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College of Life Science and Technology, Shanghai Jiaotong University, 800 Dong-Chuan Road, Shanghai 200240, China
Correspondence
Jianhua Liu
jianhualiudl{at}sjtu.edu.cn
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). RNase Hs have been classified into type 1 and type 2 RNase H, and prokaryotic RNase Hs are divided into three groups, RNase HI, HII and HIII, encoded by rnhA, rnhB and rnhC, respectively (Ohtani et al., 1999a, b). RNase HI is a type 1 RNase H, while RNase HII and HIII are both of type 2.
Although many bacteria, such as Escherichia coli, contain both type 1 and type 2 RNase H, some bacteria, such as Bacillus stearothermophilus, Streptococcus pneumoniae, Chlamydia trachomatis and Aquifex aeolicus only have two different type 2 RNase Hs (Ohtani et al., 1999a). Although the crystal structures of some RNase HI and HII enzymes have been determined, e.g. E. coli (Katayanagi et al., 1990; Yang et al., 1990), Methanococcus jannaschii (Lai et al., 2000) and Thermoccus kadakaraensis (Muroya et al., 2001), little 3D structural information on RNase HIII is available. The enzymic properties of a few RNase HIIIs such as Bacillus subtilis RNase HIII and B. stearothermophilus RNase HIII have been analysed (Ohtani et al., 1999b; Chon et al., 2004, 2006a). Except for the conserved motifs, all the described type 2 RNase Hs contain unique active-site residues, a DEDD motif (Asp-Glu-Asp-Asp) for RNase HII and a DEDE motif (Asp-Glu-Asp-Glu) for RNase HIII (Chon et al., 2006a).
Chlamydophila pneumoniae, a pathogenic eubacterial intracellular parasite for humans and some animals, infects the mucosal surfaces of the respiratory tract, causing pharyngitis, bronchitis and pneumonia (Hahn et al., 2002). The complete genome sequence of C. pneumoniae AR39 offered much genetic information about this microbe (Read et al., 2000). Two prospective RNase HII and HIII genes, CP0654 and CP0782, have been isolated from C. pneumoniae AR39 and expressed in E. coli previously (Pei et al., 2005). In this study, the enzymic properties of these two RNase Hs (Cpn-RNase HII and HIII) were analysed further and their functional complementation of RNase H-deficient E. coli mutants was demonstrated in vivo.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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lacU169 nadA : : Tn10 gal490 
cI857(cro-bioA); Yu et al., 2000] was used in this study; the plasmids used are listed in Table 1. Bacteria were cultured in Luria–Bertani (LB) medium at 32 °C and antibiotics were used as follows: ampicillin, 100 µg ml–1, kanamycin, 50 µg ml–1 and tetracycline 10 µg ml–1. Oligonucleotides were synthesized by Invitrogen. Restriction enzymes and DNA-modifying enzymes were purchased from TaKaRa. All other chemicals were analytical grade.
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). PCR reactions were carried out using LA-Taq DNA polymerase (TaKaRa) and products were recovered with a gel purification kit (Sangon). Homologous recombination was carried out as described before (Yu et al., 2000) to perform complementation assays. All constructs were confirmed by DNA sequencing (Invitrogen).
Construction of plasmids.
To achieve constitutive expression of the genes under study, the GAPDH-promoter fragment was amplified from genomic DNA with GAPDH-F/GAPDH-R (F: 5'-GGGGGGGGATCCgctcacatctcactttaatcg-3'; R: 5'-GGGGGAAGCTTGCATGCGAGCTCGTCGACGGTACCTGCAGccaccagctatttgttagtg-3'; F and R represent forward and reverse primers respectively, the underlined bases are introduced to create restriction sites for DNA cloning and the lower-case bases can match the template, but the capitalized bases are added for DNA cloning or homologous recombination), containing the GAPDH-promoter sequence, RBS and recognition sites for several enzymes (XhoI, KpnI, SacI, SphI and HindIII). The fragment was treated with BamHI/HindIII and ligated with predigested pUC18, producing pUC-G (Table 1
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Amplified rnhA and rnhB fragments from genomic DNA using primers rnhA-F/rnhA-R (F: 5'-GGGGGGCTCGAGatgcttaaacaggtagaaattttcacc-3'; R: 5'-GGGGGGGCATGCttaaacttcaacttggtagcctgtatct-3') and rnhB-F/rnhB-R (F: 5'-GGGGGGCTCGAGatgatcgaatttgtttatccgcacacgagctggttgcgg-3'; R: 5'-GGGGGGGCATGCtcaggacgcaagtcccagtgcgcgtttgaca-3'). The CP0654 and CP0782 fragments were amplified from pET28a-CP0654 and pET28a-CP0782 (Pei et al., 2005; Table 1) with primers CP0654-F/CP0654-R (F: 5'-GGGGGGCTCGAGatgaatacttctatttctgaaattc-3'; R: 5'-GGGGGGGCATGCtcatacaatagcacacatttgc-3') and CP0782-F/CP0782-R (F: 5'-GGGGGGCTCGAGatgtcctgcatgccgccacc-3'; R: 5'-GGGGGGGCATGCttatttccccgaacaaatttcgtca-3'). These fragments were treated with XhoI/SphI and ligated with predigested pUC-G, producing pUC-rnhA, pUC-rnhB, pUC-CP0654 and pUC-CP0782, respectively (Table 1). Another rnhB fragment amplified with primers rnhB-F2/rnhB-R2 (F: 5'-GGGGGGGCATGCtggtggatgatcgaatttgtttatccgcacacgcagctggttgcgg-3'; R: 5'-GGGGGGAAGCTTtcaggacgcaagtcccagtgcgcgtttgaca-3') was digested with SphI/HindIII and ligated into pUC-rnhA to produce pUC-rnhA+B (Table 1).
Complementation assay.
Plasmids pUC-rnhA, pUC-rnhB, pUC-rnhA+B, pUC-CP0654 or pUC-CP0782 were electro-transformed into DY329 and these strains were named as DY-rnhA, DY-rnhB, DY-rnhA+B, DY-CP0654 and DY-CP0782, respectively. Then electro-competent cells of these strains were made (Yu et al., 2000).
The tetracycline and kanamycin cassettes were amplified using primers Tc-F/Tc-R (F: 5'-TACAGTTCGATTCAATTACAGGAAGTCTACCAGAGatgaaatctaacaatgcgctcatcg-3'; R: 5'-GCCTGTGGTTTACGACATTGCCGGGTGGCTCCAACtcaggtcgaggtggccc-3') and Km-F/Km-R (F: 5'-TGAGCAGGCGGCACAAGCCGTTCTGGAGTTAGCACAATGAgccatattcaacggga-3'; R: 5'-ACATCTTCAGATTCCGGTTTACTTAATCTCGACACAAGAAgaaaaactcatcgagcatca-3') to replace the chromosomal rnhA and rnhB. Note that most of rnhA and rnhB, but not the whole of either gene, was substituted, as the promoters of other essential genes were located at the end of their coding sequences, which was retained in our constructed strains. Then the two cassettes were electro-transformed into DY329, DY-rnhA, DY-rnhB, DY-rnhA+B, DY-CP0654 or DY-CP0782. The transformants were screened with appropriate antibiotics and the plates were photographed with a digital camera (Kodak DX6490).
In vitro enzymic activity assays
Recombinant Cpn-RNase Hs were expressed and purified, and the enzymic activities were measured as before (Pei et al., 2005). Unless specified otherwise, assays were performed in reaction buffer containing 10 mM Tris/HCl, pH 9.0, 50 mM NaCl, 1 mM 
-mercaptoethanol, 10 mM MgCl2 and 10 µg bovine serum albumin ml–1. Reactions were stopped by adding an equal volume of stopping buffer (100 mM EDTA, 8 M urea, 0.1 % bromophenol blue and 0.1 % xylene cyanol) and analysed by electrophoresis on 20 % polyacrylamide gels (with 8 M urea), followed by measurements with an FLA-5000 phosphorimager.
Kinetic parameters.
For determination of kinetic parameters, the enzyme was used at 0.01 µM and the concentration of the 12 bp RNA–DNA substrate varied from 0.052 to 2 µM. The reaction was performed at 30 °C for 1 min and stopped by adding stopping buffer. The kinetic parameters were determined from Lineweaver–Burk plots.
pH effect.
pH effects were determined by performing the assay as above except that the reaction buffer was substituted with 10 mM imidazole/HCl (pH 5.0–6.5), 10 mM Tris/HCl (pH 7.0–9.0) or 10 mM glycine/NaOH (pH 9.0–12.0).
Temperature effect.
Recombinant Cpn-RNase Hs (2 nM) were dissolved in reaction buffer and treated at 70 °C for different times; the temperature effects were analysed by determining the remaining enzymic activities.
Substrate specificity.
A 12 bp RNA–DNA (5'-32P-cggagaugacgg-3', 3'-GCCTCTACTGCC-5'; note that ribonucleotides are shown by lower-case letters), 35 bp DNA–RNA–DNA/DNA (5'-32P-AGAGGCAGGAGAGTuGCAACGCAGCAAGAGGACGC-3', 3'-TCTCCGTCCTCTCAACGTTGCGTCGTTCTCCTGCG-5') and Ribo primer (5'-32P-GCGTCCTCTT GCTGCGTTGCaa-3', 3'-CGCAGGAGAACGACGCAACGTTGAGAGGACGGAGA-5') were used as substrates. All oligonucleotides carrying ribonucleotide(s) were 5'-end-labelled with 32P and the hybrid duplexes were prepared by standard procedures (Katayanagi et al., 1990). The cleavage assays were performed in reaction mixture containing 0.2 µM of different ribonucleotide substrates and specified amounts of Cpn-RNase Hs.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), consistent with previous reports that the active-site motif is conserved as DEDD in RNase HII, but DEDE in RNase HIII (Chon et al., 2006a).
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Effects of pH and temperature on the enzymic activity of Cpn-RNase Hs
To analyse the enzyme properties of Cpn-RNase Hs, their kinetic parameters were first determined. The Km and Vmax values of Cpn-RNase HII were 1.6 µM and 0.022 µM min–1, while those of Cpn-RNase HIII were 0.644 µM and 0.0135 µM min–1 (about 2-fold lower and 2.5-fold higher, respectively. than those of Cpn-RNase HIII). Errors were below 10 % of the reported value.
pH experiments showed that both the Cpn-RNase Hs exhibited activities at an alkaline pH, consistent with other RNase Hs (Ohtani et al., 1999b; Chon et al., 2006b). The optimal pH for Cpn-RNase HII was 9.0 and for Cpn-RNase HIII, 10.0. The enzymic activities decreased rapidly at pH values below 7.0; activities also decreased at pH values above 10.0, but slowly (Fig. 2).
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). Thus neither Cpn-RNase H was a heat-stable enzyme, consistent with a previous report that the thermostability of RNase Hs often correlates with the growth temperature of the organism (Chon et al., 2004).
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). As to Cpn-RNase HIII, only a6-u7 was the favoured cleavage site (Fig. 4b); this behaviour was quite different from that of other RNase HIIIs such as Bsu-RNase HIII (Ohtani et al., 1999b) and Bst-RNase HIII (Chon et al., 2004), both with multiple-site cleavages. When the 35 bp DNA–RNA–DNA/DNA was used as substrate, only Cpn-RNase HII could cleave it (Fig. 5). And as to the Ribo primer (one primer with two ribonucleotides at the 3'-end), no cleavage was observed with any Cpn-RNase H (data not shown) These results indicate that both the Cpn-RNase Hs could cleave RNA–DNA duplex, but could not use Ribo primer as target, and only Cpn-RNase HII could cleave a hybrid with RNA flanked by DNA.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) or B. stearothermophilus (Chon et al., 2004). Further different properties of the two proteins imply that Cpn-RNase HII may be the major RNase H of C. pneumoniae and the RNase HII/HIII combination in C. pneumoniae AR39 is not a simple substitution of E. coli RNase HI/II.
Although many organisms contain two types of RNase H genes in one cell, such as E. coli (AB type), multiple type 2 RNase H genes (BC type) have been revealed in some organisms, e.g. B. subtilis (Ohtani et al., 1999a) and B. stearothermophilus (Chon et al., 2004). Therefore, the presence of two type 2 RNase Hs in C. pneumoniae AR39 may not be unusual. Further genome database analysis also showed that the two type 2 RNase H homologues are conserved in other Chlamydophila and Chlamydia species, such as Chlamydophila abortus, Chlamydia muridarum Nigg, Chlamydophila felis Fe/C-56 and Chlamydophila caviae GPIC, with very high identities with the Cpn-RNase Hs. But up to now, except for the RNase Hs of C. pneumoniae AR39, the enzymic properties and biological function of other Chlamydophila RNase Hs have not been determined. Interestingly, A. aeolicus, a hyperthermophilic hydrogen-oxidizing bacterium similar to primordial forms of life (Deckert et al., 1998), also only contains two type 2 RNase H homologues. Considering the evolutionary position of these organisms, the RNase H genes of A. aeolicus and Chlamydophila perhaps represent an ancestral structure and the BC type is perhaps more primordial than the AB type.
The main function of RNase H is to remove RNA primers from Okazaki fragments, process R loops to modulate replication initiation and restore DNA topology (Broccoli et al., 2004; Kogoma & Foster 1998; Arudchandran et al., 2000). Recently, a mitochondrial RNase H from the parasitic protozoon Leishmania was analysed, which is essential for the parasite's survival (Misra et al., 2005). This may give a clue to the biological status of the Cpn-RNase Hs, which may play an important role on the DNA replication/translation processes of Chlamydophila and may be vital for this organism. As to the unique biochemical function of RNase Hs, any differential biochemistry and molecular biology of these enzymes in a parasite as compared to that of its host could be exploited for rational drug design against the parasite infection. Therefore, the characterization of Cpn-RNase Hs will be an important avenue toward the development of anti-Chlamydophila compounds.
Moreover, RNase Hs is a member of the ‘polynucleotide transferases' superfamily, including resolvase (Ariyoshi et al., 1994), integrase (Maignan et al., 1998), transposase (Rice & Mizuuchi, 1995), exonuclease III (Mol et al., 1995), type II restriction enzymes (Kostrewa & Winkler, 1995), Vsr endonuclease (Tsutakawa et al., 1999), RNA helicase (Nishino et al., 2003) and RNAse III (Blaszczyk et al., 2001). These enzymes have homologous active sites and are likely to share a common mechanism for catalysis. Hence, understanding of RNase H may contribute to the study of other members.
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ACKNOWLEDGEMENTS |
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Edited by: T. P. Hatch
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 13 October 2006;
revised 5 December 2006;
accepted 7 December 2006.
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) and HIII (
) activity are plotted.
