Báo cáo khoa học: Ribonuclease H: molecular diversities, substrate binding domains, and catalytic mechanism of the prokaryotic enzymes ppt

MINIREVIEW Ribonuclease H: molecular diversities, substrate binding domains, and catalytic mechanism of the prokaryotic enzymes Takashi Tadokoro and Shigenori Kanaya Department of Material and Life Science, Osaka University, Japan Identification of bacterial RNase HIII with a TBP-like substrate-binding domain RNase H is defined as an enzyme that specifically hydrolyzes the phosphodiester bonds of RNA hybrid- ized to DNA at the P–O3¢ bond. Prokaryotic RNases H, which are involved in DNA replication, repair and transcription [1,2] have been classified into RNases HI, HII and HIII based on differences in their amino acid sequences [3,4] (Fig. 1). RNase HI represents type 1 RNase H, and RNases HII and HIII represent type 2 RNase H. The genes encoding RNase HI [5] and RNase HII [6] were first cloned from the Escherichia coli genome in 1983 and 1990, respectively. The gene encoding RNase HIII was first cloned from the Bacillus subtilis genome in 1999 [3]. Two different terminologies, RNases HII and HIII, are given to type 2 RNases H because certain bacte- rial genomes, such as the B. subtilis, Streptococ- cus pneumoniae and Aquifex aeolicus genomes, contain two genes encoding type 2 RNases H. The protein that shows higher amino acid sequence similarity to E. coli RNase HII is designated as RNase HII, whereas the other is designated as RNase HIII. Unlike RNase HII, which is widely present in various organisms including bacteria and archaea, RNase HIII is present only in a limited number of bacteria [3,4]. Keywords catalytic mechanism; crystal structure; evolution; genome; hybrid binding domain; molecular diversity; prokaryote; RNase H; RNA ⁄ DNA hybrid; substrate-binding domain Correspondence S. Kanaya, Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1, Yamadaoka, Suita, Osaka 565-0871, Japan Fax ⁄ Tel: +81 6 6879 7938 E-mail: kanaya@mls.eng.osaka-u.ac.jp (Received 18 October 2008, revised 18 December 2008, accepted 12 January 2009) doi:10.1111/j.1742-4658.2009.06907.x The prokaryotic genomes, for which complete nucleotide sequences are available, always contain at least one RNase H gene, indicating that RNase H is ubiquitous in all prokaryotic cells. Coupled with its unique substrate specificity, the enzyme has been expected to play crucial roles in the biochemical processes associated with DNA replication, gene expression and DNA repair. The physiological role of prokaryotic RNases H, espe- cially of type 1 RNases H, has been extensively studied using Escherichia coli strains that are defective in RNase HI activity or overproduce RNase HI. However, it is not fully understood yet. By contrast, significant progress has been made in this decade in identifying novel RNases H with respect to their biochemical properties and structures, and elucidating catalytic mechanism and substrate recognition mechanism of RNase H. We review the results of these studies. Abbreviations Afu, Archaeoglobus fulgidus; Bha, Bacillus halodurans; Bst, Bacillus stearothermophilus; Bsu, Bacillus subtilis; Eco, Escherichia coli; Halo, Halobacterium sp. NRC-1; HBD, hybrid binding domain; HIV-1, human immunodeficiency virus type 1; Mja, Methanococcus jannaschii; MMLV, Moloney murine leukemia virus; Pae, Pyrobaculum aerophilum; RNase H, ribonuclease H; Son, Shewanella oneidensis; Sto, Sulfolobus tokodaii; TBP, TATA-box binding proteins; Tko, Thermococcus kodakaraensis; Tma, Thermotoga maritima; Tth, Thermus thermophilus. 1482 FEBS Journal 276 (2009) 1482–1493 ª 2009 The Authors Journal compilation ª 2009 FEBS The enzymatic properties of RNase HIII have been well characterized using B. subtilis RNase HIII (Bsu- RNase HIII) [3]. RNase HIII is evolutionarily more distantly related to RNase HI than to RNase HII. For example, Bsu-RNase HIII shows the poor amino acid sequence identity to E. coli RNase HI (Eco- RNase HI), whereas it shows the amino acid sequence identities of 20–21% to E. coli RNase HII (Eco- RNase HII) and B. subtilis RNase HII (Bsu-RNase HII). Nevertheless, Bsu-RNases HIII is more closely related to Eco-RNase HI than to Eco-RNase HII or Bsu-RNase HII in the enzymatic properties, such as metal ion specificities, specific activities and cleavage site specificities. These results may suggest that Bsu-RNase HIII functions as a substitute for Eco- RNase HI. However, it has been reported for Chlamydophila pneumoniae that the RNase HII ⁄ HIII combination is not a simple substitution of Eco-RNase HI ⁄ HII [7,8]. RNases HIII are characterized by the presence of a long N-terminal extension compared with other type 2 RNases H (Fig. 1). The amino acid sequences of these extensions show a significant similarity to those of TATA-box binding proteins (TBPs). The first crystal structure of RNase HIII was determined using RNase HIII from the moderate thermophile B. stearo- thermophilus (Bst-RNase HIII) [9]. Bst-RNase HIII shows the amino acid sequence identity of 47.1% to Bsu-RNase HIII and has a similar N-terminal exten- sion. Its enzymatic properties are similar to those of Bsu-RNase HIII, except that it is more stable [10]. According to the crystal structure of Bst-RNase HIII, the N-terminal extension assumes a TBP-like structure and is present as an independent domain (Fig. 2). The Fig. 1. Schematic representation of the pri- mary structures of prokaryotic RNases H. The primary structures of the representative members of type 1 (A) and type 2 (B) RNases H, which have been reported to be enzymatically active, are shown. Solid box represents the RNase H domain and gray box represents TBP-like domain, hybrid-bind- ing domain (HBD) or acid phosphatase domain. The positions of the four acidic active site residues and the histidine resi- due, which is well conserved in type 1 RNases H, are shown. The numbers repre- sent the positions of the amino acid residues relative to the initiator methionine for each protein. T. Tadokoro and S. Kanaya Prokaryotic RNases H FEBS Journal 276 (2009) 1482–1493 ª 2009 The Authors Journal compilation ª 2009 FEBS 1483 structure of the C-terminal RNase H domain is highly similar to those of archaeal RNases HII [11–13] (Fig. 2). The active site motif of RNase HIII (DEDE) is similar, but is not identical to that of RNase HI or HII (DEDD) [4] (Fig. 1). Nevertheless, the steric configurations of these four acidic active-site residues are very similar to those of RNases HI and HII. Bio- chemical characterization of the Bst-RNase HIII deriv- atives with N- and ⁄ or C-terminal truncations indicates that the N-terminal domain with a TBP-like structure and C-terminal helix are involved in substrate binding, but the former contributes to substrate binding more greatly than the latter [9]. TBP binds to the target DNA at the flat surface of the molecule [14–16]. The N-terminal domain of RNase HIII probably uses the same surface for substrate binding as TBP to bind DNA. Identification of archaeal type 1 RNases H Most of the archaeal genomes, for which the complete genome sequences are available, only contain the type 2 RNase H (RNase HII) genes. The exceptions are the Halobacterium sp. NRC-1, Sulfolobus tokodaii and Pyrobaculum aerophilum genomes, which contain the type 1 RNase H (RNase HI) genes in addition to the RNase HII genes. The first archaeal RNase HI protein, which was shown to be active both in vivo and in vitro, is RNase HI from Halobacterium sp. NRC-1 (Halo-RNase HI) [17]. Later, RNases HI from S. tokodaii (Sto-RNase HI) and P. aerophilum (Pae- RNase HI) were also shown to be enzymatically active [18]. Database searches using the Sto-RNase HI sequence indicate that not only the archaeal genomes, Fig. 1. (Continued). Prokaryotic RNases H T. Tadokoro and S. Kanaya 1484 FEBS Journal 276 (2009) 1482–1493 ª 2009 The Authors Journal compilation ª 2009 FEBS but also the bacterial and eukaryotic genomes contain the genes encoding a Sto-RNase HI homologue. Of them, the Sto-RNase HI homologue from B. subtilis (YpdQ) is inactive [3], whereas those from Strepto- myces coelicolor (SCO7284 [18] and SCO2299 [19]) and Corynebacterium glutamicum (Cg12236) [20] are active. Phylogenetic analyses of the type 1 RNase H sequences show that the Sto-RNase HI homologues form an independent domain, which is different from those of bacterial, eukaryotic and retroviral type 1 RNases H [18]. Characteristics common to the amino acid sequences of these Sto-RNase HI homologues are the lack of a basic protrusion, which has been shown to be important for substrate binding in Eco - RNase HI [21,22], and the lack of the histidine residue, which is well conserved in other type 1 RNases H and has been shown to be important for catalytic function of Eco-RNase HI [23] (Fig. 1). Sto-RNase HI consists of 149 amino acid residues and has neither an N-terminal nor C-terminal extension compared with Eco-RNase HI (Fig. 1). By contrast, Halo-RNase HI consists of 199 residues and has an N-terminal 65-residue extension (Fig. 1). This extension does not show significant amino acid sequence similar- ity to an N-terminal TBP-like domain of RNases HIII or hybrid-binding domain (HBD) of eukaryotic RNases H1. Sto-RNase HI and Halo-RNase HI exhibit unique enzymatic properties. They cleave the substrate at the RNA–DNA junction [17,18]. Interestingly, Sto - RNase HI exhibits RNase H* activity, which degrades the RNA strand of the RNA ⁄ RNA duplex [18]. It has been reported that retroviral RNases H cleave the RNA–DNA junction [24] and exhibit RNase H* activ- ity [25]. Thus, archaeal RNases HI are more closely related to retroviral RNases H than bacterial and eukaryotic type 1 RNases H in enzymatic properties. The crystal structure of Sto-RNase HI was deter- mined as the first archaeal type 1 RNase H structure [26] (Fig. 2). Despite the low amino acid sequence identity between Sto-RNase HI and other type 1 RNases H, Sto-RNase HI shows high structural simi- larity to these RNases H, including Eco-RNase HI [27,28], human immunodeficiency virus type 1 (HIV-1) RNase H [29,30] and Bacillus halodurans RNase HI (Bha-RNase HI) [31]. The steric configurations of the four acidic active-site residues are well conserved in the Sto-RNase HI structure (Fig. 3). Like other Sto-RNase HI homologues, Sto-RNase HI lacks a his- tidine residue, which is well conserved in various type 1 RNases H. However, Arg118 of Sto-RNase HI is located in the same position as His124 of Eco-RNase HI, His539 of HIV-1 RNase H and Glu188 of Bha-RNase H. Mutation of this residue to Ala considerably reduces both the RNase H and RNase H* activities without seriously affecting sub- strate binding, suggesting that Arg118 is involved in catalytic function [26]. This residue may promote prod- uct release by perturbing the coordination of the metal ion A, as proposed for Glu188 of Bha-RNase H [31]. The catalytic mechanism of RNase H is described in more detail below. As described below under ‘Substrate recognition mechanism’, RNase H has two grooves in which the RNA and DNA backbones of RNA ⁄ DNA hybrids bind. A phosphate-binding pocket and DNA-binding channel located in the DNA-binding groove are respon- sible for the specificity of RNase H for the RNA ⁄ DNA Fig. 2. Crystal structures of prokaryotic RNases H. Ribbon dia- grams of RNases HI from S. tokodaii (Sto-RNase HI) (PDB code 2EHG) and E. coli (Eco-RNase HI) (PDB code 2RN2), RNases HII from T. maritima (Tma-RNase HII) (PDB code 2ETJ) and T. kodaka- raensis (Tko-RNase HII) (PDB code 1IO2), and B. stearothermophi- lus RNase HIII (Bst-RNase HIII) (PDB code 2D0A). N and C represent the N- and C-termini. Four acidic active site residues are shown as ball-and-stick models. The basic protrusion of Eco-RNa- se HI, and TBP-like domain and C-terminal helix of Bst-RNase HIII are indicated. T. Tadokoro and S. Kanaya Prokaryotic RNases H FEBS Journal 276 (2009) 1482–1493 ª 2009 The Authors Journal compilation ª 2009 FEBS 1485 hybrid. However, the amino acid residues forming the phosphate-binding pocket are not well conserved in the Sto-RNase HI structure. In addition, the DNA-binding channel, which is formed in the basic protrusion, is not present in the Sto-RNase HI structure because Sto- RNase HI lacks a basic protrusion. By contrast, the RNA-binding groove is well conserved in the Sto- RNase HI structure. The weak conservation of the DNA-binding groove may account for the ability of Sto-RNase HI to cleave double-stranded (ds)RNA. Weaker conservation of the DNA-binding groove com- pared with that of the RNA-binding groove is also observed in HIV-1 and Moloney murine leukemia virus (MMLV) RNases H. It is noted that the structure of Sto-RNase HI is highly similar to that of the RNase H-like domain of the PIWI domain of Argonaute from A. aiolicus [32]. Unlike other Argonaute proteins, A. aeolicus Argona- ute exhibits both RNA-guided and DNA-guided RNase activities, using the RNase H-like domain of the PIWI domain [32]. Identification of bacterial RNase HI with an N-terminal hybrid binding domain HBD-RNase HI from the psychrotrophic bacterium Shewanella sp. SIB1 is the first bacterial type 1 RNase H protein containing an N-terminal HBD to be characterized biochemically [33]. Bha-RNase HI also contains HBD at the N-terminus [31]. HBD, which has previously been designated as a dsRNA binding domain, consists of  50 amino acid residues and is commonly present at the N-termini of eukary- otic type 1 RNases H [34]. This domain is renamed as HBD, because HBD of human RNase H1 binds more strongly to RNA ⁄ DNA hybrids than to dsRNA [35]. The binding mechanism of HBD and its role in enzymatic activity is discussed in more detail below, based on the co-crystal structure of HBD of human RNase H1 with the RNA ⁄ DNA hybrid [35]. HBD-RNase HI from Shewanella sp. SIB1, which has previously been designated as RBD-RNase HI [33], consists of 262 amino acid residues (Fig. 1). The Shewanella sp. SIB1 genome contains RNase HI [36] and RNase HII [37] genes as well. All three RNase H proteins are enzymatically active. Thus, this genome contains three active RNase H genes (two type 1 and one type 2). In addition to SIB1 genome, the S. frigid- imarina, S. denitrificans and Photobacterium profundum genomes contain three genes encoding RNase HI, HBD-RNase HI and RNase HII. Of these, two type 1 RNases H, HBD-RNase HI (RNase H domain) always shows lower sequence identity to Eco-RNase HI. For example, SIB1 HBD-RNase HI and SIB1 RNase HI show amino acid sequence identities of 17 and 63% to Eco-RNase HI, respectively. It has been reported that the S. coelicolor genome also contains three active RNase H genes encoding two type 1 and one type 2 RNases H [19]. However, none of them contain HBD at the N-termini. Interestingly, one of these type 1 RNases H (SCO2299) is a bifunctional enzyme consist- ing of an N-terminal RNase H domain and a C-termi- nal acid phosphatase domain [19] (Fig. 1). Catalytic mechanism of RNase H The crystal structures of eight type 1 and five type 2 RNases H have so far been determined. They are Eco-RNase HI [27,28], Thermus thermophilus RNase HI (Tth-RNase HI) [38], Bha-RNase HI [31], Shewanel- la oneidensis RNase HI (Son-RNase HI) [39], Sto-RNase HI [26], HIV-1 RNase H [29,30], MMLV RNase H [40,41] and human RNase H1 [42] for type 1 RNases H, and Methanococcus jannaschii RNase HII Fig. 3. Stereoview of the active site struc- tures of RNases H. The side chains of the active site residues of Sto-RNase HI (salmon) and HIV-1 RNase H (green) are superimposed onto those in the co-crystal structure of Bha-RNase H with the sub- strate and metal ions (yellow). The positions of the RNA strand of the substrate with the scissile phosphate group between R()1) and R(0) and two metal ions A and B are also shown. Prokaryotic RNases H T. Tadokoro and S. Kanaya 1486 FEBS Journal 276 (2009) 1482–1493 ª 2009 The Authors Journal compilation ª 2009 FEBS (Mja-RNase HII) [11], Thermococcus kodakaraensis RNase HII (Tko-RNase HII) [12], Archaeoglobus fulgi- dus RNase HII (Afu-RNase HII) [13], Bst-RNase HIII [9] and Thermotoga maritima RNase HII (Tma-RNase - HII) (PDB code 2ETJ) for type 2 RNases H. The struc- tures of the representative members of these RNases H are shown in Fig. 2. These structures share a main chain fold, termed the RNase H-fold, and steric configura- tions of the four acidic active-site residues, suggesting that their catalytic mechanisms are basically identical. It has long been controversial whether the enzyme requires one or two metal ions for activity, because the number of the metal-binding sites found in the crystal structures of the enzyme–metal ion complex formed in the absence of the substrate varies for different enzymes and different metal ions. For example, single Mg 2+ [43] and Mn 2+ [44] ions bind to the active sites of the wild-type and mutant proteins of Eco-RNa- se HI, respectively, whereas two Mn 2+ ions bind to Eco-RNase HI [45] and HIV-1 RNase H [29]. How- ever, the co-crystal structure of Bha-RNase HI with RNA ⁄ DNA substrate and Mg 2+ [31] shows that two Mg 2+ ions bind to the active site of the enzyme (Fig. 3). Both metal ions are coordinated by the acidic active site residues, scissile phosphate group of the substrate and water molecules. The distance between these two metal ions increases when the substrate is cleaved [46]. Based on these results, two-metal-ion catalysis mechanism has been proposed for RNase H [31,46,47]. According to this mechanism, metal ion A is required for substrate-assisted nucleophile formation and product release, and metal ion B is required to destabilize the enzyme–substrate complex and thereby promote the phosphoryl transfer reaction (Fig. 4). However, it remains controversial whether an active site carboxyl group directly participates in catalysis as a general base [48]. In addition to the four acidic active site residues, the histidine residue is well conserved in the type 1 RNase H sequences (His124 for Eco-RNase HI and His539 for HIV-1 RNase H) (Fig. 1). This residue is located in the flexible loop near the active site and is involved in the catalytic function, but in an auxiliary manner [23]. This residue is conserved as His264 in human RNase HI, but is replaced by Glu188 in Bha- RNase HI. Based on the co-crystal structures of human RNase H1 and Bha-RNase HI, in which this residue is conserved as His264 and replaced by Glu188, respec- tively, with the substrate and Mg 2+ , this residue has been proposed to promotes the product release by per- turbing the coordination of the metal ion A [31,42]. It is noted that a folding motif of RNases H, termed RNase H-fold, has been found in other proteins with nuclease or polynucleotidyl transferase activities, such as integrase [49], DNA transposase [50], RuvC Holli- day junction resolvase [51], and a PIWI domain of Argonaute proteins [32,52,53], which is essential for RNA-induced silencing complex-mediated mRNA cleavage. Because three or four acidic amino acid resi- dues also form the active sites of these enzymes, these enzymes may also share a catalytic mechanism with RNase H. Substrate recognition mechanism of RNase H The crystal structure of human RNase H1 in complex with the substrate and Mg 2+ highly resembles to that of Eco-RNase HI free from the substrate [42], indicating that the structure of human RNase HI is not seriously changed upon substrate binding. According to the co-crystal structure of human RNase HI with the sub- strate and Mg 2+ , the RNA ⁄ DNA hybrid binds to the protein, such that the RNA backbone fits in one groove containing the active site and the DNA backbone fits in the other groove (Fig. 5). These two grooves are sepa- rated by a ridge, which is composed of highly conserved Asn151, Asn182 and Gln183 (Asn16, Asn44 and Asn45 Fig. 4. Schematic representation of the two-metal-ion catalysis mechanism proposed for RNase H. The side chains of the first aspartate, second glutamate, third aspartate and fourth aspartate residues of the DEDD motif, which form the active site (metal-bind- ing site) of RNase H, are shown. They are Asp10, Glu48, Asp70 and Asp134 for Eco-RNase HI, Asp71, Glu109, Asp132, and Asp192 for Bha-RNase HI, Asp145, Glu186, Asp210 and Asp274 for human RNase H1, and Asp443, Glu478, Asp498 and Asp549 for HIV-1 RNase H. The fourth aspartate residue is replaced by the glutamate residue for RNase HIII. The attacking hydroxyl ion that is coordinated by metal ion A is highlighted in boldface type. T. Tadokoro and S. Kanaya Prokaryotic RNases H FEBS Journal 276 (2009) 1482–1493 ª 2009 The Authors Journal compilation ª 2009 FEBS 1487 for Eco-RNase HI), and interacts with the minor groove of the RNA ⁄ DNA hybrid. At the RNA-binding groove, the 2¢-OH groups of four consecutive ribonucleotides, two on each side of the scissile phosphate group, contact the side chain of Glu186 (Glu48 for Eco-RNase HI) and the back- bone atoms of Cys148, Ser150, Asn151 and Met212 (Cys13, Gly15, Asn16 and Gln72 for Eco-RNase HI). Similar contacts are observed between Bha-RNase HI and the RNA strand of the substrate, indicating that the mechanism for RNA strand recognition is con- served among various type 1 RNases H. The DNA- binding groove contains two DNA-binding sites. The major site is a phosphate-binding pocket, which is formed by Arg179, Thr181 and Asn240 (Arg41, Thr43 and Asn100 for Eco-RNase HI) and is conserved in the Bha-RNase HI structure. This site is responsible for anchoring the B-form DNA. The second site is a channel formed by Trp221, Trp225 and Ser233 (Trp81, Trp85 and Ala93 for Eco-RNase HI) in the basic protrusion. This site is absent in the Bha-RNase HI, because Bha-RNase HI lacks a basic protrusion. The RNA strand cannot fit in this groove, because a 2¢-OH group of RNA clashes with the indole ring of Trp221. Therefore, these two DNA binding sites probably con- tribute to the specificity for an RNA ⁄ DNA hybrid. High resemblance of the structures of human RNase H1 and Eco-RNase HI strongly suggests that these proteins recognize the substrate in a similar mechanism (Fig. 5). In fact, the mutational studies suggest that Cys13, Asn16, Gln72, Thr43 and Trp85 of Eco-RNase HI are important for substrate binding [54]. Mutational studies also suggest that basic amino acid residues clustered in the basic protrusion are important for substrate binding [21]. However, none of these basic residues makes direct contact with the sub- strate according to the co-crystal structure of human RNase H1 with the substrate. It has been suggested that these basic residues facilitate initial nonspecific interactions with the substrate and promote the forma- tion of specific enzyme–substrate complexes [42]. It is noted that type 2 RNases H differ from type 1 RNases H in the location of a domain involved in sub- strate binding. For example, Tko-RNase HII and Bst-RNase HIII have a substrate-binding domain at the C- and N-termini, respectively. Both proteins lack a basic protrusion. Further mutational and structural studies will be required to understand the substrate recognition mechanism of these type 2 RNases H. Multiplicity of the RNase H genes in the single genomes Single bacterial genomes often contain multiple RNase H genes. For example, the E. coli genome con- tains two genes encoding RNases HI and HII [6]. The B. subtilis genome contains three genes encoding one type 1 RNase H with an N-terminal HBD-like domain (RNase HI) [55] and two type 2 RNases H (RNases HII and HIII) [3]. By contrast, the archaeal genomes usually contain single RNase HII genes. The exceptions are the genomes of S. tokodaii [18] and Halobacterium sp. NRC-1 [17]. These genomes contain genes encoding RNases HI in addition to those encod- ing RNases HII. The question arises whether the number and types of the RNase H genes contained in the single bacterial genomes are correlated with the evolutionary relationships of their source organisms determined based on the 16S rRNA sequences. The answer is no. For example, Shewanella sp. SIB1 and S. oneidensis MR-1 are c-proteobacteria and evolution- arily closely related. Nevertheless, the former genome contains three genes encoding RNase HI, HBD- RNase HI and RNase HII, whereas the latter contains only two genes encoding RNases HI and HII [33]. Likewise, T. maritima and A. aeolicus are hypertherm- ophilic bacteria and evolutionarily closely related. Nevertheless, the former genome contains the RNase HI and RNase HII genes, whereas the latter contains the RNases HII and HIII genes [4]. These results suggest that RNase H genes have been Fig. 5. Crystal structure of the RNase H–substrate complex. The crystal structure of Eco-RNase HI (PDB code 2RN2) is superim- posed onto that of the human RNase H1 C-domain complexed with RNA ⁄ DNA hybrid (PDB code 2QK9). The structures of Eco-RNa- se HI and human RNase H1 C domain are shown in green and gold, respectively. The active-site residues are shown as red ball- and-stick models. The basic protrusion and the phosphate binding pocket and DNA-binding channel in the DNA-binding groove are also indicated. Prokaryotic RNases H T. Tadokoro and S. Kanaya 1488 FEBS Journal 276 (2009) 1482–1493 ª 2009 The Authors Journal compilation ª 2009 FEBS transferred horizontally among different organisms during evolutionary processes. The physiological significance of the multiplicity of RNases H in cells remains to be understood. The observation that RNase HII ⁄ H2 cleaves dsDNA containing single ribonucleotide at the DNA–RNA junction (5¢ side of the ribonucleotide) [56,57] suggests that RNase HII ⁄ H2 is involved in the excision of a Table 1. Diversity of RNase H in different organisms a . Organisms Gene type Type 1 RNase H Type 2 RNase H HI ⁄ H1 without HBD HI ⁄ H1 with HBD HII ⁄ H2 HIII Bacteria (Actinobacteria) Corynebacterium glutamicum A¢B Q79VE b Q8NNZ4 Streptomyces coelicolor AA¢B Q9X7R6 Q9L014 b O69989 (Chlamydiae) Chlamydophila pneumoniae BC Q9Z962 Q9Z6J1 (Firmicutes) Bacillus subtilis (A)BC P54162 O31744 P94541 Bacillus stearothermophilus BC AB073670 AB179782 Bacillus halodurans A¢B Q9KEI9 Q9Z9S0 Streptococcus pneumoniae BC Q04KF4 Q04M70 (Proteobacteria) Escherichia coli AB P0A7Y4 P10442 Shewanella oneidensis AB Q8EE30 Q8EGG1 Shewanella sp. SIB1 AA¢B Q8RTZ8 AB303852 Q25C12 Shewanella denitrificans AA¢B Q12MM4 Q12R78 Q12NX3 Photobacterium profundum AA¢B Q6LN65 Q6LQZ6 Q6LN38 (Others) Aquifex aeolicus BC O67768 O67644 Thermotoga maritima A¢B Q9X122 Q9X017 Thermus thermophilus AB P29253 Q5SLU5 Archaea Methanococcus jannaschii B U67470 Thermococcus kodakaraensis B AB012613 Archaeoglobus fulgidus B AE001062 Halobacterium sp. NRC-1 A¢B Q9HSF6 c Q9HNR3 Sulfolobus tokodaii AB Q973Z8 Q974Z3 Pyrobaculum aerophilum A¢B Q8ZWG9 d Q8ZXL6 Eukaryotes Homo sapiens A¢B O60930 O75792 e Mus musculus A¢B O70338 Q9CWY8 e Drosophila melanogaster A¢B O44114 Q9VPP5 e Caenorhabditis elegans Others Q9XVE6 (rnh-1.3) Q21024 (rnh-1.0) Q9U6P6 e Q09633 (rnh-1.1) Q23676 (rnh-1.2) Saccharomyces cerevisiae A¢B Q04740 P53942 e Candida albicans Others Q5AC61 (RNH11) Q5AGX6 e Q5ABY6 (RNH12) Q5ABY8 (RNH13) Retrovirus Human immunodeficiency virus type 1 A U53871 f Moloney murine leukemia virus A 0711245A f Rous sarcoma virus A AF052428 f a The accession numbers for RNases HI ⁄ H1 with and without HBD, RNases HII ⁄ H2, and RNases HIII are shown. Only the representative members of organisms with fifferent combination of the RNase H genes are listed. The genes A, A¢, B, and C represent those encoding RNases HI ⁄ H1 without HBD, RNases HI ⁄ H1 with HBD, RNases HII ⁄ H2, and RNases HIII, respectively. The accession numbers for the pro- teins which do not exhibit RNase H activity or exhibits other activities are underlined. b N-terminal domain of bi-functional enzyme. c It has an N-terminal extension with little sequence similarity to HBD and TBP. d It has a C-terminal extension. e Catalytic subunit of a heterotrimer. f C-terminal domain of reverse transcriptase. T. Tadokoro and S. Kanaya Prokaryotic RNases H FEBS Journal 276 (2009) 1482–1493 ª 2009 The Authors Journal compilation ª 2009 FEBS 1489 single ribonucleotide misincorporated into DNA. RNase HI ⁄ H1 is not involved in this process because it does not cleave this substrate. However, all RNases H endonucleolytically cleave RNA ⁄ DNA hybrids in a nonspecific manner. The T. thermophilus RNase HII orthologue does not exhibit RNase H activity but cleaves the RNA–DNA junction, and is therefore designated as junction ribonuclease [58]. Functional similarities between type 1 and type 2 RNases H may suggest that a physiological signifi- cance of the multiplicity of the RNase H genes in a single genome is the protection of cells from a lethal mutation in the RNase H gene. The E. coli [59] and B. subtilis [55] mutants that lack all RNase H genes are not lethal, but show a temperature-sensitive growth phenotype. These results indicate that RNase H activ- ity is dispensable for the growth of these micro-organ- isms, but is involved in important cellular processes. We previously showed that two RNases H greatly dif- fer in specific activities when they are simultaneously produced in single cells and are therefore classified into high- and low-activity type RNases H [37]. These types are not correlated with the RNase H families. These results suggest that bacteria evolve such that functional redundancies of the RNase H genes are eliminated. As mentioned earlier, RNases H have been transferred horizontally among different organisms. An RNase H transferred horizontally may provide a selective advan- tage to recipients. However, once a cell that already has an RNase H receives a second RNase H by lateral gene transfer, the responsibilities can be shared in ways that would not necessarily be repeated following other occurrences of transfer. In some instances, the incom- ing RNase H may retain the selective traits, whereas in others, the resident and incoming RNase H may swap some or all of their properties. Because RNases HI and HII represent high-activity type RNases H in E. coli and Shewanella sp. SIB1, respectively, these RNases H may retain the selective traits. The phylo- genetic and statistical analyses using 353 prokaryotic genomes also suggest that functional redundancy contributes to the exclusion or weakening of redundant genes from the genome [60]. It is noted that not only the prokaryotic genomes, but also the eukaryotic genomes usually contain multiple RNase H genes. This minireview is followed others which focus on RNases H from eukaryotes and retroviruses. Table 1 summarizes large diversities of RNases H in different organisms. These organisms are classified into several types based on differences in the combination of the RNase H genes. Appar- ently, these types are not correlated with the species of organisms. Perspectives Prokaryotic RNases H vary greatly in domain struc- tures and substrate specificities. For example, HBD-RNases HI and RNases HIII contain a substrate- binding domain at the N-termini. Tko-RNase HII and Eco-RNase HI contain it at the C-terminus and middle of the molecule, respectively. Sto-RNase HI contains none of these domains. Likewise, Sto-RNase HI cleaves the RNA strand of not only the RNA⁄ DNA hybrid, but also the RNA ⁄ RNA duplex. 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MINIREVIEW Ribonuclease H: molecular diversities, substrate binding domains, and catalytic mechanism of the prokaryotic enzymes Takashi Tadokoro and Shigenori Kanaya Department of Material and Life. sites of these enzymes, these enzymes may also share a catalytic mechanism with RNase H. Substrate recognition mechanism of RNase H The crystal structure of human RNase H1 in complex with the substrate. H with respect to their biochemical properties and structures, and elucidating catalytic mechanism and substrate recognition mechanism of RNase H. We review the results of these studies. Abbreviations Afu,