indicates that Dbp9p has ATPase-independent RNA helix-destabilizing activity and apparently does not need to interact with other proteins to display RNA unwinding activity like that of E. coli CsdA (7), although Dbp9p seems to interact with Dbp6p functionally in vivo (16). In the RNA unwinding assays, there must be several artificial conditions because most of the DEAD box proteins identified thus far do not show any substrate specificity. Therefore, one could speculate that the unwinding reaction mediated by Dbp9p would be nonenzymatic. However, we think this possibility is excluded by the experiments with the mutated proteins, which could not hydrolyze ATP. The mutated proteins were unable to unwind the S1 RNA substrate.
Dbp9p hydrolyzes ATP to produce free phosphate (Fig. 2B). The R414I, R414T, and R414K mutations, which have a conserved Arg residue in the RNA binding motif replaced with Ile, Thr, and Lys, respectively, impaired the ATPase activity of Dbp9p. This Dbp9p ATPase activity was inhibited by one-third with the addition of RNA and stimulated by the addition of genomic DNA. We have not tried to identify an in vivo Dbp9p substrate. We therefore could not examine whether in vivo substrate RNA also inhibits the ATPase activity of Dbp9p. The results presented above indicate that the Dbp9p ATPase has a preference for DNA. In terms of its cofactor, Drosophila Vasa (24) and HCV CAP-Rf (25), another two DEAD box proteins, also have RNA-independent ATPase activity, and the activity of CAP-Rf is even inhibited by RNA. The ATPase activity of some DExH box proteins, another RNA helicase family, is known to be stimulated with DNA. For example, ATPase activity of S. cerevisiae Mer3p, a DExH box protein, is stimulated by DNA (26). But there are few other DEAD box proteins whose ATPase activity is known to be stimulated by DNA.
The Dbp9p was able to displace the 17-nt fragment that had been annealed to the M13mp18 single-stranded circular DNA in a reaction containing ATP. Some DExH box RNA helicases, such as RNA helicase A and human G3BP, have DNA-DNA and DNA-RNA helicase activities (23, 27). Some viral helicases, such as NPHII and HCV NS3, also have DNA-DNA and RNA-RNA helicase activities (6, 28, 29). In the case of DEAD box proteins, DP103 and AtDRH1 show dsRNA unwinding and DNA-RNA helicase activities (30, 31), but no DEAD box protein has been shown to have DNA helicase activity. The DNA helicase activity of Dbp9p was found to be abolished by an amino acid substitution in its RNA-binding domain (Fig. 7). To our knowledge, this is the first study to show that a DEAD box protein has DNA helicase activity. It is not known whether the unwinding of the DNA-DNA duplex by the recombinant Dbp9p is significant in terms of its physiological function. Although the hypothesis that Dbp9p could act as a DNA helicase in addition to RNA helicase is attractive, experimental data in support of such a possibility are lacking.
NOH61 is a human ortholog of Dbp9p. Although it has not been determined whether NOH61 has helicase activity, the nucleolar localization and the participation in ribosome biogenesis are common to both proteins (32). The addition of ds and ssDNA activated the ATPase activity of NOH61 3.9- and 1.6-fold, respectively. The biochemical characteristic that ATPase activity is stimulated by the addition of DNA could also be applied to NOH61. But NOH61 cannot complement the defect of dbp9 mutation. In addition, NOH61 has a different substrate than that of Dbp9p in ribosome biogenesis. It is therefore speculated that NOH61 plays a role in the later stages of the processing that leads 65 S preribosomal particles to mature to 60 S ribosomal subunits, whereas Dbp9p seems to act at a very early stage in the formation of the 60 S equivalent. These differences might be based on the difference between the biochemical characteristics of these two orthologous proteins: the ATPase activity of Dbp9p is severely inhibited by RNA but that of NOH61 is not.
| FOOTNOTES |
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To whom correspondence should be addressed: Dept. of Biological Science and Technology, Faculty of Industrial Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba 278-8510, Japan. Tel.: 81-4-7124-1501 (ext. 4408); Fax: 81-4-7122-1360; E-mail: yasufumi{at}rs.noda.tus.ac.jp.
1 The abbreviations used are: DTT, dithiothreitol; ss, single strand; ds, double strand. ![]()
| ACKNOWLEDGMENTS |
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| REFERENCES |
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