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Biochemical Journal Genes, Whereas the Hyperthermophilic Archaeon Pyrococcus DNA Hybrids, Similarly to the Rnase H Proteins

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Biochemical Journal Genes, Whereas the Hyperthermophilic Archaeon Pyrococcus DNA Hybrids, Similarly to the Rnase H Proteins www.biochemj.org Biochem. J. (2010) 426, 337–344 (Printed in Great Britain) doi:10.1042/BJ20091553 337 Characterization of RNase HII substrate recognition using RNase HII–argonaute chimaeric enzymes from Pyrococcus furiosus Sayaka KITAMURA*†, Kosuke FUJISHIMA*, Asako SATO*, Daisuke TSUCHIYA*, Masaru TOMITA*† and Akio KANAI*†1 *Institute for Advanced Biosciences, Keio University, Tsuruoka 997-0017, Japan, and †Systems Biology Program, Graduate School of Media and Governance, Keio University, Fujisawa 252-8520, Japan RNase H (ribonuclease H) is an endonuclease that cleaves one of the conserved secondary structural regions (the fourth the RNA strand of RNA–DNA duplexes. It has been reported β-sheet and the fifth α-helix of Pf-RNase HII) contains family- that the three-dimensional structure of RNase H is similar to that specific amino acid residues. Using a series of Pf-RNase HII–Pf - of the PIWI domain of the Pyrococcus furiosus Ago (argonaute) Ago chimaeric mutants of the region, we discovered that residues protein, although the two enzymes share almost no similarity Asp110,Arg113 and Phe114 are responsible for the dsRNA (double- in their amino acid sequences. Eukaryotic Ago proteins are stranded RNA) digestion activity of Pf-RNase HII. On the basis of key components of the RNA-induced silencing complex and the reported three-dimensional structure of Ph-RNase HII from are involved in microRNA or siRNA (small interfering RNA) Pyrococcus horikoshii, we built a three-dimensional structural recognition. In contrast, prokaryotic Ago proteins show greater model of RNase HII complexed with its substrate, which suggests affinity for RNA–DNA hybrids than for RNA–RNA hybrids. that these amino acids are located in the region that discriminates Interestingly, we found that wild-type Pf-RNase HII (P. furiosus, DNA from RNA in the non-substrate strand of the duplexes. RNase HII) digests RNA–RNA duplexes in the presence of Mn2+ ions. To characterize the substrate specificity of Pf-RNase HII, we aligned the amino acid sequences of Pf-RNase HII and Pf - Key words: archaeon, argonaute, double-stranded RNA (dsRNA), Ago, based on their protein secondary structures. We found that ribonuclease H, RNA–DNA duplex, site-directed mutagenesis. INTRODUCTION abyssi and Pyrococcus horikoshii. Interestingly, several bacterial Ago proteins, such as Aa-Ago (Aquifex aeolicus Ago) and RNase H (ribonuclease H) is a ubiquitous enzyme found in all Tt-Ago (Thermus thermophilus Ago), are reported to be DNA- three kingdoms of the tree of life: archaea, bacteria and eukarya. strand-mediated site-specific RNA endonucleases [8–10]. In other Bacteria and eukaryotes contain two or more RNase H-encoding words, these Ago proteins can digest the RNA strand of RNA– Biochemical Journal genes, whereas the hyperthermophilic archaeon Pyrococcus DNA hybrids, similarly to the RNase H proteins. It has also furiosus has only one gene, designated rnhB [1]. We have pre- been reported that the archaeal PIWI protein from Archaeoglobus viously constructed a reconstitution system for Okazaki fragment fulgidus (Af -PIWI) binds to DNA more tightly than it does to processing using two P.furiosus recombinant enzymes, Pf-RNase RNA [11], suggesting an evolutionary relationship between the HII and Pf -FEN-1 (Flap endonuclease 1) [2]. We showed that both RNase H and Ago proteins in prokaryotes. enzymes are required for the effective degradation of the RNA To characterize the substrate specificity of Pf-RNase HII, we moiety of an RNA–DNA–DNA substrate (the Okazaki substrate). first aligned the amino acid sequences of Pf-RNase HII and Pf - Song et al. [3] have reported that the three-dimensional structure Ago based on the protein secondary structures. We found that of the PIWI domain of the P. furiosus Ago (argonaute) protein one of the helical regions corresponding to the same secondary is similar to that of RNase H, although the two enzymes show structure in both proteins contains family-specific conserved almost no similarity in their amino acid sequences. It has also been amino acid residues. We then used Pf -RNase HII as the basic reported that in the eukarya, the Ago proteins are key components enzyme to produce a series of chimaeric Ago–RNase HII enzymes of the RNA-induced silencing complex and are involved in in Escherichia coli that were mutated in the region of interest, microRNA or siRNA (small interfering RNA) recognition [4–7]. and characterized their specificities. As a result, we unexpectedly Biochemical studies of Ago proteins from the eukarya have shown found that wild-type Pf -RNase HII digests the RNA strand of that some have endonuclease (slicer) activity and can digest RNA–RNA duplexes. We also discovered that amino acid residues one RNA moiety of RNA–RNA duplexes. Unlike eukaryotic Asp110,Arg113 and Phe114 are responsible for the dsRNA (double- genomes, which contain one or more Ago genes (and/or Ago- stranded RNA) digestion activity of Pf-RNase HII and are located related piwi genes), few genomes of either bacterial or archaeal in the region that discriminates the non-substrate strand (the so- origin encode Ago or Ago-related PIWI proteins. For example, the called ‘guide strand’) in RNA–DNA and RNA–RNA duplexes, as P.furiosus genome has one Ago gene (Pf-Ago), whereas there is no revealed by a three-dimensional structural model of Pyrococcus such gene in the genomes of the closely related species Pyrococcus enzymes. Abbreviations used: Aa, Aquifex aeolicus; Af, Archaeoglobus fulgidus; Ago, argonaute; dsRNA, double-stranded RNA; FAM, carboxyfluorescein; FEN, Flap endonuclease; Mj, Methanococcus jannaschii; Mk, Methanopyrus kandleri; Ni-IMAC, nickel-immobilized metal-ion-affinity chromatography; Pf, Pyrococcus furiosus; Ph, Pyrococcus horikoshii; RNase H, ribonuclease H; rnhB, RNase H-encoding gene; RT, reverse transcriptase; ssDNA, single- stranded DNA; Tt, Thermus thermophilus; WT, wild-type. 1 To whom correspondence should be addressed (email [email protected]). c The Authors Journal compilation c 2010 Biochemical Society 338 S. Kitamura and others EXPERIMENTAL Table S1 for details). Standard processing assays were performed in a final volume of 20 μl which contained 20 mM Tris/HCl Dataset (pH 8.0), 1 mM dithiothreitol, 50 mM KCl, 4 mM MnCl2, The UniProt accession codes for each protein are as follows: 40 pmol of substrate and 5–100 ng of the purified recombinant Pf -RNase HII, Q8U036; Ph-RNase HII, O59351; Af -RNase Pf -RNase HII protein. The reaction mixture was incubated at HII, O29634; Mj (Methanococcus jannaschii)-RNase HII, 50 ◦C for 15 min and quenched with an equal volume of STOP Q57599; Mk (Methanopyrus kandleri)-RNase HII, Q8TYV5; solution [1 M Tris/HCl (pH 7.2), 8 M urea and a small amount Pf -Ago, Q8U3D2; Af -PIWI protein, O28951; Mj-PIWI protein of Blue Dextran (Sigma–Aldrich)]. In some experiments, MgCl2 (uncharacterized protein MJ1321), Q58717; and Mk-Ago (the was used instead of MnCl2. The samples were denatured at homologue of the eukaryotic protein), Q8TVS7. The protein 70 ◦C for 5 min and the cleavage products were separated on secondary structures of the Pf -RNase HII and the Pf -Ago PIWI a20% polyacrylamide gel containing 8 M urea. Gel images domain proteins were generated from the structures of Ph- were visualized and analysed using a Molecular Imager FX Pro RNase HII (PDB entry 1UAX) and Pf -Ago (PDB entry 1U04) apparatus (Bio-Rad Laboratories). respectively. Structural model of Pf-RNase HII complexed with a dsRNA ligand Construction of the expression vectors A structural model was constructed using the molecular graphics The Pf -RNase HII M1–M4 expression plasmids were constructed software Visual Molecular Dynamics [12] with the atomic co- by replacing the NdeI–HindIII fragment of the WT (wild- ordinates of Ph-RNase HII (PDB 1UAX) and an A-form dsRNA type) Pf -RNase HII plasmid pET-rnh2 [2] with each of the (PDB 2Q1R), which were downloaded from the Protein Data NdeI–HindIII fragments amplified using the mutant-specific Bank. The dsRNA structure was manually positioned on to the primers described in Supplementary Table S1 (available at RNase HII model, with reference to the crystal structure of http://www.BiochemJ.org/bj/426/bj4260337add.htm). The Pf - the complex between Bacillus halodurans RNase H and an RNA– RNase HII M5–M9 plasmids were constructed using the DNA hybrid (PDB 1ZBI). QuikChange® site-directed mutagenesis kit (Stratagene) and specific primers (Supplementary Table S1) with pET-rnh2 as the template. The resulting vectors, pET-rnhM1–M9, encoded each of RESULTS the Pf -RNase HII mutant proteins with a His6 tag at the C-terminal ends. The nucleotide sequences of the expression plasmids were Alignment of the amino acid residues of Pf-RNase HII and the PIWI verified by sequencing. domain of Pf-Ago, based on their secondary structures As several bacterial Ago proteins (e.g. Aa-Ago and Tt-Ago) Expression and purification of His6-tagged recombinant proteins are reportedly DNA-strand-mediated site-specific RNA endo- E. coli strain BL21(DE3) was transformed with the expression nucleases [8–10], and because the archaeal Af -PIWI protein plasmids. The transformants were grown exponentially in Luria– binds to DNA more tightly than to RNA [11], we first focused Bertani medium containing 50 μg/ml ampicillin and 0.4 mM on the biochemical properties of recombinant Pf-Ago protein. ◦ IPTG (isopropyl β-D-thiogalactoside) at 37 C. After 14–16 h of We used a gel-shift assay to confirm that the purified Pf-Ago further growth at 30 ◦C, the cells were harvested by centrifugation protein preferentially binds to ssDNA (single-stranded DNA) (5000 g for 5 min at 4 ◦C) and the recombinant proteins were over dsDNA (double-stranded DNA) or RNA–DNA duplexes, extracted by sonication with a Handy Sonic model UR-20P and obtained no clear band-shift for dsRNA (results not shown).
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