1 National Health Research Institutes, 3F No. 109, Section 6, Min-Chuan East Road, Taipei 114, Taiwan
2 Graduate Institute of Microbiology, College of Medicine, National Taiwan University, No. 1, Section 1, Jen-Ai Road, Taipei 100, Taiwan
Correspondence
Jen-Yang Chen
cjy{at}nhri.org.tw
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ABSTRACT |
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INTRODUCTION |
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Herpesvirus DNases are conserved, and alignments of amino acid sequences reveal that the DNases share several conserved regions (Martinez et al., 1996; Goldstein & Weller, 1998
; Liu et al., 1998
; see Fig. 1
). Furthermore, human cytomegalovirus (HCMV) DNase can substitute for HSV-1 DNase and complement the growth of an HSV-1 DNase deletion mutant (Gao et al., 1998
). In the absence of structural information from crystallographic studies, little is known about the cleavage mechanism and active site(s) for catalysis by herpesvirus DNases. In previous studies, we tried to locate the regions important for DNase activity by deleting amino acid residues serially from both ends of EBV DNase; a drastic loss of DNase activity was observed in mutants with small deletions and substitutions (Lin et al., 1994
; Liu et al., 1998
). We extended our mutational analysis to internal regions, including four conserved regions and two non-conserved regions of herpesvirus DNases. DNA-binding and nuclease activities were abolished in all six internal deletion mutants, except that one mutant, with a deletion of residues 138152, retained an intermediate ability to bind DNA (Liu et al., 1998
). It is difficult to propose a mechanism for DNA cleavage by EBV DNase and to assign amino acid residues for an active centre(s) in catalysis simply from analyses of deletion and substitution mutations.
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METHODS |
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Expression of the DNase protein in E. coli and assay for nuclease activity.
Expression of pDNase5 and its mutated derivatives was carried out as described previously (Lin et al., 1994). Briefly, a 2 ml culture of E. coli BL21(DE3) pLysS (Novagen) harbouring a particular plasmid was grown to exponential phase (OD600=0·40·6) and induced for 2 h with 1 mM IPTG. Cells were harvested and lysed with 250 µl 50 mM Tris/HCl, pH 8·0, 50 mM NaCl, 1 mM DTT and 10 % glycerol. After sonication, 5 µl of prepared lysate was assayed at various dilutions for nuclease activity (Chen et al., 1982
; Lin et al., 1994
). The reaction mixture contained 50 mM Tris/HCl, pH 8·0, 4 mM
-mercaptoethanol, 4 mM MgCl2 and 3 µg E. coli [14C]DNA (2x104 c.p.m.). The reaction mixture was incubated at 37 °C for 1 h and then stopped by the addition of 25 µl sheared calf thymus DNA (2 mg ml-1) followed by 25 µl 50 % trichloroacetic acid. DNase activity was defined as the amount of enzyme that converted [14C]DNA to acid-soluble material. The relative nuclease activity was normalized with the DNase protein concentration, as described previously (Liu et al., 1998
). The nuclease activity of each clone minus that of cells harbouring pET3a gave the net nuclease activity. The specific nuclease activity was calculated by dividing the net activity of each clone by the intensity of each expressed protein in enhanced chemiluminescence (ECL) Western blot analysis (Tsai et al., 1997
), which was quantified by scanning with a densitometer. The relative nuclease activity is the specific nuclease activity of each clone divided by that of pDNase5.
Expression of recombinant EBV DNase and purification of His-tagged DNase.
Purification of wild-type and mutant proteins was carried out as follows. E. coli BL21(DE3) pLysS harbouring pET-DNase15b or its mutated derivatives was grown to exponential phase (OD600=0·40·6) and induced for 2 h with 1 mM IPTG. Cells were harvested by centrifugation and then pellets were resuspended in binding buffer (5 mM imidazole, 500 mM NaCl, 20 mM Tris/HCl, pH 8·0, 0·1 % NP-40 and 10 % glycerol). The lysate was sonicated and clarified by centrifugation at 39 000 g for 20 min. The supernatant was used as starting material for DNase purification. The starting material prepared above was applied to a His-Bind column (Novagen). The column was washed sequentially with 10 bed vols of TNGI-5 (20 mM Tris/HCl, pH 8·0, 500 mM NaCl, 10 % glycerol, 5 mM imidazole), 5 bed vols of TGI-30 (20 mM Tris/HCl, pH 8·0, 10 % glycerol, 30 mM imidazole) and 5 bed vols of TGI-60 (20 mM Tris/HCl, pH 8·0, 10 % glycerol, 60 mM imidazole). The bound proteins were then eluted with 5 bed vols of TGI-100 (20 mM Tris/HCl, pH 8·0, 10 % glycerol, 100 mM imidazole). The collected TGI-100 eluate was sequentially collected into four fractions (TGI-100-1, -2, -3, -4). The protein in each fraction was analysed by 10 % SDS-PAGE.
Plasmid DNA cleavage assay.
For the plasmid DNA cleavage assay, reaction mixtures contained 125 ng of purified DNase, 0·5 µg of pEGFP-C1 plasmid DNA (Invitrogen), 50 mM Tris/HCl, pH 8·0, 4 mM -mercaptoethanol and 4 mM MgCl2 or 4 mM MnCl2. The reactions were incubated at 37 °C, aliquots were taken at the indicated times and analysed by agarose gel electrophoresis. Similar assays were carried out using linear plasmid DNA, which was cut by HindIII.
Expression of the DNase protein by in vitro transcription/translation and assay of DNA-binding activity.
For measuring the DNA-binding ability of EBV DNase, [35S]methionine-labelled DNase and dsDNA cellulose chromatography were used as described previously (Liu et al., 1998). Using a TNT T7 coupled reticulocyte lysate system (Promega), DNA templates of pDNase5 and its mutants were transcribed with T7 RNA polymerase and translated in a rabbit reticulocyte lysate. Each 0·25 µg of purified plasmid DNA was used as an in vitro transcription/translation (IVT) template in a final volume of 12·5 µl. The IVT-expressed polypeptides were radiolabelled in the reaction mixture containing [35S]methionine. After 90 min of in vitro translation at 30 °C, the [35S]methionine-labelled polypeptides were diluted 1 : 10 in buffer A (50 mM Tris/HCl, pH 8·0, 1 mM DTT, 1 mM PMSF, 10 % glycerol) containing 100 µg RNase A. An aliquot (12·5 µl) was retained as starting material and the remainder was applied to a 200 µl bed volume of dsDNAcellulose (Sigma) in a Bio-Spin column (Bio-Rad) equilibrated in buffer A. The column was washed with 300 µl buffer A and then eluted with 400 µl step gradients of 100, 200, 300, 400 and 500 mM NaCl in buffer A. Five µl of starting material and 20 µl of eluates were mixed with SDS-PAGE sample buffer (100 mM Tris/HCl, pH 6·8, 4 % SDS, 5 %
-mercaptoethanol, 20 % glycerol, 0·05 % bromophenol blue) and analysed by 10 % SDS-PAGE and fluorography.
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RESULTS |
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Analysis of the nuclease activity of EBV DNase mutants
To investigate the possible functional roles of the three conserved residues, each was replaced by residues with different properties using site-directed mutagenesis. Residue D203 was changed to Asn, Lys, Gln, Glu or His; residue E225 was changed to either Asp or Gln and residue K227 was changed to Glu, Asp, Gly, Asn or Arg (Fig. 1a). In addition, the other acidic residues (D195, E209, D213, D219, E238 and D240) within conserved regions also were replaced by Asn or Gln (Fig. 1a
). Wild-type and mutant DNases were cloned into the pET3a vector and expressed in E. coli BL21(DE3) pLysS. The recombinant EBV DNase was intact and contained no additional domains derived from the vector. The nuclease activities of wild-type, mutants and the vector control were measured. All mutants with substitutions of D203, E225 and K227 showed drastically reduced nuclease activity (Fig. 2
). Five of the six other acidic residue mutants, E209Q, D213N, D219N, E238Q and D240N, retained their nuclease activities (>75 %) and only the mutant D195N lost nuclease activity (3 %) (Fig. 2
). Residue D195 was not conserved in the alignment of herpesvirus DNases but a conserved Asp residue was found nearby in other herpesvirus DNases (Fig. 1a
). Based on the analysis of nuclease activity, we found that four charged residues of EBV DNase, D195, D203, E225 and K227, may play important roles in catalysis.
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DISCUSSION |
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To test this hypothesis, each of the three conserved residues and flanking acidic residues were replaced by site-directed mutagenesis. Residue D203 was changed to Asn, Lys, Gln, Glu or His, residue E225 was changed to Asp or Gln, and residue K227 was changed to Glu, Asp, Gly, Asn or Arg. All of these mutants exhibited significantly reduced nuclease activities (Fig. 2), underlining the importance of these three residues. Similar mutagenesis studies have been performed on EcoRI, EcoRV, BamHI and PvuII (Selent et al., 1992
; Dorner & Schildkraut, 1994
; Grabowski et al., 1995
; Nastri et al., 1997
). Mutation of any of the three corresponding residues of these restriction enzymes abolished nuclease activity. Mutant D430E of HSV-1 alkaline nuclease, corresponding to the D203E mutant of EBV DNase, lost exonuclease activity but retained endonuclease activity (Goldstein & Weller, 1998
). According to crystallographic studies, the two acidic residues form a binding site for the catalytically essential metal ion and the Lys residue orients the water and assists in the nucleophile attack on the phosphorus atom (Pingound & Jeltsch, 1997
; Kovall & Matthews, 1999
). Corresponding amino acid residues D203 and E225 of EBV DNase were proposed to bind the metal ion. In a plasmid DNA cleavage assay, Mn2+ could activate the E225D mutant (Fig. 4c
), indicating a change of preference for divalent cations from Mg2+ to Mn2+ and implying that the E225 residue is involved in the formation of the metal binding site. Because the radial size of Mn2+ is larger than Mg2+ and the side chain of Asp is shorter than that of Glu, the change in cation preference of the E225D mutant is most likely attributable to the short side chain of Asp forming a larger metal binding site, fitting Mn2+ better than Mg2+. Unlike E225D, D203E and K227E were not activated by Mn2+. Interestingly, a K92E mutant of EcoRV also became active in the presence of Mn2+ (Selent et al., 1992
) and an E98Q mutant of MunI endonuclease switched its cofactor requirement from Mg2+ to Mn2+ (Lagunavicius & Siksnys, 1997
). Unlike the K92E mutant of EcoRV, K227E of EBV DNase lost nuclease activity, even in the presence of Mn2+, which is similar to a K113E mutant of EcoRI (Grabowski et al., 1995
).
In addition to D203, E225 and K227, six other acidic residues near the D...EXK motif were also replaced by Asn or Gln. Mutational analysis showed that five of the six acidic residue mutants retained, and only mutant D195N lost, nuclease activity (Fig. 2). Although the D195 residue of EBV DNase is not conserved, a conserved Asp residue was aligned nearby in other herpesvirus DNases (Fig. 1a
). The E238 residue of EBV DNase is partially conserved (Asp or Glu in the other herpesvirus DNases, shown in Fig. 1a
), but the E238Q mutant retained nuclease activity, indicating that the carboxylate group of E238 was not required for catalysis. Many metal-dependent nucleases contain more than two catalytic acidic residues (Asp or Glu) to form metal binding sites at their active sites, such as type II restriction endonucleases (Pingound & Jeltsh, 1997
), the 3'
5' exonuclease activity of the Klenow fragment (Bernad et al., 1989
), T4 RNase H and RAD2 (Mueser et al., 1996
) and hFEN-1 (Shen et al., 1997
). In the alignment of human herpesvirus DNases, wholly conserved acidic residues include E107, E166, D203 and E225 in the EBV sequence. The role of E166 in EBV DNase was predicted to involve catalysis (Aravind et al., 2000
). The residue E107 could not be predicted in the absence of homology. However, the presence of a D...(D\E)XK motif in the active sites of type II restriction endonucleases and other nucleases provided the information to predict the roles of D203 and E225. Although amino acid sequences of type II endonucleases and other nucleases did not exhibit any significant sequence identity, local structures, particularly in catalytic sites, showed striking resemblance (Kovall & Matthews, 1999
). It was suggested that these nucleases with structural homology were diverged from a common ancestor (Kovall & Matthews, 1999
). Furthermore, Glu113 of BamHI substitutes for the conserved Lys and the active site was characterized as a D...EXE motif (Newman et al., 1994
). The (D\E)X(K\D\E) motif may exhibit a common function for nucleases and many nucleases contain such a motif in their active sites, such as the 3'
5' exonucleases of prokaryotic and eukaryotic DNA polymerases, T4 RNase H and RAD2, bacterial RecJ exonucleases, hFEN-1 and Holliday junction resolvases and related nucleases (Bernad et al., 1989
; Morrison et al., 1991
; Mueser et al., 1996
; Moser et al., 1997
; Shen et al., 1997
, 1998
; Aravind & Koonin, 1998
; Aravind et al., 2000
). These similarities indicate that the catalytic (D\E)X(K\D\E) motif is conserved in the evolution of nucleases. Corresponding to this concept, NaeI was found to be a likely evolutionary bridge between DNA endonuclease and topoisomerase (Huai et al., 2000
).
Double-stranded DNAcellulose chromatography was used to determine the DNA-binding characteristics of the mutants. Wild-type EBV DNase, expressed by IVT, bound efficiently to dsDNA and was eluted with 500 mM NaCl (Fig. 6). The DNA-binding characteristics of the D203E, E225D and E225Q mutants were similar to that of wild-type (Fig. 6
). The D203 and E225 mutants of EBV DNase bound but failed to cleave DNA, similar to D91A and E111A of EcoRI, D74A and D90A of EcoRV, and D94A and E111A of BamHI (Selent et al., 1992
; Dorner & Schildkraut, 1994
; Grabowski et al., 1995
). K227 mutants of EBV DNase showed variable ability to bind DNA: K227G and K227N mutants retained, K227E and K227D had reduced and K227R lost DNA-binding ability, indicating that the K227 residue may be involved in catalysis and that a positive charge for K227 is not required for DNA binding. Of the corresponding substitutions in other nucleases, K113A of EcoRI, K92A of EcoRV and K70A of PvuII lost nuclease activity but bound to DNA (Selent et al., 1992
; Grabowski et al., 1995
; Nastri et al., 1997
), similar to the K227G mutant of EBV DNase. However, the K227R mutant of EBV DNase lost the ability to bind DNA, similar to K70R of PvuII (Nastri et al., 1997
). Mutational analysis of K227 mutants of EBV DNase demonstrated that biochemical alteration of K227 mutants was similar to their counterparts in EcoRI and PvuII, underlining the similarity of these residues. Based on the studies of site-directed mutagenesis and biochemical properties of corresponding substitutions, it is suggested that the conserved motif D...EXK of herpesvirus DNases is most likely the putative catalytic centre, and is homologous to those of type II restriction endonucleases.
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ACKNOWLEDGEMENTS |
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Received 30 July 2002;
accepted 25 October 2002.
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