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The N-terminal hybrid binding domain of RNase HI from Thermotoga maritima is important for substrate binding and Mg 2+ -dependent activity Nujarin Jongruja 1 , Dong-Ju You 1 , Eiko Kanaya 1 , Yuichi Koga 1 , Kazufumi Takano 1,2 and Shigenori Kanaya 1 1 Department of Material and Life Science, Graduate School of Engineering, Osaka University, Japan 2 CRESTO, JST, Osaka, Japan Introduction Ribonuclease H (RNase H; EC 3.1.26.4) is an enzyme that specifically cleaves RNA of RNA⁄ DNA hybrids [1]. It requires divalent metal ions, such as Mg 2+ and Mn 2+ , for activity. RNase H is widely present in bac- teria, archaea and eukaryotes. These RNase H are involved in DNA replication, repair and transcription Keywords cleavage site specificity; hybrid binding domain; metal preference; RNase H; substrate binding affinity; Thermotoga maritima Correspondence S. Kanaya, Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1, Yamadaoka, Suita, Osaka 565-0871, Japan Fax: +81 6 6879 7938 Tel.: +81 6 6879 7938 E-mail: kanaya@mls.eng.osaka-u.ac.jp (Received 18 June 2010, revised 6 August 2010, accepted 27 August 2010) doi:10.1111/j.1742-4658.2010.07834.x Thermotoga maritima ribonuclease H (RNase H) I (Tma-RNase HI) con- tains a hybrid binding domain (HBD) at the N-terminal region. To analyze the role of this HBD, Tma-RNase HI, Tma-W22A with the single mutation at the HBD, the C-terminal RNase H domain (Tma-CD) and the N-termi- nal domain containing the HBD (Tma-ND) were overproduced in Escheri- chia coli, purified and biochemically characterized. Tma-RNase HI prefers Mg 2+ to Mn 2+ for activity, and specifically loses most of the Mg 2+ -depen- dent activity on removal of the HBD and 87% of it by the mutation at the HBD. Tma-CD lost the ability to suppress the RNase H deficiency of an E. coli rnhA mutant, indicating that the HBD is responsible for in vivo RNase H activity. The cleavage-site specificities of Tma-RNase HI are not significantly changed on removal of the HBD, regardless of the metal cofactor. Binding analyses of the proteins to the substrate using surface plasmon resonance indicate that the binding affinity of Tma-RNase HI is greatly reduced on removal of the HBD or the mutation. These results indicate that there is a correlation between Mg 2+ -dependent activity and substrate binding affinity. Tma-CD was as stable as Tma-RNase HI, indicating that the HBD is not important for stability. The HBD of Tma-RNase HI is important not only for substrate binding, but also for Mg 2+ -dependent activity, probably because the HBD affects the interaction between the substrate and enzyme at the active site, such that the scissile phosphate group of the substrate and the Mg 2+ ion are arranged ideally. Abbreviations Bha-RNase HI, Bacillus halodurans RNase HI; Bst-RNase HIII, Bacillus stearothermophilus RNase HIII; Bsu-RNase HII, Bacillus subtilis RNase HII; D13-R4-D12 ⁄ D29, 29 bp DNA 13 -RNA 4 -DNA 12 ⁄ DNA duplex; D15-R1-D13 ⁄ D29, 29 bp DNA 15 -RNA 1 -DNA 13 ⁄ DNA duplex; Eco-RNase HI, Escherichia coli RNase HI; Eco-RNase HII, Escherichia coli RNase HII; GdnHCl, guanidine hydrochloride; HBD, hybrid binding domain; HIV-1 RNase H, RNase H domain of HIV-1 reverse transcriptase; Hsa-RNase H1, human RNase H1; IPTG, isopropyl thio-b- D-galactoside; MMLV RNase H, Moloney murine leukemia virus reverse transcriptase; R12 ⁄ D12, 12 bp RNA ⁄ DNA hybrid; R29 ⁄ D29, 29 bp RNA ⁄ DNA hybrid; R9-D9 ⁄ D18, 18 bp RNA 9 -DNA 9 ⁄ DNA duplex; RNase H, ribonuclease H; Sce-RNase H1, Saccharomyces cerevisiae RNase H1; Sto-RNase HI, Sulfolobus tokodaii RNase HI; Tma-CD, C-terminal catalytic domain (residues 64–223) of Tma-RNase HI; Tma-ND, N-terminal domain (residues 1–63) of RNase HI from Thermotoga maritima containing HBD; Tk-RNase HII, Thermococcus kodakaraensis RNase HII. 4474 FEBS Journal 277 (2010) 4474–4489 ª 2010 The Authors Journal compilation ª 2010 FEBS [2–6]. In antisense therapy, RNase H is involved in the recognition and cleavage of a disease-causative mRNA [7]. Mutations in human RNase H2, which do not necessarily significantly affect the activity [8], cause severe neurological disorder termed Aicardi–Goutieres syndrome [9]. RNase H is also present in retroviruses as a C-terminal domain of reverse transcriptase. Retro- viral RNases H are required for the conversion of sin- gle-stranded genomic RNA into double-stranded DNA, which is an initial step of viral proliferation, and are therefore regarded as one of the targets for AIDS therapy [10]. RNases H are classified into two major families (type 1 and type 2 RNases H) based on the difference in their amino acid sequences [11]. Four acidic active- site residues are fully conserved in these RNases H, except that from compost metagenome [12], and their geometrical configurations are well conserved [13]. According to the crystal structures of the C-terminal catalytic domains of Bacillus halodurans RNase HI (Bha-RNase HI) [14] and human RNase H1 [15] in complex with the RNA ⁄ DNA substrate, type 1 RNase H binds to the minor groove of the substrate, such that one depression containing the active site interacts with the RNA backbone and the other depression con- taining the phosphate-binding pocket interacts with the DNA backbone. These two depressions are sepa- rated by a ridge, which is composed of three highly conserved Asn ⁄ Gln residues. Because two metal ions are coordinated by the four acidic active site residues, the scissile phosphate group of the substrate and water molecules, a two-metal ion catalysis mechanism has been proposed for RNase H [14,16,17]. According to this mechanism, one metal ion is required for sub- strate-assisted nucleophile formation and product release, whereas the other is required to destabilize the enzyme–substrate complex and thereby promote the phosphoryl transfer reaction. Thermotoga maritima is a strictly anaerobic, extre- mely thermophilic eubacterium, isolated from various geothermally heated locales on the sea floor, and grows in the temperature range 55–90 °C with an opti- mum at 80 °C [18]. Its genome sequence has been determined previously [19]. The genome contains single rnhA and single rnhB genes, encoding type 1 (Tma- RNase HI; accession no. AAD36370) and type 2 (Tma-RNase HII; accession no. AAD35996) RNases H, respectively. Tma-RNase HI is composed of 223 amino acid residues and contains a hybrid binding domain (HBD) at the N-terminal region. Without this domain, Tma-RNase HI shows relatively low (£ 20%) amino acid sequence identities to any one of the repre- sentative members of type 1 RNases H, which have been biochemically characterized. Therefore, it would be informative to characterize Tma-RNase HI and compare its biochemical properties with those of other type 1 RNases H. HBD, previously termed as double-stranded RNA and hybrid binding domain [20], consists of approxi- mately 40 amino acid residues and is commonly present at the N-terminal regions of eukaryotic type 1 RNases H (RNase H1) [21]. According to the crystal structure of the HBD of human RNase H1 in complex with the RNA ⁄ DNA substrate, HBD consists of a three-stranded anti-parallel b-sheet (b1–b3) and two helices (aA and aB) [22]. It binds to the minor groove of the substrate, such that a loop between aA and b3 interacts with the RNA backbone and a positively- charged depression interacts with the DNA backbone. The importance of HBD with respect to substrate binding has been reported for yeast [20,23], mouse [24] and human [22,25,26] RNases H1. The requirement of HBD for processivity [24] and positional preference [25,26] has also been reported for mouse and human RNases H1, respectively. HBD is also present in several bacterial type 1 RNases H, including Tma-RNase HI, Bha-RNase HI and RBD-RNase HI from Shewanella sp. SIB1 [13]. However, it remains to be determined whether these HBDs have a role similar to those of eukaryotic RNases H1, although the isolated HBD from SIB1 RBD-RNases HI, which is renamed as SIB1 HBD- RNase HI in the present study, has been reported to bind to the RNA ⁄ DNA substrate [27]. Attempts to overproduce the SIB1 HBD-RNase HI derivative lack- ing the HBD have so far been unsuccessful, probably as a result of the instability of the protein (T. Tadok- oro, unpublished data). In the present study, we over- produced, purified and biochemically characterized Tma-RNase HI and its derivatives lacking the HBD or RNase H domain. On the basis of the results obtained, we discuss the role of the HBD from Tma-RNase HI. Results Protein preparations The amino acid sequence of Tma-RNase HI is com- pared with those of the representative members of type 1 RNases H, Bha-RNase HI, HBD-RNase HI from Shewanella sp. SIB1, Saccharomyces cerevisiae RNase H1 (Sce-RNase H1), human RNase H1 (Hsa-RNase H1), E. coli RNase HI (Eco-RNase HI) and the RNase H domain of HIV-1 reverse transcriptase (HIV- 1 RNase H) in Fig. 1. The HBD of Tma-RNase HI shows relatively high amino acid sequence identities of N. Jongruja et al. Role of HBD from T. maritima RNase HI FEBS Journal 277 (2010) 4474–4489 ª 2010 The Authors Journal compilation ª 2010 FEBS 4475 43%, 42%, 33% and 32% with respect to those of Sce-RNase H1, Bha-RNase HI, SIB1 HBD-RNase HI and Hsa-RNase H1, whereas the RNase H domain of Tma-RNase HI shows relatively low amino acid sequence identities of 20% to Hsa-RNase H1, 19% to Eco-RNase HI, 18% to SIB1 HBD-RNase HI, Sce- RNase H1 and Bha-RNase HI, and 17% to HIV-1 RNase H. Nevertheless, all active-site residues (four acidic and one histidine residues) are fully conserved in Tma-RNase HI as Asp71, Glu111, Asp135, His179 and Asp189. To analyze the role of the HBD of Tma-RNase HI, the Tma-RNase HI derivatives lacking either the HBD (Tma-CD, residues 64–223) or the RNase H domain (Tma-ND, residues 1–63) were constructed. Tma-ND contains the entire HBD of Tma-RNase HI (Fig. 1). Tma-RNase HI, Tma-CD and Tma-ND were over- produced in the rnhA deficient strain E. coli MIC3001(DE3) to avoid a contamination of host- derived RNase HI. Upon overproduction, these pro- teins accumulated in E. coli cells in a soluble form and were purified to give a single band on SDS ⁄ PAGE (Fig. 2). The amount of the protein purified from 1L culture was approximately 35 mg for Tma-RNase HI, 15 mg for Tma-CD and 30 mg for Tma-ND. The molecular masses of these proteins were estimated to be 28 kDa for Tma-RNase HI, 16 kDa for Tma-CD and 8 kDa for Tma-ND by gel filtration column chromato- graphy. These values are comparable to those calculated from the amino acid sequences (25 967 for Tma-RNase Fig. 1. Alignment of the amino acid sequences. The amino acid sequence of Tma-RNase HI (Tma) is compared with those of Bha-RNase HI (Bha), SIB1 HBD-RNase HI (SIB1HBD), Sce-RNase H1 (Sce), Hsa-RNase H1 (Hsa), Eco-RNase HI (Eco) and HIV-1 RNase H (HIV1). The accession numbers are AAD36370 for Tma-RNase HI, BAF73617 for SIB1 HBD-RNase HI, DAA10134 for Sce-RNase H1, EAX01061 for Hsa- RNase H1, P0A7Y4 for Eco-RNase HI and ABU62661 for HIV-1 RNase H. The ranges of the secondary structures of Hsa-RNase H1 are shown above the sequence, based on the crystal structures of its HBD (Protein Data Bank code: 3BSU) and RNase H domain (Protein Data Bank code: 2KQ9), which were independently determined in complex with the substrate. The range of HBD is also shown. The amino acid residues, which are conserved in at least three (for HBD) or four (for RNase H domain) different proteins, are highlighted in black. The five active-site residues are denoted by filled circles above the sequences. The amino acid residues that contact the substrate in the co-crystal structure of the HBD of Hsa-RNase H1 with the substrate are also denoted by open circles above the sequence. The amino acid residue that is mutated in the present study is indicated by an arrow. Gaps are denoted by dashes. The numbers represent the positions of the amino acid residues relative to the initiator methionine for each protein. Role of HBD from T. maritima RNase HI N. Jongruja et al. 4476 FEBS Journal 277 (2010) 4474–4489 ª 2010 The Authors Journal compilation ª 2010 FEBS HI, 18 860 for Tma-CD and 7107 for Tma-ND), sug- gesting that all proteins exist as a monomer in solution. CD spectra The far- and near-UV CD spectra of Tma-RNase HI, Tma-CD and Tma-ND were measured at 20 °C and pH 9.0, and comparisons are shown in Fig. 3. The far- and near-UV CD spectra of Tma-CD are similar to those of Tma-RNase HI, suggesting that removal of the HBD does not significantly affect the structure of the RNase H domain of Tma-RNase HI. The far- and near-UV CD spectra of Tma-ND were sig- nificantly different from those of Tma-RNase HI, probably because the secondary structure contents and environment of the aromatic residues are different in these proteins. According to the crystal structures of the HBD [22] and RNase H domain [15] of Hsa- RNase HI, the b-strand contents are 37% for HBD and 21% for RNase H domain, whereas the a-helix contents of these domains are similar to each other (39% for HBD and 38% for RNase H domain). Enzymatic activity The dependencies of the Tma-RNase HI and Tma- CD activities on pH, salt and metal ion were analyzed at 30 °C by changing one of the conditions used for assay [10 mm Tris ⁄ HCl, 1 mm MgCl 2 , 50 mm KCl (pH 9.0) for Tma-RNase HI, and 10 mm Tris ⁄ HCl, 1 mm MnCl 2 ,10mm KCl (pH 9.0) for Tma-CD]. The M13 DNA ⁄ RNA hybrid was used as a substrate. The enzymatic activities of these proteins were determined at the temperature (30 °C), which could be much lower than the optimum one because the substrate used for assay is not fully stable at ‡ 60 °C. When the enzymatic activity was determined over the range pH 5–12, both proteins exhibited the highest activities at around pH 9.0 (data not shown). They exhibited approximately 50% of the maximal activities at pH 7.0 and 11.0. When the enzymatic activity was determined in the presence of various concentrations of NaCl or KCl, Tma-RNase HI exhibited the highest activity in the presence of 50 mm KCl, whereas Tma-CD exhibited it in the presence of 10 mm KCl (Fig. 4). Their enzymatic Fig. 2. SDS ⁄ PAGE of Tma-RNase HI and its derivatives. The purified proteins of Tma-RNase HI (lane 1), Tma-CD (lane 2) and Tma-ND (lane 3) were subjected to electrophoresis on a 15% poly- acrylamide gel in the presence of SDS. After electrophoresis, the gel was stained with Coomassie Brilliant Blue. Lane M, a low- molecular-weight marker kit (GE Healthcare, Tokyo, Japan). Fig. 3. CD spectra of Tma-RNase HI and its derivatives. Far-UV (A) and near-UV (B) CD spectra of Tma-RNase HI (thick solid dark line), Tma-W22A (thin solid dark line), Tma-CD (dashed dark line) and Tma-ND (thick solid gray line) are shown. These spectra were mea- sured at pH 9.0 and 20 °C, as described in the Experimental proce- dures. N. Jongruja et al. Role of HBD from T. maritima RNase HI FEBS Journal 277 (2010) 4474–4489 ª 2010 The Authors Journal compilation ª 2010 FEBS 4477 activities decreased to a large extent at higher (‡ 0.2 m) salt concentrations. When the enzymatic activity was determined in the presence of various concentrations of MgCl 2 , MnCl 2 , NiCl 2 , ZnCl 2 , CoCl 2 or CaCl 2 , both Tma-RNase HI and Tma-CD exhibited the highest activities in the presence of 1mm MgCl 2 and 0.1–5 mm MnCl 2 (Fig. 5). Both proteins exhibited little activity (less than 0.01% of the maximal activity) in the presence of NiCl 2 , ZnCl 2 , CoCl 2 or CaCl 2 . The maximal Mg 2+ - and Mn 2+ - dependent activities of these proteins are summarized in Table 1. Tma-RNase HI prefers Mg 2+ to Mn 2+ because its maximal Mg 2+ -dependent activity is higher than its maximal Mn 2+ -dependent activity by 16-fold. By contrast, Tma-CD prefers Mn 2+ to Mg 2+ because its maximal Mn 2+ -dependent activity is higher than its maximal Mg 2+ -dependent activity by 69-fold. Interestingly, the maximal Mn 2+ -dependent activity of Tma-CD is comparable to that of Tma- RNase HI. These results indicate that removal of the HBD severely reduces the Mg 2+ -dependent activity of Tma-RNase HI without significantly affecting its Mn 2+ -dependent activity. The kinetic parameters of Tma-CD were determined at 30 °C in the presence of 1 mm MgCl 2 or MnCl 2 and compared with those of Tma-RNase HI (Table 1). The V max values of Tma-CD determined in the presence of 1mm MgCl 2 and MnCl 2 were 410-fold lower and 1.6-fold higher than those of Tma-RNase HI. The K m values of Tma-CD determined in the presence of 1 mm MgCl 2 and MnCl 2 were 5.1- and 6.8-fold higher than those of Tma-RNase HI. These results indicate that the substrate binding affinity of Tma-RNase HI is reduced by five- to seven-fold on removal of the HBD, regardless of the metal cofactor, and the large reduc- tion in Mg 2+ -dependent activity on removal of the HBD is not a result of the marked decrease in substrate binding affinity. Fig. 4. Salt dependencies of Tma-RNase HI and Tma-CD. The enzy- matic activities of Tma-RNase HI (A) and Tma-CD (B) were deter- mined at 30 °Cin10m M Tris ⁄ HCl (pH 9.0) containing 1 mM MgCl 2 (Tma-RNase HI) or 1 mM MnCl 2 (Tma-CD), 1 mM b-mercaptoe- thanol, 50 lgÆmL )1 BSA, and various concentrations of NaCl (open circle) or KCl (closed circle), using M13 DNA ⁄ RNA hybrid as a sub- strate. Experiments were carried out at least twice and the average values are shown together with the errors. Fig. 5. Metal ion dependencies of Tma-RNase HI and Tma-CD. The enzymatic activities of Tma-RNase HI (A) and Tma-CD (B) were determined at 30 °Cin10m M Tris ⁄ HCl (pH 9.0) containing 50 m M KCl (Tma-RNase HI) or 10 mM KCl (Tma-CD), 1 mM b-mercaptoethanol, 50 lgÆmL )1 BSA, and various concentrations of MgCl 2 (open circle) or MnCl 2 (closed circle), using M13 DNA ⁄ RNA hybrid as a substrate. Experiments were carried out at least twice and the average values are shown together with the errors. Role of HBD from T. maritima RNase HI N. Jongruja et al. 4478 FEBS Journal 277 (2010) 4474–4489 ª 2010 The Authors Journal compilation ª 2010 FEBS Complementation assay E. coli MIC3001 shows an RNase H-dependent temperature-sensitive growth phenotype [28]. E. coli MIC3001(DE3) also displays this phenotype. To exam- ine whether the genes encoding Tma-RNase HI and Tma-CD complement the temperature-sensitive growth phenotype of MIC3001(DE3), E. coli MIC3001(DE3) transformants for overproduction of these proteins were grown in the absence of isopropyl thio-b-d-galac- toside (IPTG) at permissive (30 °C) and nonpermissive (42 °C) temperatures. The results showed that the Tma-RNase HI gene complements the temperature- sensitive growth phenotype of MIC3001(DE3), whereas the Tma-CD gene does not (data not shown). These results suggest that HBD is required for in vivo function of Tma-RNase HI. It is unlikely that Tma- CD is not produced or produced in a nonfunctional form in E. coli cells in the absence of IPTG because the protein is overproduced in a soluble and functional form upon overproduction, as noted above. Cleavage-site specificity The cleavage-site specificities of Tma-RNase HI and Tma-CD were analyzed by using 12 bp RNA ⁄ DNA hybrid (R12 ⁄ D12), 29 bp DNA 13 -RNA 4 -DNA 12 ⁄ DNA duplex (D13-R4-D12 ⁄ D29), 29 bp DNA 15 - RNA 1 -DNA 13 ⁄ DNA duplex (D15-R1-D13 ⁄ D29) and 18 bp RNA 9 -DNA 9 ⁄ DNA duplex (R9-D9 ⁄ D18). For comparative purposes, these substrates were cleaved by Eco-RNase HI, Sulfolobus tokodaii RNase HI (Sto-RNase HI) and Thermococcus kodakaraensis RNase HII (Tk-RNase HII) as well. D13-R4-D12 and D15-R1-D13 are the chimeric oligonucleotides, in which four and single ribonucleotides are flanked by 12–15 bp of DNA at both sides. R9-D9 ⁄ D18 is a Okazaki fragment-like substrate, in which the 18 base chimeric oligonucleotide (RNA 9 -DNA 9 ) is hybridized to the 18 base complementary DNA. Cleavage of the R12 ⁄ D12 substrate with various RNase H enzymes is summarized in Fig. 6A,B. Tma- RNase HI, Eco-RNase HI, Sto-RNase HI and Tk- RNase HII cleaved this substrate at multiple sites, although with different site specificities. Tma-RNase HI cleaved this substrate slightly more efficiently in the presence of Mg 2+ than in the presence of Mn 2+ . Tma- CD cleaved this substrate with much less and compa- rable efficiencies compared to those of Tma-RNase HI in the presence of Mg 2+ and Mn 2+ , respectively. These results are consistent with those obtained by using M13 DNA ⁄ RNA as a substrate. The cleavage sites of the R12 ⁄ D12 substrate with Tma-CD are simi- lar to those with Tma-RNase HI, regardless of the metal cofactor, although their preferable cleavage sites are slightly different with each other. The cleavage sites of this substrate and their susceptibilities to cleav- age with Eco-RNase HI, Sto-RNase HI, and Tk- RNase HII are essentially the same as those reported previously [27–30]. Cleavage of the D13-R4-D12 ⁄ D29 substrate with various RNase H enzymes is summarized in Fig. 6C,D. Tma-RNase HI, Eco-RNase HI, Sto-RNase HI and Tk-RNase HII cleaved this substrate most preferably at a16-a17, a15-a16, a14-a15 and a16-a17, respectively. The cleavage sites of this substrate with Eco-RNase HI and Tk-RNase HII are the same as those reported previously [30]. The a16-a17 site has been reported to be exclusively cleaved only by type 2 RNases H, except for bacterial RNases HIII [31,32]. Therefore, Tma- RNase HI is the first type 1 RNase H enzyme that exclusively cleaves this substrate at this site. Tma-CD also cleaved this substrate at a16-a17 with a similar efficiency to that of Tma-RNase HI. However, these enzymes cleaved this substrate only in the presence of Mn 2+ . Table 1. Specific activities and kinetic parameters of Tma-RNase HI and its derivatives. Hydrolysis of the M13 DNA ⁄ RNA hybrid by the enzyme was carried out at 30 °C under the conditions described in the Experimental procedures. ND, not determined. Protein Metal Salt Specific activity (UÆmg )1 ) Relative activity a (%) K m (lM) V max (UÆmg )1 ) Tma-RNase HI 1 m M MgCl 2 50 mM KCl 3.6 ± 0.52 100 0.39 ± 0.064 7.3 ± 1.4 1m M MnCl 2 10 mM KCl 0.23 ± 0.026 6.4 0.25 ± 0.042 0.62 ± 0.071 Tma-W22A 1 m M MgCl 2 50 mM KCl 0.48 ± 0.057 13 ND ND 1m M MnCl 2 10 mM KCl 0.35 ± 0.039 9.7 ND ND Tma-CD 1 m M MgCl 2 50 mM KCl 0.0048 ± 0.00068 0.13 2.0 ± 0.24 0.018 ± 0.0042 1m M MnCl 2 10 mM KCl 0.33 ± 0.0081 9.2 1.7 ± 0.33 1.0 ± 0.14 Eco-RNase HI 10 m M MgCl 2 50 mM NaCl 8.3 ± 0.22 231 ND ND a The specific activities of the proteins relative to that of Tma-RNase HI determined in the presence of 1 mM MgCl 2 and 50 mM KCl. N. Jongruja et al. Role of HBD from T. maritima RNase HI FEBS Journal 277 (2010) 4474–4489 ª 2010 The Authors Journal compilation ª 2010 FEBS 4479 Role of HBD from T. maritima RNase HI N. Jongruja et al. 4480 FEBS Journal 277 (2010) 4474–4489 ª 2010 The Authors Journal compilation ª 2010 FEBS The D15-R1-D13 ⁄ D29 substrate was used to con- firm that Tma-RNase HI and Tma-CD do not cleave the DNA-RNA-DNA ⁄ DNA substrate containing a single ribonucleotide. This substrate is not cleaved by type 1 RNases H but is cleaved by type 2 RNases H, except for bacterial RNases HIII, at the DNA-RNA junction [21,27,32]. As expected, this substrate was not cleaved with Tma-RNase HI, Sto-RNase HI and Eco- RNase HI, although it was cleaved with Tk-RNase HII at the DNA-RNA junction (data not shown). These results exclude the possibility that the cleavage of the D13-R4-D12 ⁄ D29 substrate with Tma-RNase HI at a16-a17 is caused by the contamination of a type 2 RNase H enzyme. Cleavage of the R9-D9 ⁄ D18 substrate with various RNase H enzymes is summarized in Fig. 6E,F. Tma- RNase HI and Tma-CD cleaved this substrate most preferably at g7-c8 and c8-c9, and much less preferably at u6-g7 and c9-T10 in the presence of Mn 2+ . They cleaved this substrate with similar site specificities in the presence of Mg 2+ . However, their abilities to cleave this substrate are greatly reduced in the presence of Mg 2+ by more than 100-fold. Eco-RNase HI and Sto-RNase HI cleaved this substrate at all sites between a5 and c9 and between a5 and T10, respec- tively, as reported previously [33]. However, both enzymes showed a preference for the sites far from the RNA-DNA junction (a5-u6, u6-g7 and g7-c8 for Eco- RNase HI, and u5-a6 and u6-g7 for Sto-RNase HI). Tk-RNase HII cleaved this substrate almost exclusively at c8-c9. Eco-RNase HI and Tk-RNase HII cleaved the RNA-DNA junction (c9-T10) as well, although with very poor efficiency. It has been demonstrated for mouse RNase H1 that the HBD is required for processivity of the enzyme [24]. Tma-RNase HI did not show the processivity for cleavage of the R12 ⁄ D12 substrate (Fig. 6). How- ever, this result does not necessarily indicate that Tma-RNase HI shows no processivity because mouse RNase H1 shows the processivity only for long RNA ⁄ DNA substrates. Therefore, it would be infor- mative to examine whether Tma-RNase HI shows processivity for long RNA ⁄ DNA substrates and loses this processivity on removal of the HBD. Binding to substrate To examine whether the HBD of Tma-RNase HI is important for substrate binding, the binding affinities of Tma-RNase HI, Tma-CD and Tma-ND to the 29 bp RNA ⁄ DNA hybrid (R29 ⁄ D29) were analyzed in the absence of the metal cofactor using surface plas- mon resonance. These proteins were injected onto the sensor chip, on which the R29 ⁄ D29 substrate was immobilized. The sensorgrams obtained by injecting 1 lm of these proteins are shown in Fig. 7. The disso- ciation constants, K D , of the proteins for binding to the R29 ⁄ D29 substrate, which were determined by measuring the equilibrium-binding responses at various concentrations of the proteins, are summarized in Table 2. The K D value of Tma-ND was higher than (although comparable to) that of Tma-RNase HI. By Fig. 7. Binding of Tma-RNase HI and its derivatives to the sub- strate. Sensorgrams from Biacore X showing binding of 1 l M of Tma-RNase HI (thick solid dark line), Tma-W22A (thin solid dark line), Tma-CD (dashed dark line) and Tma-ND (thick solid gray line) to the immobilized R29 ⁄ D29 substrate are shown. Injections were performed at time zero for 60 s. Fig. 6. Cleavage of various oligomeric substrates with various RNases H. (A, C, E) Separation of the hydrolysates by urea gel. The 5¢-end labeled R12 ⁄ D12 (A), 5¢-end labeled D13-R4-D12 ⁄ D29 (C) and 3¢-end labeled R9-D9 ⁄ D18 (E) were hydrolyzed by the enzyme at 30 °C for 15 min and the hydrolysates were separated on a 20% polyacrylamide gel containing 7 M urea, as described in the Experimental procedures. The concentration of the substrate was 1.0 l M. The amount of the enzyme added to the reaction mixture (10 lL) is indicated above each lane. The metal cofactors used to cleave these substrates with Tma-RNase HI and Tma-CD are also shown above the gel together with their concentrations. The complete sequence of R12 (A) as well as the partial sequences of D13-R4-D12 (C) and R9-D9 (E) are indicated along the gel. (B, D, F) Schematic representation of the sites and extents of cleavage by various RNases H. Cleavage sites of R12 ⁄ D12 (B), D13-R4- D12 ⁄ D29 (D) and R9-D9 ⁄ D18 (F) by the enzyme are shown by arrows. In these panels, only the sequences of the oligonucleotides cleaved by the enzyme are shown. The differences in the lengths of the arrows reflect relative cleavage intensities at the position indicated. These lengths do not necessarily reflect the amount of the products accumulated upon complete hydrolysis of the substrate. Deoxyribonucleotides are indicated by capital letters and ribonucleotides are indicated by lowercase letters. N. Jongruja et al. Role of HBD from T. maritima RNase HI FEBS Journal 277 (2010) 4474–4489 ª 2010 The Authors Journal compilation ª 2010 FEBS 4481 contrast, the K D value of Tma-CD was considerably higher than that of Tma-RNase HI by 49-fold. These results indicate that the HBD of Tma-RNase HI is important for substrate binding. When binding of Tma-RNase HI to the R29 ⁄ D29 substrate was analyzed in the presence of 0.5 m NaCl, only a small positive signal was observed, even when 4 lm of the protein was injected, indicating that the binding affin- ity of Tma-RNase HI to the substrate is severely decreased at high salt concentration. Thermal stability To examine whether removal of the N- or C-terminal domain affects the stability of Tma-RNase HI, the thermal stabilities of Tma-RNase HI, Tma-CD and Tma-ND were determined by monitoring changes of the CD values at 222 nm. In the presence of 3 m guanidine hydrochloride (GdnHCl) and 10 mm dith- iothreitol at pH 9, all proteins unfolded in a single cooperative fashion in a reversible manner. The ther- mal denaturation curves of these proteins are com- pared with one another in Fig. 8. The parameters characterizing the thermal denaturation of these pro- teins are summarized in Table 2. A comparison of these parameters indicates that Tma-CD and Tma-ND are less stable than Tma-RNase HI by 1.3 and 10.8 °C in T m , respectively. These results suggest that the inter- actions between the N- and C-terminal domains of Tma-RNase HI do not significantly contribute to the stabilization of its C-terminal domain but contribute to the stabilization of its N-terminal domain. Tma- RNase HI is thermally denatured in a single coopera- tive fashion, probably because its N-terminal domain is denatured immediately after its C-terminal RNase H domain is denatured. It is noted that the DH m and DS m values of Tma-CD are considerably higher than those of Tma-RNase HI and Tma-ND, which are comparable to each other. The reason why the DH m and DS m values of Tma-RNase HI increase on removal of the N-terminal domain remains to be clarified. Analysis for interaction between two domains To examine whether the HBD of Tma-RNase HI strongly interacts with the RNase H domain, Tma-ND was mixed with Tma-CD in a 1 : 1 molar ratio and applied to gel filtration column chromatography. Both proteins were eluted from the column as two inde- pendent peaks (data not shown), indicating that Tma-ND does not form a stable complex with Table 2. Dissociation constants and parameters characterizing thermal denaturation of Tma-RNase HI and its derivatives. ND, not determined. Protein K D a (lM) T m b (°C) DT m b (°C) DH m b (kJÆmol )1 ) DS m b (kJ.mol )1 ÆK )1 ) Tma-RNase HI 0.16 ± 0.013 67.0 ± 0.83 – 115.9 ± 11.1 0.34 ± 0.032 Tma-W22A 3.3 ± 0.54 ND ND ND ND Tma-CD 7.8 ± 0.47 65.7 ± 4.3 )1.3 205.7 ± 22.3 0.58 ± 0.067 Tma-ND 0.40 ± 0.083 56.2 ± 3.2 )10.8 111.7 ± 7.43 0.33 ± 0.038 a Dissociation constant of the protein for binding to the R29 ⁄ D29 substrate was determined by measuring equilibrium-binding responses at various concentrations of the protein using surface plasmon resonance (Biacore) as described in the Experimental procedures. b Parameters characterizing thermal denaturation of the proteins were determined from the thermal denaturation curves shown in Fig. 8. The melting tem- perature (T m ) is temperature of the midpoint of the thermal denaturation transition. DT m is the difference in T m between the intact and trun- cated proteins and is calculated as: T m (truncated) ) T m (intact). DH m and DS m are the enthalpy and entropy changes of unfolding at T m calculated by van’t Hoff analysis. Fig. 8. Thermal denaturation curves. Thermal denaturation curves of Tma-RNase HI (closed circl), Tma-CD (open square) and Tma-ND (closed triangle) are shown. These curves were obtained at pH 9.0 in the presence of 3 M GdnHCl and 10 mM dithiothreitol by monitor- ing the change in the CD value at 222 nm, as described in the Experimental procedures. The theoretical curves are drawn on the assumption that the proteins are denatured via a two-state mechanism. Role of HBD from T. maritima RNase HI N. Jongruja et al. 4482 FEBS Journal 277 (2010) 4474–4489 ª 2010 The Authors Journal compilation ª 2010 FEBS Tma-CD. In addition, the Mg 2+ -dependent activity of Tma-CD was not significantly changed in the presence of a 10–1000 molar excess of Tma-ND, indicating that the Mg 2+ -dependent activity of Tma-CD is not restored in the presence of Tma-ND. It has been pro- posed for eukaryotic type 1 RNases H that the HBD and RNase H domain are separated by a flexible linker and move rather freely [21]. The HBD of Tma-RNase HI also may not strongly interact with the RNase H domain. Biochemical properties of Tma-W22A According to the crystal structure of the HBD of Hsa- RNase H1 in complex with the substrate, Tyr29, Trp43, Phe58, Lys59 and Lys60 interact with the DNA strand of the substrate [22]. These residues, except for Lys60, are well conserved in various HBDs, suggesting that the HBDs of other type 1 RNases H bind to the substrate in a manner similar to the interaction of the HBD of Hsa-RNase H1. The mutation of Trp43 to Ala greatly reduces both the substrate binding affinities and enzymatic activities of Hsa-RNase H1 [22,26] and mouse RNase H1 [24]. To examine whether the corre- sponding tryptophan residue (Trp22) is important for substrate binding and enzymatic activity of Tma- RNase HI, the mutant protein, Tma-W22A, was con- structed, overproduced and purified. The production level and purification yield of Tma-W22A were compa- rable to those of Tma-RNase HI. The far- and near- UV CD spectra of Tma-W22A are similar to those of Tma-RNase HI (Fig. 3), suggesting that the mutation at Trp22 does not significantly affect the structure of Tma-RNase HI. The pH, salt and metal ion dependencies of Tma- W22A were similar to those of Tma-RNase HI (data not shown). Its maximal Mn 2+ -dependent activity was also similar to that of Tma-RNase HI (Table 1). How- ever, its maximal Mg 2+ -dependent activity was lower than that of Tma-RNase HI by 7.5-fold (Table 1), indicating that the mutation of Trp22 to Ala con- siderably reduces the Mg 2+ -dependent activity of Tma-RNase HI without significantly affecting the Mn 2+ -dependent activity. The binding affinity of Tma-W22A to the R29 ⁄ D29 substrate was analyzed in the absence of the metal cofactor using surface plas- mon resonance and compared with that of Tma-RNase HI. The K D value of Tma-W22A was higher than that of Tma-RNase HI by 21-fold, suggesting that Trp22 of Tma-RNase HI is involved in substrate binding. These results suggest that the HBD of Tma-RNase HI inter- acts with the substrate in a manner similar to the inter- action of the HBD of Hsa-RNase H1. The cleavage site specificities of Tma-W22A were not analyzed because the cleavage site specificities of Tma-RNase HI are not significantly changed even when its N-terminal domain is removed, and therefore it is highly likely that the cleavage site specificities of Tma-W22A are similar to those of Tma-RNase HI. Likewise, the stability of Tma-W22A was not analyzed because the stability of Tma-W22A is not significantly changed even when the HBD is completely removed, and therefore it is highly likely that Tma-W22A is as stable as Tma-RNase HI. Discussion Importance of HBD for substrate binding In the present study, we showed that the HBD of Tma-RNase HI is important for substrate binding. However, on removal of the HBD, the K m value of Tma-RNase HI increases by 5–7-fold, whereas its K D value increases by 49-fold. Because the K m and K D val- ues are determined in the presence and absence of the metal cofactor, these results suggest that the difference in substrate binding affinity between Tma-RNase HI and Tma-CD determined in the presence of the metal cofactor is smaller than that determined in its absence. Presumably, the HBD governs binding of Tma-RNase HI to the substrate and its substrate binding affinity is not significantly changed either in the presence or absence of the metal cofactor. By contrast, the sub- strate binding affinity of the RNase H domain proba- bly increases in the presence of the metal cofactor compared to that in its absence. The cleavage-site spec- ificity of Tma-RNase HI is not significantly changed on removal of the HBD, probably because the HBD of Tma-RNase HI facilitates initial nonsite-specific interactions with the substrate and promotes the site- specific interactions between the RNase H domain of Tma-RNase HI and substrate. Importance of HBD for Mg 2+ -dependent activity Removal of the HBD severely reduces the Mg 2+ - dependent activity of Tma-RNase HI by 750-fold without significantly affecting the Mn 2+ -dependent activity. Similarly, single mutation at the HBD (Trp22 to Ala) reduces the Mg 2+ -dependent activity of Tma-RNase HI by 7.5-fold without significantly affecting the Mn 2+ -dependent activity. Removal of the HBD and single mutation at the HBD reduces the binding affinity of Tma-RNase HI by 49- and 21- fold, respectively. Thus, there is a correlation between the Mg 2+ -dependent activity of Tma-RNase HI and N. Jongruja et al. Role of HBD from T. maritima RNase HI FEBS Journal 277 (2010) 4474–4489 ª 2010 The Authors Journal compilation ª 2010 FEBS 4483 [...]...Role of HBD from T maritima RNase HI N Jongruja et al the substrate binding affinity of the HBD High similarity in the conformation of the active site between the Hsa -RNase H1 derivative lacking the HBD and Eco -RNase HI [15] suggests that the conformation of the active site is not significantly changed on removal of the HBD Because the HBD is important for substrate binding, the HBD may affect the interaction... site of Tma -RNase HI and 5¢-end of the RNA ⁄ DNA hybrid region is five bases for the R12 ⁄ D12 substrate (Fig 6B), which can be effectively cleaved by the enzyme in the presence of Mg2+, and seven or eight bases for the R9-D9 ⁄ D18 substrate (Fig 6F), whereas the distance between this cleavage site and the 3¢-end of the RNA ⁄ DNA hybrid region is seven bases for the R12 ⁄ D12 substrate (Fig 6B) and one... bases for the R9-D9 ⁄ D18 substrate (Fig 6F) These results suggest that the HBD of Tma -RNase HI binds to the downstream region of the substrate from the scissile bond Tma -RNase HI cannot effectively cleave the D13-R4-D12 ⁄ D29 and R9-D9 ⁄ D18 substrates in the presence of Mg2+, probably because the HBD cannot bind to double-stranded DNA, which is located downstream from the scissile bond of the substrate. .. between the enzyme and substrate at the active site Because not only the active-site residues, but also the substrate provide ligands for coordination of the metal ion [14], removal of the HBD or mutation at the HBD may alter the interaction between the substrate and metal ion, such that the scissile phosphate group of the substrate and the Mg2+ ion are arranged ideally The effect of this alteration on the. .. Mg2+ to Mn2+, whereas MMLV RNase H prefer Mn2+ to Mg2+, although all of them specifically lose or greatly reduce Mg2+-dependent activity by deletion of the basic protrusion (for Eco -RNase HI and MMLV RNase H) or removal of the N-terminal substrate binding domain (for Bst -RNase HIII) The metal ion preference of Hsa -RNase HI is also changed on removal of the HBD because Hsa -RNase HI prefers Mg2+ to Mn2+... Glu478 for HIV-1 RNase H) Eco -RNase HI specifically loses the Mg2+dependent activity by deletion of the last helix [39] In all cases, the conformation of the metal binding site is probably slightly changed, so that it becomes unfavorable for binding of Mg2+ but is kept favorable for binding of Mn2+ The finding that the D13-R4-D12 ⁄ D29 and R9D9 ⁄ D18 substrates cannot be effectively cleaved by Tma -RNase HI. .. in the presence of Mg2+ suggests that the RNA ⁄ DNA hybrid region in these substrates is too short to accommodate both the HBD and the RNase H domain According to the crystal structure 4484 of the catalytic domain of human RNase H1 in complex with the substrate [15], the enzyme interacts with several RNA residues preceding the scissile bond The distance between the most preferable cleavage site of. .. differ in Mg2+-dependent activity, the Mg2+dependent activity of Tma -RNase HI may be responsible for its RNase H activity in vivo It has been reported that E coli RNase HII (Eco -RNase HII) [40] and Bacillus subtilis RNase HII (Bsu -RNase HII) [41], which prefer Mn2+ to Mg2+ for activity, complement the temperature-sensitive growth phenotype of E coli MIC3001 Both proteins exhibit the highest Mn2+dependent... decrease sharply with the increase of the concentration of salt beyond the optimum one (Fig 4) The surface plasmon resonance analyses for binding to the R29 ⁄ D29 substrate indicate that the activities of these proteins greatly decrease at high salt concentrations because the binding affinities of these proteins to the substrate greatly decrease However, the highest activities of Tma -RNase HI and Tma-CD are... conserved in the HBD of Tma -RNase HI, Tma -RNase HI probably requires 50 mm KCl for maximal activity to overcome these nonspecific interactions Role of HBD from T maritima RNase HI Tma-ND Primers 1 and 3, primers 2 and 3, and primers 1 and 4 were used to amplify the genes encoding Tma -RNase HI, Tma-CD and Tma-ND, respectively The resultant DNA fragments were digested with NdeI and HindIII or EcoRI, and ligated . The N-terminal hybrid binding domain of RNase HI from Thermotoga maritima is important for substrate binding and Mg 2+ -dependent activity Nujarin. loses this processivity on removal of the HBD. Binding to substrate To examine whether the HBD of Tma -RNase HI is important for substrate binding, the binding

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