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 the RNA strand of RNA–DNA duplexes. It has been reported that the three-dimensional structure of RNase H is similar to that of the PIWI domain of the Pyrococcus furiosus Ago (argonaute) protein, although the two enzymes share almost no similarity in their amino acid sequences. Eukaryotic Ago proteins are key components of the RNA-induced silencing complex and are involved in microRNA or siRNA (small interfering RNA) recognition. In contrast, prokaryotic Ago proteins show greater affinity for RNA–DNA hybrids than for RNA–RNA hybrids. Interestingly, we found that wild-type Pf-RNase HII (P. furiosus, RNase HII) digests RNA–RNA duplexes in the presence of Mn 2+ ions. To characterize the substrate specificity of Pf-RNase HII, we aligned the amino acid sequences of Pf-RNase HII and Pf - Ago, based on their protein secondary structures. We found that one of the conserved secondary structural regions (the fourth β -sheet and the fifth α-helix of Pf-RNase HII) contains family- specific amino acid residues. Using a series of Pf-RNase HII–Pf - Ago chimaeric mutants of the region, we discovered that residues Asp 110 , Arg 113 and Phe 114 are responsible for the dsRNA (double- stranded RNA) digestion activity of Pf-RNase HII. On the basis of the reported three-dimensional structure of Ph-RNase HII from Pyrococcus horikoshii, we built a three-dimensional structural model of RNase HII complexed with its substrate, which suggests that these amino acids are located in the region that discriminates DNA from RNA in the non-substrate strand of the duplexes. Key words: archaeon, argonaute, double-stranded RNA (dsRNA), ribonuclease H, RNA–DNA duplex, site-directed mutagenesis. INTRODUCTION RNase H (ribonuclease H) is a ubiquitous enzyme found in all three kingdoms of the tree of life: archaea, bacteria and eukarya. Bacteria and eukaryotes contain two or more RNase H-encoding genes, whereas the hyperthermophilic archaeon Pyrococcus furiosus has only one gene, designated rnhB [1]. We have pre- viously constructed a reconstitution system for Okazaki fragment processing using two P. furiosus recombinant enzymes, Pf-RNase HII and Pf -FEN-1 (Flap endonuclease 1) [2]. We showed that both enzymes are required for the effective degradation of the RNA moiety of an RNA–DNA–DNA substrate (the Okazaki substrate). Song et al. [3] have reported that the three-dimensional structure of the PIWI domain of the P. furiosus Ago (argonaute) protein is similar to that of RNase H, although the two enzymes show almost no similarity in their amino acid sequences. It has also been reported that in the eukarya, the Ago proteins are key components of the RNA-induced silencing complex and are involved in microRNA or siRNA (small interfering RNA) recognition [4–7]. Biochemical studies of Ago proteins from the eukarya have shown that some have endonuclease (slicer) activity and can digest one RNA moiety of RNA–RNA duplexes. Unlike eukaryotic genomes, which contain one or more Ago genes (and/or Ago- related piwi genes), few genomes of either bacterial or archaeal origin encode Ago or Ago-related PIWI proteins. For example, the P. furiosus genome has one Ago gene (Pf-Ago), whereas there is no such gene in the genomes of the closely related species Pyrococcus abyssi and Pyrococcus horikoshii. Interestingly, several bacterial Ago proteins, such as Aa-Ago (Aquifex aeolicus Ago) and Tt-Ago (Thermus thermophilus Ago), are reported to be DNA- strand-mediated site-specific RNA endonucleases [8–10]. In other words, these Ago proteins can digest the RNA strand of RNA– DNA hybrids, similarly to the RNase H proteins. It has also been reported that the archaeal PIWI protein from Archaeoglobus fulgidus (Af -PIWI) binds to DNA more tightly than it does to RNA [11], suggesting an evolutionary relationship between the RNase H and Ago proteins in prokaryotes. To characterize the substrate specificity of Pf-RNase HII, we first aligned the amino acid sequences of Pf-RNase HII and Pf - Ago based on the protein secondary structures. We found that one of the helical regions corresponding to the same secondary structure in both proteins contains family-specific conserved amino acid residues. We then used Pf -RNase HII as the basic enzyme to produce a series of chimaeric Ago–RNase HII enzymes in Escherichia coli that were mutated in the region of interest, and characterized their specificities. As a result, we unexpectedly found that wild-type Pf -RNase HII digests the RNA strand of RNA–RNA duplexes. We also discovered that amino acid residues Asp 110 , Arg 113 and Phe 114 are responsible for the dsRNA (double- stranded RNA) digestion activity of Pf-RNase HII and are located in the region that discriminates the non-substrate strand (the so- called ‘guide strand’) in RNA–DNA and RNA–RNA duplexes, as revealed by a three-dimensional structural model of 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 akio@sfc.keio.ac.jp). c  The Authors Journal compilation c  2010 Biochemical Society www.biochemj.org Biochemical Journal