Journal of Biotechnology 184 (2014) 39–46
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Mutations in the palm subdomain of Twa DNA polymerase to enhance PCR efficiency and its function analysis Sung Suk Cho, Mi Yu, Suk-Tae Kwon ∗ Department of Genetic Engineering, Sungkyunkwan University, 300 Chunchun-dong, Jangan-gu, Suwon 440-746, Republic of Korea
a r t i c l e
i n f o
Article history: Received 26 December 2013 Received in revised form 19 April 2014 Accepted 12 May 2014 Available online 24 May 2014 Keywords: Thermococcus waiotapuensis (Twa) Twa DNA polymerase Twa N501R DNA polymerase PCR amplification Forked-point
a b s t r a c t Among the family B DNA polymerases, the Twa DNA polymerase from T. wiotapuensis, a hyperthermophilic archaeon, has exceedingly high fidelity. For applications in PCR, however, the enzyme is limited by its low extension rate and processivity. To resolve these weaknesses, we focused on two amino acid residues (A381 and N501) located at the palm subdomain of Twa DNA polymerase. Following replacement of these residues by site-directed mutagenesis, Twa N501R DNA polymerase showed significantly improved polymerase function compared to the wild-type enzyme in terms of processivity (3-fold), extension rate (2-fold) and PCR efficiency. Kinetic analysis using DNA as template revealed that the kcat value of the Twa N501R mutant was similar to that of wild-type, but the Km of the Twa N501R mutant was about 1.5-fold lower than that of the wild-type. These results suggest that a positive charge at residue 501 located in the forked-point does not impede catalytic activity of the polymerase domain but stabilizes interactions between the polymerase domain and the DNA template. © 2014 Elsevier B.V. All rights reserved.
1. Introduction DNA polymerases are widely used as in vitro applications and some of them are well studied because of their academic and scientific importance. In particular, use of thermostable Taq DNA polymerase from Thermus aquaticus in PCR has revolutionized molecular biology (Saiki et al., 1988). Recently, the family B DNA polymerases from hyperthermophilic archaea (Pyrococcus furiosus, Thermococcus litoralis) have gained attention for their high fidelity in PCR based on their strong 3 → 5 exonuclease (proof-reading) activity (Lundberg et al., 1991; Mattila et al., 1991). However, these DNA polymerases generally exhibit lower processivity and extension rates than Taq DNA polymerase (Barnes, 1994). An improved processivity of DNA polymerase was reportedly achieved through fusion of the ds DNA-binding protein identified in Sulfolobus solfataricus (Sso7d) to the thermostable polymerase (Wang et al., 2004; Lee et al., 2010a) and fusion of multiple helix-hairpin-helix motifs identified in DNA topoisomerase V (Pavlov et al., 2002). In alternative different approaches, an auxiliary enzyme such as dUTPase may be added to the PCR assay (Hogrefe et al., 2001; Cho et al., 2012b) and mutant polymerases may be obtained via site-directed mutagenesis (Song et al., 2010; Lee et al., 2010b).
∗ Corresponding author. Tel.: +82 31 290 7863; fax: +82 31 290 7870. E-mail address: [email protected] (S.-T. Kwon). http://dx.doi.org/10.1016/j.jbiotec.2014.05.007 0168-1656/© 2014 Elsevier B.V. All rights reserved.
Most family B DNA polymerases from hyperthermophilic archaea consist of several distinct domains, including an N-terminal domain, exonuclease domain and a polymerase domain with palm, fingers and thumb subdomains (Hopfner et al., 1999; Rodriguez et al., 2000; Hashimoto et al., 2001). The arginine residues (R243, R247, R264, R266, R346, R381 and R501) of the exonuclease and palm subdomains of the forked-point in KOD DNA polymerase provide a basic environment (Fig. 1A). Structural studies suggested that basic residues interact with the phosphate group of the DNA strand and stabilize the melted DNA strands at the forked-point (Hashimoto et al., 2001). Several arginine residues at the forked-point in most family B DNA polymerases from hyperthermophilic archaea are conserved (Hashimoto et al., 2001). KOD DNA polymerase exhibits a five-fold higher extension rate (100–130 nucleotides/s) and 10–15-fold higher processivity (>300 bases) than Pfu DNA polymerase (Takagi et al., 1997). However, the fidelity of KOD DNA polymerase was similar to that of Pfu DNA polymerase. It is considered that the substitution of arginine residues around the forked-point may affect the difference of the enzymatic characteristics between KOD and Pfu DNA polymerases (Hashimoto et al., 2001). In the forked-point of Pfu DNA polymerase, R247, R265 and R501 residues are replaced by M247, T265 and K502 residue, respectively. Biochemical characterization of the T. waiotapuensis (Twa) DNA polymerase (Cho et al., 2012a) revealed the high fidelity of this enzyme associated with its 3 → 5 exonuclease-dependent proofreading activity. The fidelity of Twa DNA polymerase was higher
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Fig. 1. (A) Tertiary structure of KOD DNA polymerase model showing the arginine residues in forked-point (PBD ID: 1WNS) (Hashimoto et al., 2001). Tertiary structure was shown using the 3D Molecule Viewer program of Vector NTI AdvanceTM 9.1 (Invitrogen, USA). (B) Amino acid sequence alignment of archaeal family B DNA polymerase. Multiple alignments were produced using the AlignX software (Invitrogen, USA): Twa (T. waiotapuensis, GeneBank accession no. JQ399904), KOD1 (P. kodakaraensis KOD1, GeneBank accession no. D29671), Pfu (P. furiosus, GeneBank accession no. D12983), 9◦ N-7 (Thermococcus sp. 9◦ N-7 GeneBank accession no. AAA88769), Tgo (T. gorgonarius, GeneBank accession no. P56689). Shaded amino acid residues indicate identical and conserved residues matched with basic residues of fork-point.
than that of other family B DNA polymerase, including the Pfu, KOD and Vent. However, Twa DNA polymerase has a lower extension rate and processivity than Taq DNA polymerase (Cho et al., 2012a). In the Twa DNA polymerase, the R381 and R501 residues in the forked-point of KOD DNA polymerase are replaced by A381 and N501 residues, respectively (Fig. 1B). This replacements of Twa DNA polymerase may have low extension rate and processivity as above described. Therefore, we replaced the two amino acid residues (A381 and N501) located at the forked-point of Twa DNA polymerase with other amino acid residues using site-directed mutagenesis. The mutant Twa N501R DNA polymerase showed significantly improved polymerase function compared to wild-type Twa DNA polymerase in terms of processivity, extension rate and PCR efficiency. 2. Materials and methods 2.1. Construction of mutated Twa DNA polymerase genes Point mutations were introduced into the wild-type Twa DNA polymerase gene using the QuikChange site-directed mutagenesis method (Stratagen, USA) with the Twa DNA polymerase gene subcloned into pET-20b(+) as a template (Cho et al., 2012a). The primers used for mutations at specific residues are listed in Table 1. Table 1 The positions of amino acid substitution and the primer sequences for mutation. Name of mutants
Name of primers
Mutation primer sequence
A381R
F-A381R R-A381R F-N501R R-N501R F-N501A R-N501A F-N501D R-N501D F-N501E R-N501E F-N501K R-N501K
5 -GAGTTAGCAAGGAGACGGGAGAGCTACGCG-3 5 -CGCGTAGCTCTCCCGTCTCCTTGCTAACTC-3 5 -CTACGGCTACGCACGTGCCCGCTGG-3 5 -CCAGCGGGCACGTGCGTAGCCGTAG-3 5 -CTACGGCTACGCAGCTGCCCGCTGG-3 5 -CCAGCGGGCAGCTGCGTAGCCGTAG-3 5 -CTACGGCTACGCAGATGCCCGCTGG-3 5 -CCAGCGGGCATCTGCGTAGCCGTAG-3 5 -CTACGGCTACGCAGAAGCCCGCTGG-3 5 -CCAGCGGGCTTCTGCGTAGCCGTAG-3 5 -CTACGGCTACGCAAAGGCCCGCTGG-3 5 -CCAGCGGGCCTTTGCGTAGCCGTAG -3
N501R N501A N501D N501E N501K
–, The underlined triplets signify the mutated codons.
All of the mutant plasmid DNA was synthesized by PCR amplification, and subsequently digested with the restriction enzyme DpnI to remove unmutated parental methylated plasmid DNA. The mutated plasmids DNA were transformed into E. coli DH5␣ for nick repair. Introduced mutations were confirmed by sequencing each of the Twa DNA polymerase mutant genes. 2.2. Expression and purification of mutant DNA polymerases The expression plasmids were transformed into E. coli strain Rosetta(DE3)pLysS cells, and the wild-type and mutant Twa DNA polymerase genes were induced to overexpress through the addition of isopropyl--d-thiogalactopyranoside (IPTG) to a final concentration 0.2 mM at the mid-exponential growth phase (O.D.600 0.6), followed by a 12-h incubation at 25 ◦ C. Cells were harvested by centrifugation (5590 × g) at 4 ◦ C, 20 min and resuspended in buffer A (20 mM Tris–HCl buffer, pH 7.4, containing 0.5 M NaCl and 1 mM phenylmethylsulfonyl fluoride). Cells were disrupted by sonication, cellular debris was removed by centrifugation (18,700 × g) at 4 ◦ C for 15 min, and the supernatant was heat-treated for 30 min at 80 ◦ C. Following centrifugation, the heattreated supernatant was loaded onto a HisTrapTM HP column (GE Healthcare, UK) pre-equilibrated with buffer A. The column was washed with buffer A and proteins were eluted with a linear gradient of 0–500 mM imidazole in buffer B. The peak fractions containing each of the Twa DNA polymerases were pooled and dialyzed against buffer C (20 mM sodium phosphate, pH 6.0, and 0.1 M NaCl), and loaded on to a HiTrapTM SP HP column (GE Healthcare) equilibrated with buffer C and washed with the same buffer. Bound proteins were eluted with a linear gradient of 0.1 M to 1.0 M NaCl prepared in buffer C. Protein concentrations were determined by the Bradford assay (Bradford, 1976). Purified proteins were dialyzed against 20 mM Tris–HCl (pH 7.4), 0.1 mM EDTA, 0.1% Tween 20, 0.5% Nonidet P40, 50 mM KCl, 1.0 mM DTT and 50% glycerol, and stored at −20 ◦ C. Protein purity was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). 2.3. DNA polymerase activity assay The DNA polymerase activity of the purified enzyme was determined as previously described (Choi et al., 2006; Cho et al., 2012a).
S.S. Cho et al. / Journal of Biotechnology 184 (2014) 39–46
The basic reaction mixture (50 l) contained 50 mM Tris–HCl (pH 8.6), 2 mM MgCl2 , 0.01% Triton X-100, and 0.005% BSA, 100 M each of dATP, dCTP and dGTP, 10 M dTTP, 0.5 Ci of [methyl3 H]thymidine 5 -triphosphate (68 Ci/mmol, ARC), 3 g of activated calf thymus DNA, and enzyme solution. One unit of polymerase activity was defined as the amount required to catalyze the incorporation of 10 nmol of dNTPs into an acid-insoluble form at 75 ◦ C in 30 min. 2.4. Exonuclease activity assay An exonuclease activity assay was performed as previously described with slight modification (Song et al., 2007). The 3 -endlabeled DNA substrate was prepared by filling DNA linearized by XapI with Klenow fragment in the presence of [methyl-3 H] thymidine 5 -triphosphate (GE Healthcare). After labeling, the DNA substrate was purified by gel filtration, followed by ethanol precipitation. The exonuclease activity of the purified Twa DNA polymerase and mutants were analyzed in 50 l containing 50 mM Tris–HCl (pH 7.5), 14 mM MgCl2 , 80 mM KCl, 0.01% BSA, enzyme solution and end-labeled DNA substrate at 75 ◦ C for 40 min in the presence and absence of dNTPs, as described previously (Song et al., 2007). 2.5. PCR efficiency assay Fragments 2-kb in length were amplified using anchor- F and -2 R primers (Table S1) with wild-type, mutant Twa DNA polymerase (N501R, N501K, N501D, N501E and N501A) and other commercial DNA polymerase including Pfu (Promega, USA), Vent (NEB, USA) and Taq (Takara, Japan). PCR amplification was initiated with one denaturation step at 94 ◦ C for 30 s followed by 30 cycles of 94 ◦ C for 30 s and then 72 ◦ C for 10, 20, 30, 60 and 90 s using -phage DNA as a template. Each 50 l PCR mixture contained 25 ng of -phage DNA, 20 pmol of each primer, 1.0–1.5 U of DNA polymerases (an amount of 1.4 U (200 ng) of Twa, 1.25 U of Pfu (Promega, USA), 1U of Vent (NEB, USA), and 1.5 U of Taq (Takara, Japan) as defined in the manufacturer’s protocol), 1× optimal PCR buffer for Twa [50 mM Tris–HCl (pH 8.6), 2 mM MgCl2 , 0.01% Triton X-100, and 0.005% BSA] or the PCR buffer supplied by the manufacturer, and 0.2 mM of dNTPs. Plasmid pTYB1 DNA (NEB, USA) template was also used to measure PCR efficiency of the Twa wildtype, Twa N501R mutant, Pfu, Vent and Taq DNA polymerases. PCR amplification was initiated with one denaturation step at 94 ◦ C for 30 s followed by 30 cycles of 94 ◦ C for 30 s, 60 ◦ C for 30 s and 72 ◦ C for 30 s using pTYB1 plasmid DNA as a template. To test the extension efficiency of the DNA polymerases, primers (Table S1) were used to amplify DNA fragments of 2, 4, 6, 8, 10 and 12 kb from DNA in a 50 l reaction. Reaction conditions were as follows: one initial denaturation step at 94 ◦ C for 3 min followed by 30 cycles of amplification at 94 ◦ C for 30 s; 62 ◦ C, 30 s; 72 ◦ C, 1 min/kb. PCR products were analyzed by electrophoresis on a 0.8% standard agarose gel. 2.6. Extension rate assay The extension rate was determined from the product length synthesized during a fixed reaction time (Takagi et al., 1997). M13mp18 ssDNA (NEB, USA) primed with an M13 primer (5 GCATCGGAACGAGGGTAGCAACGG-3 ) was used as the substrate. Each 60-l reaction contained 1.62 ng of M13 ssDNA, 10 pmol of primer, dATP, dTTP and dGTP each at 200 M, dCTP at 50 M, 1 Ci of [␣-32 P] dCTP (3000 Ci/mmol, PerkinElmer), 0.2 g of the DNA polymerase and the optimized buffer for the polymerase. Assays were incubated at 75 ◦ C for the indicated times, then stopped by addition of an equal volume solution consisting of 60 mM EDTA
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and 60 mM NaOH. A 10-l aliquot of each sample was analyzed by agarose gel electrophoresis. 2.7. Processivity assay Processivity of the mutated DNA polymerase was measured as previously described (Kim et al., 2007). The reaction mixture, containing 400 fmol of 5 Hex-labeled M13-primer (5 GCATCGGAACGAGGGTAGCAACGG-3 ), 200 fmol of M13 ssDNA (NEB), a buffer specific for the DNA polymerase and 0.2 mM dNTP, was pre-heated at 95 ◦ C for 1 min and then incubated at 62 ◦ C for 1 min for primer annealing. Each enzyme was added at a final concentration of 4 pmol to the mixture and incubated at 75 ◦ C for 10 s. The resulting DNA fragments were analyzed on an ABI3100 automated sequencer. To ensure that multiple binding did not occur on any primer-template complex, both the polymerase concentration and the reaction time were varied and the major peak equal to the median product length was determined for each reaction (Wang et al., 2004). Processivity may be expressed as a probability (Von Hippel et al., 1994). To describe the difference in processivity between Twa and Twa N501R, we measured the probability of chain elongation per nucleotide, defined as the microscopic processivity parameter (P) (Von Hippel et al., 1994). Peaks of each enzyme were plotted on a log scale according to the method of McClure and Chow (1980). The slope of the line is equal to P and an average primer extension length was calculated from 1/1 − P. 2.8. Steady-state kinetic analysis Steady-state kinetic analyses were performed as previously described (Kong et al., 1993). Assays for Km and kcat were carried out in polymerase optimized buffers containing variable amounts of dNTPs and pre-annealed primed M13mp18 DNA template. Each reaction contained 2 pmol (180 ng) of enzyme. To determine dNTP parameters, 24 nM of M13mp18 DNA template was annealed with the primer (5 -GCATCGGAACGAGGGTAGCAACGG-3 ) by heating at 95 ◦ C for 3 min followed by slow cooling to room temperature. PCR activity was measured at seven different dNTP concentrations ranging 20–140 M. To determine DNA template parameters, dNTP concentration was each maintained at 200 M. Pre-annealed primed M13 ssDNA template was used at concentrations ranging from 1 to 12 nM. [methyl-3 H] dTTP was used to radiolabel a dNTP mix. For example, a 100 M dNTP mixture contained 100 M each of dATP, dCTP and dGTP and 10 M dTTP including 0.5 M [methyl3 H] dTTP (68 Ci/mmol, ARC). After a 3 min reaction at 75 ◦ C, an aliquot was evenly spotted onto a 23 mm diameter DE81 filterpaper disc (Whatman, UK) and the dried disc was washed and re-dried. Incorporated radioactivity was counted using a Tri-Carb Liquid Scintillation Analyzer (PerkinElmer, USA). Plots of the reaction time course were analyzed to determine initial rates and kinetic parameters were obtained using a Lineweaver–Burk plot. 2.9. Fidelity assay PCR fidelity assays were as previously described (Lee et al., 2010c). pJR2-lacZ (5.7 kb), an expression plasmid containing the entire lacZ gene, was used as the template. An 832-bp fragment containing the 5 region of the lacZ gene was amplified using primers Lac-B (5 –NNNNGGATCCAATGATAGATCCCGTCGTTTTAC–3 ) and Lac-C (5 –NNNNATCGATAATTTCACCGCCGAAAGGCGC–3 ), in which the BamHI and ClaI sites, respectively, are underlined. PCR was performed with Twa, Twa N501R, Taq and Pfu DNA polymerases, optimized buffer or the buffer supplied by the manufacturer for 3 min at 94 ◦ C; 30 cycles of 30 s at 94 ◦ C, 30 s at 60 ◦ C and 1 min at 72 ◦ C; and 10 min at 72 ◦ C. PCR products were digested with BamHI and ClaI, purified, and ligated with
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Fig. 2. Comparisons of DNA polymerase activities and PCR efficiencies. (A) Results are presented as a percentage of radioactivity incorporated by the wild-type polymerase percentage of relative incorporated radioactivity compared to wild-type. Error bars represent standard deviation calculated from three replicates. (B) Experiments for comparing PCR efficiency for 2-kb fragments. PCR condition were 94 ◦ C for 3 min followed by 30 cycles of 94 ◦ C for 30 s, 60 ◦ C for 30 s and 72 ◦ C for 60 s.
the 4.9 kb BamHI/ClaI pJR2-lacZ fragment. E. coli DH5␣ cells were transformed with ligation mix with heat shock and plated on LB agar plates containing 100 g/ml ampicillin, 0.2 mM IPTG, and 20 g/ml X-gal. Pale blue and white colonies were formed by cells transformed with mutated plasmids, while blue colonies were formed by cells containing intact plasmids. 3. Results and discussion
Fig. 3. SDS-PAGE analysis of purified wild-type and mutant Twa DNA polymerases. Electrophoresis was performed on a vertical gel of 10% polyacrylamide, and the gel that is shown was stained with Coomassie brilliant blue R-250. Lane M, low molecular mass markers (molecular masses are indicated at the left); lane 1, wildtype Twa DNA polymerase; lane 2, A381R mutant; lane 3, N501R mutant; lane 4, N501K; lane 5, N501D mutant; lane 6, N501E mutant; lane 7, N501A mutant.
polymerase activities and 3 → 5 exonuclease activities of wildtype and mutant Twa DNA polymerases were determined presented in Table 2. While substitutions with basic amino acids (Arg and Lys) at position 501 increased the polymerase activity, the substitutions with acidic amino acids decreased. The 3 → 5 exonuclease activities of the mutant enzymes did not differ from wild-type, except for that of the N501E mutant.
3.1. Design and purification of mutant Twa DNA polymerases 3.3. PCR efficiency of the wild-type and mutant DNA polymerases Based on structural data of KOD DNA polymerase (Hashimoto et al., 2001) and amino acid sequence alignment of family B DNA polymerase, the A381 and N501 residues of Twa DNA polymerase are located in the palm subdomain at the forked-point of the enzyme (Fig. 1B). Using site-directed mutagenesis, we firstly replaced A381 and N501 with arginine. The N501R mutant showed significantly improved polymerase function compared to wild-type Twa DNA polymerase in terms of activity and PCR efficiency (Fig. 2). Therefore, we produced four additional mutants by exchanging N501 for Lys, Glu, Asp and Ala respectively. Wild-type and mutant Twa DNA polymerase were expressed and then purified through heat treatment followed by using His-tag affinity (HisTrapTM HP) and cation exchange (HiTrapTM SP HP) chromatography. Wild-type and mutant Twa DNA polymerase showed the same mobilities, and their molecular masses were estimated to be 90 kDa (Fig. 3). As a result, six mutant proteins (A381R, N501R, N501K, N501D, N501E and N501A) were obtained for assays. 3.2. Activities of mutant DNA polymerases The DNA polymerase activities of the wild-type and mutant enzymes were initially compared by measuring radioactivity incorporated into DNA and expressing this as a percentage of wild-type incorporation (Fig. 2A). Activity of the Twa A381R mutant (99.8%) was similar to that of wild-type polymerase and activity of the Twa N501R mutant (114.9%) was higher. PCR efficiency, tested using 2-kb lambda DNA fragments, was substantially higher with the N501R mutant than with the wild-type, A381R mutant polymerases, as expected (Fig. 2B). These findings indicate that the charge of the amino acid at position 501 significantly influences PCR efficiency. To verify this, we tested the four other mutants obtained by exchanging N501 for a basic amino acid (Lys), acidic amino acids (Glu and Asp), a neutral amino acid (Ala). The
To test PCR efficiency of wild-type and N501 mutants, 2-kb lambda DNA fragments were amplified using each polymerase for various extension times without annealing times. The wild-type only amplified a 2-kb target gene with a 60 s extension time, but the N501R, N501K and N501A mutants successfully amplified a 2-kb target gene with a 10 s extension time (Fig. 4A). The N501R and N501K mutants were also quantitatively more productive than either the wild-type or any other mutant. In comparing the amplification rates of mutant Twa DNA polymerases and other commercial DNA polymerase, we used the Twa N501R mutant because the DNA polymerase activity of the N501R mutant was higher than that of the N501K mutant (Table 2). To measure these amplification rates, lambda DNA targets were amplified using selected extension times (5, 10, 30 and 60 s). The amplification rate of the Twa N501R mutant exceeded rates of Pfu and Taq DNA polymerases (Fig. 4B). Moreover, using pTYB1 plasmid targets (1–4 kb), the Twa N501R mutant was able to amplify a 4-kb target in a 30 s extension time (Fig. 4C). Reportedly, the high fidelity DNA polymerase does not maintain long-range accuracy (LA) in PCR because of their strong associated exonuclease activity (Barnes, 1994). High accuracy in the Table 2 Comparison of the polymerase activities and 3 → 5 exonuclease activities of wildtype and mutated Twa DNA polymerases. DNA polymerase
Relative polymerase activity (%)
WT N501R N501K N501D N501E N501A
100 114.9 113.2 72.6 79.6 85.3
± ± ± ± ± ±
1.7 0.9 1.2 2.0 2.5 3.3
Relative 3 → 5 exonuclease activity (%) 100 101.3 100.7 101 87.5 96.2
± ± ± ± ± ±
2.4 3.2 2.3 2.1 3.8 3.9
S.S. Cho et al. / Journal of Biotechnology 184 (2014) 39–46
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Fig. 4. Comparison of the wild-type and mutant Twa DNA polymerases, Pfu, Vent and Taq in time-saving PCR. (A) Comparison of the wild-type and mutant Twa DNA polymerases. Extension times used for PCR amplification of a 2-kb fragment are indicated top each lane. (B) Comparison of the wild-type, Twa N501R, Pfu, Vent and Taq DNA polymerases. (C) pTYB1 plasmid DNA was used as the template and amplicon sizes are indicated top of lanes. PCR condition were 94 ◦ C for 3 min followed by 30 cycles of 94 ◦ C for 30 s and 72 ◦ C for 30 s. Lane M, GeneRuler 1 kb DNA Ladder (Thermo Scientific #SM0311).
amplification of long sequences is increasingly important for current PCR applications (Nishioka et al., 2001). In comparison of extension efficiencies, the Twa DNA polymerase could amplify DNA an 8-kb DNA fragment, but the Twa N501R DNA polymerase could amplify a 10-kb DNA fragment (Fig. 5). Thus the N501R mutant displayed a significant improvement in PCR extension efficiency.
rates of the wild-type and Twa N501R DNA polymerase, based on the length of DNA synthesized in each fixed time window (30, 60, 90 and 120 s). By this measure, the extension rate was determined as about 6 nt/s for Twa DNA polymerase and about 11 nt/s for Twa N501R DNA polymerase. Twa N501R DNA polymerase was 2-fold faster than Twa DNA polymerase (Fig. 6).
3.4. Extension rates
3.5. Processivity
The specific extension rate represents the number of dNTPs polymerized per second per molecule of DNA polymerase. Extension rates depend notably on the reaction media and DNA templates (Andrey et al., 2004). To understand the basis for improved PCR performance of the N501R DNA polymerase, we compared extension
The concept of processivity was introduced as the probability that, following attachment of a nucleotide, a polymerase will not dissociate from the DNA when translocating to the next position (Fairfield et al., 1983). Like the extension rate, processivity depends on the reaction medium and DNA templates (Andrey et al.,
Fig. 5. Comparison of wild-type and Twa N501R DNA polymerase in long-range PCR. DNA was used as the template and amplicon sizes are indicated top each lane. Extension times used for PCR amplification were 1 min/kb. Lane M, GeneRuler 1 kb DNA Ladder (Thermo Scientific #SM0311).
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S.S. Cho et al. / Journal of Biotechnology 184 (2014) 39–46 Table 3 Summary of the processivity parameters.
Twa Twa N501R
Microscopic processivity (P)
Average primer extension length (nt) [1/(1 − P)]
0.9636 ± 0.004 0.9874 ± 0.001
27 ± 3 79 ± 7
Table 4 Summary of the steady-state kinetics analysis. Enzyme
Fig. 6. Comparison of extension rates wild-type and Twa N501R DNA polymerase. The extension rates of the wild-type and Twa N501R DNA polymerase were calculated from the length of DNA synthesized versus each extension time indicated at the top of the figure.
Twa Twa N501R KOD a b
2004). To analyze the superior PCR performance of the mutant Twa DNA polymerases, a processivity assay was performed. In electropherogram trace for wild-type and N501R DNA polymerases (Fig. 7A), peaks correspond to single primer extension products. Single primer extension products in the Fig. 7A are plotted as long (Ant/At) versus the length of extended primer and fitted by linear regression (Fig. 7B). Each value of processivity (P) was obtained from the slope. The processivity factors are summarized in Table 3. Wild-type exhibits a processivity (P) of 0.9636, which correlates to
DNAa
dNTPb −1
Km (nM)
Kcat (s
)
10.3 ± 0.5 6.6 ± 0.3 0.3 ± 0.1
2.6 ± 0.2 2.3 ± 0.16 0.8 ± 0.1
Km (M)
Kcat (s−1 )
0.3 ± 0.02 0.3 ± 0.02 0.5 ± 0.02
3.9 ± 0.3 4.5 ± 0.3 20.3 ± 0.5
Moles of template, in the presence of an excess of annealed primer. Moles of each nucleotide in an equimolar mixture of the four nucleotides.
an average primer extension length of 27 nt. In contrast, the Twa N501R DNA polymerase exhibits higher processivity with a P of 0.9874 and an average extension length 79 nt. Pfu DNA polymerase of lysine at residue 501 exhibits a P of 0.9777, which correlates to an average primer extension length of 45 nt (Kim et al., 2007). Thus, substitution of arginine at residue 501 increased the processivity of the DNA polymerase.
Fig. 7. Processivity analysis of wild-type and Twa N501R DNA polymerase. (A) Peaks in electropherogram traces of Twa and Twa N501R DNA polymerases correspond to single primer extension products. (B) Single primer extension products in the Fig. 7A are plotted as long (Ant /AT ) versus the length of extended primer and fitted by linear regression. Ant is equal to the total extended fragments greater than each product length which is the defined length of zero. AT is the sum of the length of extended primers (Bibillo and Eickbush, 2002).
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Table 5 Comparison of Taq, Pfu, Twa and Twa N501R DNA polymerase fidelities. Number of colonies
Blue Taq Pfu Twa Twa 501R
7690 7753 7404 8271
Mutation frequencya
Template doublingsb
Error ratec (× 10−5 )
Fold improvement over Taq
White and Pale blue ± ± ± ±
148 169 143 154
1398 403 267 390
± ± ± ±
35 21 20 23
0.15 0.05 0.03 0.05
± ± ± ±
0.04 0.001 0.006 0.003
7.5 6.5 6.2 7.5
± ± ± ±
0.2 0.3 0.2 0.3
2.45 0.91 0.67 0.72
± ± ± ±
0.5 0.008 0.006 0.02
1.0 2.7 3.6 3.4
a
Mutation frequency was expressed as the proportion of mutant colonies relative to the total number of colonies. Template doublings were calculated using the equation 2d = (amount of PCR product)/(amount of starting target) c Error rate was determined using the equation ER = mf/(bp × d), where mf is the mutation frequency, bp is the lacZ target size (832 bp), and d is the number of template doublings. b
3.6. Determination of kinetic constants
4. Conclusion
To study the effects of mutation of N501 on the catalytic rate of nucleotide incorporation, steady-state kinetic parameters were determined for wild-type, Twa N501R and KOD DNA polymerases. The rate of primer extension was measured at different primer-template concentrations in the presence of a fixed concentration (0.2 mM) of dNTPs and at different dNTPs concentrations in the presence of a fixed concentration (0.24 nM) of primer template. Plots of the reaction time course were analyzed to determine initial rates and kinetic parameters were obtained using a Lineweaver–Burk plot. Interestingly, the Twa N501R DNA polymerase had a 1.5-fold lower Km (DNA) value compared to wild-type and a nearly identical Km (dNTP) value compared to wild-type. However Twa and Twa N501R DNA polymerases have nearly identical turnover rates (kcat ) (Table 4). The R501 residue of KOD DNA polymerase, which correspond to the R501 residue of Twa N501R DNA polymerase are located in the palm subdomain at the forkedpoint of the enzyme (Hashimoto et al., 2001) (Fig. 1). The KOD DNA polymerase had also a 22-fold lower Km (DNA) value compared to Twa N501R DNA polymerase and a nearly identical Km (dNTP) value compared to Twa N501R DNA polymerase. Although Km is a direct measurement of the equilibrium binding of these enzymes to DNA, the difference observed is consistent with the notion that the mutant N501R stabilizes interactions between the polymerase domain and the DNA template (Wang et al., 2004). The results from the steady state kinetic analysis demonstrate that, while the N501R mutant significantly increases PCR efficiency and processivity, it does not negatively effect on the catalytic activity of the polymerase domain.
In this work, we have focused on two amino acid residues (A381 and N501) in forked-point of Twa DNA polymerase. These two residues were replaced by site-directed mutagenesis and the enzymatic properties of the mutants were analyzed. Here, Twa N501R DNA polymerase showed significantly improved polymerase function compared to wild-type Twa DNA polymerase in terms of processivity (3-fold), extension rate (2-fold) and PCR efficiency. Kinetic analysis using DNA as template revealed that positive charge of N501R exhibits a stabilizing effect on the interactions between the polymerase domain and the DNA template. These results clearly demonstrate that the Twa N501R DNA polymerase showed a significant improvement in PCR efficiency without loss of wild-type fidelity. Twa N501R DNA polymerase might be useful in high-fidelity DNA amplification and various PCR-based applications. Acknowledgement This work was supported by the Marine and Extreme Genome Research Center Program of the Ministry of Maritime Affairs and Fisheries, Republic of Korea. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jbiotec.2014. 05.007. References
3.7. PCR fidelity assay Fidelity is the frequency of correct nucleotide insertion per incorrect insertion and an inherent property of DNA polymerase (Pavlov et al., 2002). The use of high-fidelity DNA polymerase is necessary to reduce errors in PCR amplification during cloning and site-directed mutagenesis (Zheng et al., 2001). The fidelity of Twa N501R DNA polymerase was compared with wild-type, Taq, Pfu DNA polymerases by measuring the error frequency of each enzyme in a lacZ forward mutation assay. Wild-type showed a high fidelity with an error rate of 0.67 × 10−5 and Twa N501R DNA polymerase exhibited an error rate of 0.72 × 10−5 (Table 5). The fidelity of Twa N501R DNA polymerase was approximately 3.4-fold higher than Taq DNA polymerase and 1.2-fold higher than Pfu DNA polymerase. Thus, substitution of arginine at residue 501 does not negatively effect on fidelity of Twa DNA polymerase. These enzymes had excellent fidelity and are thus attractive for molecular biology applications.
Andrey, P., Nadejda, V.P., Sergei, A.K., Alexei, I.S., 2004. Recent developments in the optimization of thermostable DNA polymerase for efficient applications. Trends Biotechnol. 22, 253–260. Barnes, W.M., 1994. PCR amplification of up to 35-kb DNA with high fidelity and high yield from bacteriophage templates. Proc. Natl. Acad. Sci. U.S.A. 91, 2216–2220. Bibillo, A., Eickbush, T.H., 2002. High processivity of the reverse transcriptase from a non-long terminal repeat retrotransposon. J. Biol. Chem. 277, 34836–34845. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Cho, S.S., Kim, K.P., Lee, K.K., Youn, M.H., Kwon, S.T., 2012a. Characterization and PCR application of a new high-fidelity DNA polymerase from Thermococcus waiotapuensis. Enzyme Microb. Technol. 51, 334–341. Cho, S.S., Sun, Y., Yu, M., Kwon, S.H., Kwon, S.T., 2012b. Characterization and PCR application of dUTPase from hyperthermophilic euryarchaeon Thermococcus pacificus. Enzyme Microb. Technol. 51, 342–347. Choi, J.J., Nam, K.H., Min, B., Kim, S.J., Söll, D., Kwon, S.T., 2006. Protein transsplicing and characterization of a split family B-type DNA polymerase from the hyperthermophilic archaeal parasite Nanoarchaeum equitans. J. Mol. Biol. 356, 1093–1106. Fairfield, F.R., Newport, J.W., Dolejsi, M.K., von Hippel, P.H., 1983. On the processivity of DNA replication. J. Biomol. Struct. Dyn. 1, 715–727.
46
S.S. Cho et al. / Journal of Biotechnology 184 (2014) 39–46
Hashimoto, H., Nishioka, M., Fujiwara, S., Takagi, M., Imanaka, T., Inoue, T., Kai, Y., 2001. Crystal structure of DNA polymerase from hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1. J. Mol. Biol. 306, 469–477. Hogrefe, H.H., Hansen, C.J., Scott, B.R., Nelson, K.B., 2001. Archaeal dUTPase enhances PCR amplifications with archaeal DNA polymerases by preventing dUTP incorporation. Proc. Natl. Acad. Sci. U.S.A. 99, 596–601. Hopfner, K.P., Eichinger, A., Engh, R.A., Laue, F., Ankenbauer, W., Huber, R., Angerer, B., 1999. Crystal structure of a thermostable type B DNA polymerase from Thermococcus gorgonarius. Proc. Natl. Acad. Sci. U.S.A. 96, 3600–3605. Kim, Y.J., Lee, H.S., Bae, S.S., Jeon, J.H., Lim, J.K., Cho, Y., Nam, K.H., Kang, S.G., Kim, S.J., Kwon, S.T., Lee, J.-H., 2007. Cloning, purification, and characterization of a new DNA polymerase from a hyperthermophilic archaeon, Thermococcus sp. NA1. J. Microbiol. Biotechnol. 17, 1090–1097. Kong, H., Kucera, R.B., Jack, W.E., 1993. Characterization of a DNA polymerase from the hyperthermophile archaea Thermococcus litoralis. Vent DNA polymerase, steady state kinetics, thermal stability, processivity, strand displacement, and exonuclease activities. J. Biol. Chem. 268, 1965–1975. Lee, J.I., Cho, S.S., Kil, E.J., Kwon, S.T., 2010a. Characterization and PCR application of a thermostable DNA polymerase from Thermococcus pacificus. Enzyme Microb. Technol. 47, 147–152. Lee, J.H., Kang, S.G., Kim, S.J., Kwon, K.K., Lee, H.S., Kim, Y.J., Ryu, Y.G., Bae, S.S., Lim, J.K., Jeon, J.H., Cho, Y.N., 2010b. Mutant DNA polymerases and their genes from Thermococcus. US Patent Publication No. 2010/0297706 A1. Lee, J.I., Kim, Y.J., Bae, H., Cho, S.S., Lee, J.H., Kwon, S.T., 2010c. Biochemical properties and PCR performance of a family B DNA polymerase from hyperthermophilic euryarchaeon Thermococcus peptonophilus. Appl. Biochem. Biotechnol. 160, 1585–1599. Lundberg, K.S., Shoemaker, D.D., Adams, M.W., Short, J.M., Sorge, J.A., Mathur, E.J., 1991. High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus. Gene 108, 1–6. Mattila, P., Korpela, J., Tenkanen, T., Pitkanen, K., 1991. Fidelity of DNA synthesis by the Thermococcus litoralis DNA polymerase, an extremely heat stable enzyme with proofreading activity. Nucleic Acids Res. 19, 4967–4973. McClure, W.R., Chow, Y., 1980. The kinetics and processivity of nucleic acid polymerase. Methods Enzymol. 64, 277–297.
Nishioka, M., Mizuguchi, H., Fujiwara, S., Komatsubara, S., Kitabayashi, M., Uemura, H., Takagi, M., Imanaka, T., 2001. Long and accurate PCR with a mixture of KOD DNA polymerase and its exonuclease deficient mutant enzyme. J. Biotechnol. 88, 141–149. Pavlov, A.R., Belova, G.I., Kozyavkin, S.A., Slesarev, A.I., 2002. Helix-hairpin-helix motifs confer salt resistance and processivity on chimeric DNA polymerase. Proc. Natl. Acad. Sci. U.S.A. 99, 13510–13515. Rodriguez, A.C., Park, H.W., Mao, C., Beese, L.S., 2000. Crystal structure of a pol ␣ family DNA polymerase from the hyperthermophilic archaeon Thermococcus sp. 9◦ N-7. J. Mol. Biol. 299, 447–462. Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B., Erlich, H.A., 1988. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487–491. Song, J.M., Choi, J.J., Kim, T.O., Seo, M.S., Lee, M.S., Kim, H.K., Kwon, S.T., 2007. Characterization and PCR performance of a family B-type DNA polymerase from the hyperthermophilic crenarchaeon Staphylothermus marinus. Enzyme Microb. Technol. 40, 1475–1483. Song, J.G., Kil, E.J., Cho, S.S., Kim, I.H., Kwon, S.T., 2010. An amino acid residue in the middle of the fingers subdomain is involved in Neq DNA polymerase processivity: enhanced processivity of engineered Neq DNA polymerase and its PCR application. Protein Eng. Des. Sel. 23, 835–842. Takagi, M., Nishioka, M., Kakihara, H., Kitabayashi, M., Inoue, H., Kawakami, B., Oka, M., Imanaka, T., 1997. Characterization of DNA polymerase from Pyrococcus sp. strain KOD1 and its application to PCR. Appl. Environ. Micobiol. 63, 4504–4510. Von Hippel, P.H., Fairfield, F.R., Dolejsi, M.K., 1994. On the processivity of polymerases. Ann. N. Y. Acad. Sci. 726, 118–131. Wang, Y., Dennis, E.P., Li, M., John, C.S., Michael, F., Peter, B.V.H., 2004. A novel strategy to engineer DNA polymerase for enhanced processivity and improved performance in vitro. Nucleic Acids Res. 32, 1197–1207. Zheng, R., Matsui, E., Shen, Y., Musti, K.V., Feng, Y., Darnis, S., Kawarabayasi, Y., Kikuchi, H., Harata, K., Matsui, I., 2001. The novel function of a short region K253 XRXXXD259 conserved in the exonuclease domain of hyperthermostable DNA polymerase I from Pyrococcus horikoshii. Extremophiles 5, 111–117.
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Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Mutations in the palm subdomain of Twa DNA polymerase to enhance PCR efficiency and its function analysis Sung Suk Cho, Mi Yu, Suk-Tae Kwon ∗ Department of Genetic Engineering, Sungkyunkwan University, 300 Chunchun-dong, Jangan-gu, Suwon 440-746, Republic of Korea
a r t i c l e
i n f o
Article history: Received 26 December 2013 Received in revised form 19 April 2014 Accepted 12 May 2014 Available online 24 May 2014 Keywords: Thermococcus waiotapuensis (Twa) Twa DNA polymerase Twa N501R DNA polymerase PCR amplification Forked-point
a b s t r a c t Among the family B DNA polymerases, the Twa DNA polymerase from T. wiotapuensis, a hyperthermophilic archaeon, has exceedingly high fidelity. For applications in PCR, however, the enzyme is limited by its low extension rate and processivity. To resolve these weaknesses, we focused on two amino acid residues (A381 and N501) located at the palm subdomain of Twa DNA polymerase. Following replacement of these residues by site-directed mutagenesis, Twa N501R DNA polymerase showed significantly improved polymerase function compared to the wild-type enzyme in terms of processivity (3-fold), extension rate (2-fold) and PCR efficiency. Kinetic analysis using DNA as template revealed that the kcat value of the Twa N501R mutant was similar to that of wild-type, but the Km of the Twa N501R mutant was about 1.5-fold lower than that of the wild-type. These results suggest that a positive charge at residue 501 located in the forked-point does not impede catalytic activity of the polymerase domain but stabilizes interactions between the polymerase domain and the DNA template. © 2014 Elsevier B.V. All rights reserved.
1. Introduction DNA polymerases are widely used as in vitro applications and some of them are well studied because of their academic and scientific importance. In particular, use of thermostable Taq DNA polymerase from Thermus aquaticus in PCR has revolutionized molecular biology (Saiki et al., 1988). Recently, the family B DNA polymerases from hyperthermophilic archaea (Pyrococcus furiosus, Thermococcus litoralis) have gained attention for their high fidelity in PCR based on their strong 3 → 5 exonuclease (proof-reading) activity (Lundberg et al., 1991; Mattila et al., 1991). However, these DNA polymerases generally exhibit lower processivity and extension rates than Taq DNA polymerase (Barnes, 1994). An improved processivity of DNA polymerase was reportedly achieved through fusion of the ds DNA-binding protein identified in Sulfolobus solfataricus (Sso7d) to the thermostable polymerase (Wang et al., 2004; Lee et al., 2010a) and fusion of multiple helix-hairpin-helix motifs identified in DNA topoisomerase V (Pavlov et al., 2002). In alternative different approaches, an auxiliary enzyme such as dUTPase may be added to the PCR assay (Hogrefe et al., 2001; Cho et al., 2012b) and mutant polymerases may be obtained via site-directed mutagenesis (Song et al., 2010; Lee et al., 2010b).
∗ Corresponding author. Tel.: +82 31 290 7863; fax: +82 31 290 7870. E-mail address: [email protected] (S.-T. Kwon). http://dx.doi.org/10.1016/j.jbiotec.2014.05.007 0168-1656/© 2014 Elsevier B.V. All rights reserved.
Most family B DNA polymerases from hyperthermophilic archaea consist of several distinct domains, including an N-terminal domain, exonuclease domain and a polymerase domain with palm, fingers and thumb subdomains (Hopfner et al., 1999; Rodriguez et al., 2000; Hashimoto et al., 2001). The arginine residues (R243, R247, R264, R266, R346, R381 and R501) of the exonuclease and palm subdomains of the forked-point in KOD DNA polymerase provide a basic environment (Fig. 1A). Structural studies suggested that basic residues interact with the phosphate group of the DNA strand and stabilize the melted DNA strands at the forked-point (Hashimoto et al., 2001). Several arginine residues at the forked-point in most family B DNA polymerases from hyperthermophilic archaea are conserved (Hashimoto et al., 2001). KOD DNA polymerase exhibits a five-fold higher extension rate (100–130 nucleotides/s) and 10–15-fold higher processivity (>300 bases) than Pfu DNA polymerase (Takagi et al., 1997). However, the fidelity of KOD DNA polymerase was similar to that of Pfu DNA polymerase. It is considered that the substitution of arginine residues around the forked-point may affect the difference of the enzymatic characteristics between KOD and Pfu DNA polymerases (Hashimoto et al., 2001). In the forked-point of Pfu DNA polymerase, R247, R265 and R501 residues are replaced by M247, T265 and K502 residue, respectively. Biochemical characterization of the T. waiotapuensis (Twa) DNA polymerase (Cho et al., 2012a) revealed the high fidelity of this enzyme associated with its 3 → 5 exonuclease-dependent proofreading activity. The fidelity of Twa DNA polymerase was higher
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Fig. 1. (A) Tertiary structure of KOD DNA polymerase model showing the arginine residues in forked-point (PBD ID: 1WNS) (Hashimoto et al., 2001). Tertiary structure was shown using the 3D Molecule Viewer program of Vector NTI AdvanceTM 9.1 (Invitrogen, USA). (B) Amino acid sequence alignment of archaeal family B DNA polymerase. Multiple alignments were produced using the AlignX software (Invitrogen, USA): Twa (T. waiotapuensis, GeneBank accession no. JQ399904), KOD1 (P. kodakaraensis KOD1, GeneBank accession no. D29671), Pfu (P. furiosus, GeneBank accession no. D12983), 9◦ N-7 (Thermococcus sp. 9◦ N-7 GeneBank accession no. AAA88769), Tgo (T. gorgonarius, GeneBank accession no. P56689). Shaded amino acid residues indicate identical and conserved residues matched with basic residues of fork-point.
than that of other family B DNA polymerase, including the Pfu, KOD and Vent. However, Twa DNA polymerase has a lower extension rate and processivity than Taq DNA polymerase (Cho et al., 2012a). In the Twa DNA polymerase, the R381 and R501 residues in the forked-point of KOD DNA polymerase are replaced by A381 and N501 residues, respectively (Fig. 1B). This replacements of Twa DNA polymerase may have low extension rate and processivity as above described. Therefore, we replaced the two amino acid residues (A381 and N501) located at the forked-point of Twa DNA polymerase with other amino acid residues using site-directed mutagenesis. The mutant Twa N501R DNA polymerase showed significantly improved polymerase function compared to wild-type Twa DNA polymerase in terms of processivity, extension rate and PCR efficiency. 2. Materials and methods 2.1. Construction of mutated Twa DNA polymerase genes Point mutations were introduced into the wild-type Twa DNA polymerase gene using the QuikChange site-directed mutagenesis method (Stratagen, USA) with the Twa DNA polymerase gene subcloned into pET-20b(+) as a template (Cho et al., 2012a). The primers used for mutations at specific residues are listed in Table 1. Table 1 The positions of amino acid substitution and the primer sequences for mutation. Name of mutants
Name of primers
Mutation primer sequence
A381R
F-A381R R-A381R F-N501R R-N501R F-N501A R-N501A F-N501D R-N501D F-N501E R-N501E F-N501K R-N501K
5 -GAGTTAGCAAGGAGACGGGAGAGCTACGCG-3 5 -CGCGTAGCTCTCCCGTCTCCTTGCTAACTC-3 5 -CTACGGCTACGCACGTGCCCGCTGG-3 5 -CCAGCGGGCACGTGCGTAGCCGTAG-3 5 -CTACGGCTACGCAGCTGCCCGCTGG-3 5 -CCAGCGGGCAGCTGCGTAGCCGTAG-3 5 -CTACGGCTACGCAGATGCCCGCTGG-3 5 -CCAGCGGGCATCTGCGTAGCCGTAG-3 5 -CTACGGCTACGCAGAAGCCCGCTGG-3 5 -CCAGCGGGCTTCTGCGTAGCCGTAG-3 5 -CTACGGCTACGCAAAGGCCCGCTGG-3 5 -CCAGCGGGCCTTTGCGTAGCCGTAG -3
N501R N501A N501D N501E N501K
–, The underlined triplets signify the mutated codons.
All of the mutant plasmid DNA was synthesized by PCR amplification, and subsequently digested with the restriction enzyme DpnI to remove unmutated parental methylated plasmid DNA. The mutated plasmids DNA were transformed into E. coli DH5␣ for nick repair. Introduced mutations were confirmed by sequencing each of the Twa DNA polymerase mutant genes. 2.2. Expression and purification of mutant DNA polymerases The expression plasmids were transformed into E. coli strain Rosetta(DE3)pLysS cells, and the wild-type and mutant Twa DNA polymerase genes were induced to overexpress through the addition of isopropyl--d-thiogalactopyranoside (IPTG) to a final concentration 0.2 mM at the mid-exponential growth phase (O.D.600 0.6), followed by a 12-h incubation at 25 ◦ C. Cells were harvested by centrifugation (5590 × g) at 4 ◦ C, 20 min and resuspended in buffer A (20 mM Tris–HCl buffer, pH 7.4, containing 0.5 M NaCl and 1 mM phenylmethylsulfonyl fluoride). Cells were disrupted by sonication, cellular debris was removed by centrifugation (18,700 × g) at 4 ◦ C for 15 min, and the supernatant was heat-treated for 30 min at 80 ◦ C. Following centrifugation, the heattreated supernatant was loaded onto a HisTrapTM HP column (GE Healthcare, UK) pre-equilibrated with buffer A. The column was washed with buffer A and proteins were eluted with a linear gradient of 0–500 mM imidazole in buffer B. The peak fractions containing each of the Twa DNA polymerases were pooled and dialyzed against buffer C (20 mM sodium phosphate, pH 6.0, and 0.1 M NaCl), and loaded on to a HiTrapTM SP HP column (GE Healthcare) equilibrated with buffer C and washed with the same buffer. Bound proteins were eluted with a linear gradient of 0.1 M to 1.0 M NaCl prepared in buffer C. Protein concentrations were determined by the Bradford assay (Bradford, 1976). Purified proteins were dialyzed against 20 mM Tris–HCl (pH 7.4), 0.1 mM EDTA, 0.1% Tween 20, 0.5% Nonidet P40, 50 mM KCl, 1.0 mM DTT and 50% glycerol, and stored at −20 ◦ C. Protein purity was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). 2.3. DNA polymerase activity assay The DNA polymerase activity of the purified enzyme was determined as previously described (Choi et al., 2006; Cho et al., 2012a).
S.S. Cho et al. / Journal of Biotechnology 184 (2014) 39–46
The basic reaction mixture (50 l) contained 50 mM Tris–HCl (pH 8.6), 2 mM MgCl2 , 0.01% Triton X-100, and 0.005% BSA, 100 M each of dATP, dCTP and dGTP, 10 M dTTP, 0.5 Ci of [methyl3 H]thymidine 5 -triphosphate (68 Ci/mmol, ARC), 3 g of activated calf thymus DNA, and enzyme solution. One unit of polymerase activity was defined as the amount required to catalyze the incorporation of 10 nmol of dNTPs into an acid-insoluble form at 75 ◦ C in 30 min. 2.4. Exonuclease activity assay An exonuclease activity assay was performed as previously described with slight modification (Song et al., 2007). The 3 -endlabeled DNA substrate was prepared by filling DNA linearized by XapI with Klenow fragment in the presence of [methyl-3 H] thymidine 5 -triphosphate (GE Healthcare). After labeling, the DNA substrate was purified by gel filtration, followed by ethanol precipitation. The exonuclease activity of the purified Twa DNA polymerase and mutants were analyzed in 50 l containing 50 mM Tris–HCl (pH 7.5), 14 mM MgCl2 , 80 mM KCl, 0.01% BSA, enzyme solution and end-labeled DNA substrate at 75 ◦ C for 40 min in the presence and absence of dNTPs, as described previously (Song et al., 2007). 2.5. PCR efficiency assay Fragments 2-kb in length were amplified using anchor- F and -2 R primers (Table S1) with wild-type, mutant Twa DNA polymerase (N501R, N501K, N501D, N501E and N501A) and other commercial DNA polymerase including Pfu (Promega, USA), Vent (NEB, USA) and Taq (Takara, Japan). PCR amplification was initiated with one denaturation step at 94 ◦ C for 30 s followed by 30 cycles of 94 ◦ C for 30 s and then 72 ◦ C for 10, 20, 30, 60 and 90 s using -phage DNA as a template. Each 50 l PCR mixture contained 25 ng of -phage DNA, 20 pmol of each primer, 1.0–1.5 U of DNA polymerases (an amount of 1.4 U (200 ng) of Twa, 1.25 U of Pfu (Promega, USA), 1U of Vent (NEB, USA), and 1.5 U of Taq (Takara, Japan) as defined in the manufacturer’s protocol), 1× optimal PCR buffer for Twa [50 mM Tris–HCl (pH 8.6), 2 mM MgCl2 , 0.01% Triton X-100, and 0.005% BSA] or the PCR buffer supplied by the manufacturer, and 0.2 mM of dNTPs. Plasmid pTYB1 DNA (NEB, USA) template was also used to measure PCR efficiency of the Twa wildtype, Twa N501R mutant, Pfu, Vent and Taq DNA polymerases. PCR amplification was initiated with one denaturation step at 94 ◦ C for 30 s followed by 30 cycles of 94 ◦ C for 30 s, 60 ◦ C for 30 s and 72 ◦ C for 30 s using pTYB1 plasmid DNA as a template. To test the extension efficiency of the DNA polymerases, primers (Table S1) were used to amplify DNA fragments of 2, 4, 6, 8, 10 and 12 kb from DNA in a 50 l reaction. Reaction conditions were as follows: one initial denaturation step at 94 ◦ C for 3 min followed by 30 cycles of amplification at 94 ◦ C for 30 s; 62 ◦ C, 30 s; 72 ◦ C, 1 min/kb. PCR products were analyzed by electrophoresis on a 0.8% standard agarose gel. 2.6. Extension rate assay The extension rate was determined from the product length synthesized during a fixed reaction time (Takagi et al., 1997). M13mp18 ssDNA (NEB, USA) primed with an M13 primer (5 GCATCGGAACGAGGGTAGCAACGG-3 ) was used as the substrate. Each 60-l reaction contained 1.62 ng of M13 ssDNA, 10 pmol of primer, dATP, dTTP and dGTP each at 200 M, dCTP at 50 M, 1 Ci of [␣-32 P] dCTP (3000 Ci/mmol, PerkinElmer), 0.2 g of the DNA polymerase and the optimized buffer for the polymerase. Assays were incubated at 75 ◦ C for the indicated times, then stopped by addition of an equal volume solution consisting of 60 mM EDTA
41
and 60 mM NaOH. A 10-l aliquot of each sample was analyzed by agarose gel electrophoresis. 2.7. Processivity assay Processivity of the mutated DNA polymerase was measured as previously described (Kim et al., 2007). The reaction mixture, containing 400 fmol of 5 Hex-labeled M13-primer (5 GCATCGGAACGAGGGTAGCAACGG-3 ), 200 fmol of M13 ssDNA (NEB), a buffer specific for the DNA polymerase and 0.2 mM dNTP, was pre-heated at 95 ◦ C for 1 min and then incubated at 62 ◦ C for 1 min for primer annealing. Each enzyme was added at a final concentration of 4 pmol to the mixture and incubated at 75 ◦ C for 10 s. The resulting DNA fragments were analyzed on an ABI3100 automated sequencer. To ensure that multiple binding did not occur on any primer-template complex, both the polymerase concentration and the reaction time were varied and the major peak equal to the median product length was determined for each reaction (Wang et al., 2004). Processivity may be expressed as a probability (Von Hippel et al., 1994). To describe the difference in processivity between Twa and Twa N501R, we measured the probability of chain elongation per nucleotide, defined as the microscopic processivity parameter (P) (Von Hippel et al., 1994). Peaks of each enzyme were plotted on a log scale according to the method of McClure and Chow (1980). The slope of the line is equal to P and an average primer extension length was calculated from 1/1 − P. 2.8. Steady-state kinetic analysis Steady-state kinetic analyses were performed as previously described (Kong et al., 1993). Assays for Km and kcat were carried out in polymerase optimized buffers containing variable amounts of dNTPs and pre-annealed primed M13mp18 DNA template. Each reaction contained 2 pmol (180 ng) of enzyme. To determine dNTP parameters, 24 nM of M13mp18 DNA template was annealed with the primer (5 -GCATCGGAACGAGGGTAGCAACGG-3 ) by heating at 95 ◦ C for 3 min followed by slow cooling to room temperature. PCR activity was measured at seven different dNTP concentrations ranging 20–140 M. To determine DNA template parameters, dNTP concentration was each maintained at 200 M. Pre-annealed primed M13 ssDNA template was used at concentrations ranging from 1 to 12 nM. [methyl-3 H] dTTP was used to radiolabel a dNTP mix. For example, a 100 M dNTP mixture contained 100 M each of dATP, dCTP and dGTP and 10 M dTTP including 0.5 M [methyl3 H] dTTP (68 Ci/mmol, ARC). After a 3 min reaction at 75 ◦ C, an aliquot was evenly spotted onto a 23 mm diameter DE81 filterpaper disc (Whatman, UK) and the dried disc was washed and re-dried. Incorporated radioactivity was counted using a Tri-Carb Liquid Scintillation Analyzer (PerkinElmer, USA). Plots of the reaction time course were analyzed to determine initial rates and kinetic parameters were obtained using a Lineweaver–Burk plot. 2.9. Fidelity assay PCR fidelity assays were as previously described (Lee et al., 2010c). pJR2-lacZ (5.7 kb), an expression plasmid containing the entire lacZ gene, was used as the template. An 832-bp fragment containing the 5 region of the lacZ gene was amplified using primers Lac-B (5 –NNNNGGATCCAATGATAGATCCCGTCGTTTTAC–3 ) and Lac-C (5 –NNNNATCGATAATTTCACCGCCGAAAGGCGC–3 ), in which the BamHI and ClaI sites, respectively, are underlined. PCR was performed with Twa, Twa N501R, Taq and Pfu DNA polymerases, optimized buffer or the buffer supplied by the manufacturer for 3 min at 94 ◦ C; 30 cycles of 30 s at 94 ◦ C, 30 s at 60 ◦ C and 1 min at 72 ◦ C; and 10 min at 72 ◦ C. PCR products were digested with BamHI and ClaI, purified, and ligated with
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Fig. 2. Comparisons of DNA polymerase activities and PCR efficiencies. (A) Results are presented as a percentage of radioactivity incorporated by the wild-type polymerase percentage of relative incorporated radioactivity compared to wild-type. Error bars represent standard deviation calculated from three replicates. (B) Experiments for comparing PCR efficiency for 2-kb fragments. PCR condition were 94 ◦ C for 3 min followed by 30 cycles of 94 ◦ C for 30 s, 60 ◦ C for 30 s and 72 ◦ C for 60 s.
the 4.9 kb BamHI/ClaI pJR2-lacZ fragment. E. coli DH5␣ cells were transformed with ligation mix with heat shock and plated on LB agar plates containing 100 g/ml ampicillin, 0.2 mM IPTG, and 20 g/ml X-gal. Pale blue and white colonies were formed by cells transformed with mutated plasmids, while blue colonies were formed by cells containing intact plasmids. 3. Results and discussion
Fig. 3. SDS-PAGE analysis of purified wild-type and mutant Twa DNA polymerases. Electrophoresis was performed on a vertical gel of 10% polyacrylamide, and the gel that is shown was stained with Coomassie brilliant blue R-250. Lane M, low molecular mass markers (molecular masses are indicated at the left); lane 1, wildtype Twa DNA polymerase; lane 2, A381R mutant; lane 3, N501R mutant; lane 4, N501K; lane 5, N501D mutant; lane 6, N501E mutant; lane 7, N501A mutant.
polymerase activities and 3 → 5 exonuclease activities of wildtype and mutant Twa DNA polymerases were determined presented in Table 2. While substitutions with basic amino acids (Arg and Lys) at position 501 increased the polymerase activity, the substitutions with acidic amino acids decreased. The 3 → 5 exonuclease activities of the mutant enzymes did not differ from wild-type, except for that of the N501E mutant.
3.1. Design and purification of mutant Twa DNA polymerases 3.3. PCR efficiency of the wild-type and mutant DNA polymerases Based on structural data of KOD DNA polymerase (Hashimoto et al., 2001) and amino acid sequence alignment of family B DNA polymerase, the A381 and N501 residues of Twa DNA polymerase are located in the palm subdomain at the forked-point of the enzyme (Fig. 1B). Using site-directed mutagenesis, we firstly replaced A381 and N501 with arginine. The N501R mutant showed significantly improved polymerase function compared to wild-type Twa DNA polymerase in terms of activity and PCR efficiency (Fig. 2). Therefore, we produced four additional mutants by exchanging N501 for Lys, Glu, Asp and Ala respectively. Wild-type and mutant Twa DNA polymerase were expressed and then purified through heat treatment followed by using His-tag affinity (HisTrapTM HP) and cation exchange (HiTrapTM SP HP) chromatography. Wild-type and mutant Twa DNA polymerase showed the same mobilities, and their molecular masses were estimated to be 90 kDa (Fig. 3). As a result, six mutant proteins (A381R, N501R, N501K, N501D, N501E and N501A) were obtained for assays. 3.2. Activities of mutant DNA polymerases The DNA polymerase activities of the wild-type and mutant enzymes were initially compared by measuring radioactivity incorporated into DNA and expressing this as a percentage of wild-type incorporation (Fig. 2A). Activity of the Twa A381R mutant (99.8%) was similar to that of wild-type polymerase and activity of the Twa N501R mutant (114.9%) was higher. PCR efficiency, tested using 2-kb lambda DNA fragments, was substantially higher with the N501R mutant than with the wild-type, A381R mutant polymerases, as expected (Fig. 2B). These findings indicate that the charge of the amino acid at position 501 significantly influences PCR efficiency. To verify this, we tested the four other mutants obtained by exchanging N501 for a basic amino acid (Lys), acidic amino acids (Glu and Asp), a neutral amino acid (Ala). The
To test PCR efficiency of wild-type and N501 mutants, 2-kb lambda DNA fragments were amplified using each polymerase for various extension times without annealing times. The wild-type only amplified a 2-kb target gene with a 60 s extension time, but the N501R, N501K and N501A mutants successfully amplified a 2-kb target gene with a 10 s extension time (Fig. 4A). The N501R and N501K mutants were also quantitatively more productive than either the wild-type or any other mutant. In comparing the amplification rates of mutant Twa DNA polymerases and other commercial DNA polymerase, we used the Twa N501R mutant because the DNA polymerase activity of the N501R mutant was higher than that of the N501K mutant (Table 2). To measure these amplification rates, lambda DNA targets were amplified using selected extension times (5, 10, 30 and 60 s). The amplification rate of the Twa N501R mutant exceeded rates of Pfu and Taq DNA polymerases (Fig. 4B). Moreover, using pTYB1 plasmid targets (1–4 kb), the Twa N501R mutant was able to amplify a 4-kb target in a 30 s extension time (Fig. 4C). Reportedly, the high fidelity DNA polymerase does not maintain long-range accuracy (LA) in PCR because of their strong associated exonuclease activity (Barnes, 1994). High accuracy in the Table 2 Comparison of the polymerase activities and 3 → 5 exonuclease activities of wildtype and mutated Twa DNA polymerases. DNA polymerase
Relative polymerase activity (%)
WT N501R N501K N501D N501E N501A
100 114.9 113.2 72.6 79.6 85.3
± ± ± ± ± ±
1.7 0.9 1.2 2.0 2.5 3.3
Relative 3 → 5 exonuclease activity (%) 100 101.3 100.7 101 87.5 96.2
± ± ± ± ± ±
2.4 3.2 2.3 2.1 3.8 3.9
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Fig. 4. Comparison of the wild-type and mutant Twa DNA polymerases, Pfu, Vent and Taq in time-saving PCR. (A) Comparison of the wild-type and mutant Twa DNA polymerases. Extension times used for PCR amplification of a 2-kb fragment are indicated top each lane. (B) Comparison of the wild-type, Twa N501R, Pfu, Vent and Taq DNA polymerases. (C) pTYB1 plasmid DNA was used as the template and amplicon sizes are indicated top of lanes. PCR condition were 94 ◦ C for 3 min followed by 30 cycles of 94 ◦ C for 30 s and 72 ◦ C for 30 s. Lane M, GeneRuler 1 kb DNA Ladder (Thermo Scientific #SM0311).
amplification of long sequences is increasingly important for current PCR applications (Nishioka et al., 2001). In comparison of extension efficiencies, the Twa DNA polymerase could amplify DNA an 8-kb DNA fragment, but the Twa N501R DNA polymerase could amplify a 10-kb DNA fragment (Fig. 5). Thus the N501R mutant displayed a significant improvement in PCR extension efficiency.
rates of the wild-type and Twa N501R DNA polymerase, based on the length of DNA synthesized in each fixed time window (30, 60, 90 and 120 s). By this measure, the extension rate was determined as about 6 nt/s for Twa DNA polymerase and about 11 nt/s for Twa N501R DNA polymerase. Twa N501R DNA polymerase was 2-fold faster than Twa DNA polymerase (Fig. 6).
3.4. Extension rates
3.5. Processivity
The specific extension rate represents the number of dNTPs polymerized per second per molecule of DNA polymerase. Extension rates depend notably on the reaction media and DNA templates (Andrey et al., 2004). To understand the basis for improved PCR performance of the N501R DNA polymerase, we compared extension
The concept of processivity was introduced as the probability that, following attachment of a nucleotide, a polymerase will not dissociate from the DNA when translocating to the next position (Fairfield et al., 1983). Like the extension rate, processivity depends on the reaction medium and DNA templates (Andrey et al.,
Fig. 5. Comparison of wild-type and Twa N501R DNA polymerase in long-range PCR. DNA was used as the template and amplicon sizes are indicated top each lane. Extension times used for PCR amplification were 1 min/kb. Lane M, GeneRuler 1 kb DNA Ladder (Thermo Scientific #SM0311).
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S.S. Cho et al. / Journal of Biotechnology 184 (2014) 39–46 Table 3 Summary of the processivity parameters.
Twa Twa N501R
Microscopic processivity (P)
Average primer extension length (nt) [1/(1 − P)]
0.9636 ± 0.004 0.9874 ± 0.001
27 ± 3 79 ± 7
Table 4 Summary of the steady-state kinetics analysis. Enzyme
Fig. 6. Comparison of extension rates wild-type and Twa N501R DNA polymerase. The extension rates of the wild-type and Twa N501R DNA polymerase were calculated from the length of DNA synthesized versus each extension time indicated at the top of the figure.
Twa Twa N501R KOD a b
2004). To analyze the superior PCR performance of the mutant Twa DNA polymerases, a processivity assay was performed. In electropherogram trace for wild-type and N501R DNA polymerases (Fig. 7A), peaks correspond to single primer extension products. Single primer extension products in the Fig. 7A are plotted as long (Ant/At) versus the length of extended primer and fitted by linear regression (Fig. 7B). Each value of processivity (P) was obtained from the slope. The processivity factors are summarized in Table 3. Wild-type exhibits a processivity (P) of 0.9636, which correlates to
DNAa
dNTPb −1
Km (nM)
Kcat (s
)
10.3 ± 0.5 6.6 ± 0.3 0.3 ± 0.1
2.6 ± 0.2 2.3 ± 0.16 0.8 ± 0.1
Km (M)
Kcat (s−1 )
0.3 ± 0.02 0.3 ± 0.02 0.5 ± 0.02
3.9 ± 0.3 4.5 ± 0.3 20.3 ± 0.5
Moles of template, in the presence of an excess of annealed primer. Moles of each nucleotide in an equimolar mixture of the four nucleotides.
an average primer extension length of 27 nt. In contrast, the Twa N501R DNA polymerase exhibits higher processivity with a P of 0.9874 and an average extension length 79 nt. Pfu DNA polymerase of lysine at residue 501 exhibits a P of 0.9777, which correlates to an average primer extension length of 45 nt (Kim et al., 2007). Thus, substitution of arginine at residue 501 increased the processivity of the DNA polymerase.
Fig. 7. Processivity analysis of wild-type and Twa N501R DNA polymerase. (A) Peaks in electropherogram traces of Twa and Twa N501R DNA polymerases correspond to single primer extension products. (B) Single primer extension products in the Fig. 7A are plotted as long (Ant /AT ) versus the length of extended primer and fitted by linear regression. Ant is equal to the total extended fragments greater than each product length which is the defined length of zero. AT is the sum of the length of extended primers (Bibillo and Eickbush, 2002).
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Table 5 Comparison of Taq, Pfu, Twa and Twa N501R DNA polymerase fidelities. Number of colonies
Blue Taq Pfu Twa Twa 501R
7690 7753 7404 8271
Mutation frequencya
Template doublingsb
Error ratec (× 10−5 )
Fold improvement over Taq
White and Pale blue ± ± ± ±
148 169 143 154
1398 403 267 390
± ± ± ±
35 21 20 23
0.15 0.05 0.03 0.05
± ± ± ±
0.04 0.001 0.006 0.003
7.5 6.5 6.2 7.5
± ± ± ±
0.2 0.3 0.2 0.3
2.45 0.91 0.67 0.72
± ± ± ±
0.5 0.008 0.006 0.02
1.0 2.7 3.6 3.4
a
Mutation frequency was expressed as the proportion of mutant colonies relative to the total number of colonies. Template doublings were calculated using the equation 2d = (amount of PCR product)/(amount of starting target) c Error rate was determined using the equation ER = mf/(bp × d), where mf is the mutation frequency, bp is the lacZ target size (832 bp), and d is the number of template doublings. b
3.6. Determination of kinetic constants
4. Conclusion
To study the effects of mutation of N501 on the catalytic rate of nucleotide incorporation, steady-state kinetic parameters were determined for wild-type, Twa N501R and KOD DNA polymerases. The rate of primer extension was measured at different primer-template concentrations in the presence of a fixed concentration (0.2 mM) of dNTPs and at different dNTPs concentrations in the presence of a fixed concentration (0.24 nM) of primer template. Plots of the reaction time course were analyzed to determine initial rates and kinetic parameters were obtained using a Lineweaver–Burk plot. Interestingly, the Twa N501R DNA polymerase had a 1.5-fold lower Km (DNA) value compared to wild-type and a nearly identical Km (dNTP) value compared to wild-type. However Twa and Twa N501R DNA polymerases have nearly identical turnover rates (kcat ) (Table 4). The R501 residue of KOD DNA polymerase, which correspond to the R501 residue of Twa N501R DNA polymerase are located in the palm subdomain at the forkedpoint of the enzyme (Hashimoto et al., 2001) (Fig. 1). The KOD DNA polymerase had also a 22-fold lower Km (DNA) value compared to Twa N501R DNA polymerase and a nearly identical Km (dNTP) value compared to Twa N501R DNA polymerase. Although Km is a direct measurement of the equilibrium binding of these enzymes to DNA, the difference observed is consistent with the notion that the mutant N501R stabilizes interactions between the polymerase domain and the DNA template (Wang et al., 2004). The results from the steady state kinetic analysis demonstrate that, while the N501R mutant significantly increases PCR efficiency and processivity, it does not negatively effect on the catalytic activity of the polymerase domain.
In this work, we have focused on two amino acid residues (A381 and N501) in forked-point of Twa DNA polymerase. These two residues were replaced by site-directed mutagenesis and the enzymatic properties of the mutants were analyzed. Here, Twa N501R DNA polymerase showed significantly improved polymerase function compared to wild-type Twa DNA polymerase in terms of processivity (3-fold), extension rate (2-fold) and PCR efficiency. Kinetic analysis using DNA as template revealed that positive charge of N501R exhibits a stabilizing effect on the interactions between the polymerase domain and the DNA template. These results clearly demonstrate that the Twa N501R DNA polymerase showed a significant improvement in PCR efficiency without loss of wild-type fidelity. Twa N501R DNA polymerase might be useful in high-fidelity DNA amplification and various PCR-based applications. Acknowledgement This work was supported by the Marine and Extreme Genome Research Center Program of the Ministry of Maritime Affairs and Fisheries, Republic of Korea. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jbiotec.2014. 05.007. References
3.7. PCR fidelity assay Fidelity is the frequency of correct nucleotide insertion per incorrect insertion and an inherent property of DNA polymerase (Pavlov et al., 2002). The use of high-fidelity DNA polymerase is necessary to reduce errors in PCR amplification during cloning and site-directed mutagenesis (Zheng et al., 2001). The fidelity of Twa N501R DNA polymerase was compared with wild-type, Taq, Pfu DNA polymerases by measuring the error frequency of each enzyme in a lacZ forward mutation assay. Wild-type showed a high fidelity with an error rate of 0.67 × 10−5 and Twa N501R DNA polymerase exhibited an error rate of 0.72 × 10−5 (Table 5). The fidelity of Twa N501R DNA polymerase was approximately 3.4-fold higher than Taq DNA polymerase and 1.2-fold higher than Pfu DNA polymerase. Thus, substitution of arginine at residue 501 does not negatively effect on fidelity of Twa DNA polymerase. These enzymes had excellent fidelity and are thus attractive for molecular biology applications.
Andrey, P., Nadejda, V.P., Sergei, A.K., Alexei, I.S., 2004. Recent developments in the optimization of thermostable DNA polymerase for efficient applications. Trends Biotechnol. 22, 253–260. Barnes, W.M., 1994. PCR amplification of up to 35-kb DNA with high fidelity and high yield from bacteriophage templates. Proc. Natl. Acad. Sci. U.S.A. 91, 2216–2220. Bibillo, A., Eickbush, T.H., 2002. High processivity of the reverse transcriptase from a non-long terminal repeat retrotransposon. J. Biol. Chem. 277, 34836–34845. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Cho, S.S., Kim, K.P., Lee, K.K., Youn, M.H., Kwon, S.T., 2012a. Characterization and PCR application of a new high-fidelity DNA polymerase from Thermococcus waiotapuensis. Enzyme Microb. Technol. 51, 334–341. Cho, S.S., Sun, Y., Yu, M., Kwon, S.H., Kwon, S.T., 2012b. Characterization and PCR application of dUTPase from hyperthermophilic euryarchaeon Thermococcus pacificus. Enzyme Microb. Technol. 51, 342–347. Choi, J.J., Nam, K.H., Min, B., Kim, S.J., Söll, D., Kwon, S.T., 2006. Protein transsplicing and characterization of a split family B-type DNA polymerase from the hyperthermophilic archaeal parasite Nanoarchaeum equitans. J. Mol. Biol. 356, 1093–1106. Fairfield, F.R., Newport, J.W., Dolejsi, M.K., von Hippel, P.H., 1983. On the processivity of DNA replication. J. Biomol. Struct. Dyn. 1, 715–727.
46
S.S. Cho et al. / Journal of Biotechnology 184 (2014) 39–46
Hashimoto, H., Nishioka, M., Fujiwara, S., Takagi, M., Imanaka, T., Inoue, T., Kai, Y., 2001. Crystal structure of DNA polymerase from hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1. J. Mol. Biol. 306, 469–477. Hogrefe, H.H., Hansen, C.J., Scott, B.R., Nelson, K.B., 2001. Archaeal dUTPase enhances PCR amplifications with archaeal DNA polymerases by preventing dUTP incorporation. Proc. Natl. Acad. Sci. U.S.A. 99, 596–601. Hopfner, K.P., Eichinger, A., Engh, R.A., Laue, F., Ankenbauer, W., Huber, R., Angerer, B., 1999. Crystal structure of a thermostable type B DNA polymerase from Thermococcus gorgonarius. Proc. Natl. Acad. Sci. U.S.A. 96, 3600–3605. Kim, Y.J., Lee, H.S., Bae, S.S., Jeon, J.H., Lim, J.K., Cho, Y., Nam, K.H., Kang, S.G., Kim, S.J., Kwon, S.T., Lee, J.-H., 2007. Cloning, purification, and characterization of a new DNA polymerase from a hyperthermophilic archaeon, Thermococcus sp. NA1. J. Microbiol. Biotechnol. 17, 1090–1097. Kong, H., Kucera, R.B., Jack, W.E., 1993. Characterization of a DNA polymerase from the hyperthermophile archaea Thermococcus litoralis. Vent DNA polymerase, steady state kinetics, thermal stability, processivity, strand displacement, and exonuclease activities. J. Biol. Chem. 268, 1965–1975. Lee, J.I., Cho, S.S., Kil, E.J., Kwon, S.T., 2010a. Characterization and PCR application of a thermostable DNA polymerase from Thermococcus pacificus. Enzyme Microb. Technol. 47, 147–152. Lee, J.H., Kang, S.G., Kim, S.J., Kwon, K.K., Lee, H.S., Kim, Y.J., Ryu, Y.G., Bae, S.S., Lim, J.K., Jeon, J.H., Cho, Y.N., 2010b. Mutant DNA polymerases and their genes from Thermococcus. US Patent Publication No. 2010/0297706 A1. Lee, J.I., Kim, Y.J., Bae, H., Cho, S.S., Lee, J.H., Kwon, S.T., 2010c. Biochemical properties and PCR performance of a family B DNA polymerase from hyperthermophilic euryarchaeon Thermococcus peptonophilus. Appl. Biochem. Biotechnol. 160, 1585–1599. Lundberg, K.S., Shoemaker, D.D., Adams, M.W., Short, J.M., Sorge, J.A., Mathur, E.J., 1991. High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus. Gene 108, 1–6. Mattila, P., Korpela, J., Tenkanen, T., Pitkanen, K., 1991. Fidelity of DNA synthesis by the Thermococcus litoralis DNA polymerase, an extremely heat stable enzyme with proofreading activity. Nucleic Acids Res. 19, 4967–4973. McClure, W.R., Chow, Y., 1980. The kinetics and processivity of nucleic acid polymerase. Methods Enzymol. 64, 277–297.
Nishioka, M., Mizuguchi, H., Fujiwara, S., Komatsubara, S., Kitabayashi, M., Uemura, H., Takagi, M., Imanaka, T., 2001. Long and accurate PCR with a mixture of KOD DNA polymerase and its exonuclease deficient mutant enzyme. J. Biotechnol. 88, 141–149. Pavlov, A.R., Belova, G.I., Kozyavkin, S.A., Slesarev, A.I., 2002. Helix-hairpin-helix motifs confer salt resistance and processivity on chimeric DNA polymerase. Proc. Natl. Acad. Sci. U.S.A. 99, 13510–13515. Rodriguez, A.C., Park, H.W., Mao, C., Beese, L.S., 2000. Crystal structure of a pol ␣ family DNA polymerase from the hyperthermophilic archaeon Thermococcus sp. 9◦ N-7. J. Mol. Biol. 299, 447–462. Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B., Erlich, H.A., 1988. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487–491. Song, J.M., Choi, J.J., Kim, T.O., Seo, M.S., Lee, M.S., Kim, H.K., Kwon, S.T., 2007. Characterization and PCR performance of a family B-type DNA polymerase from the hyperthermophilic crenarchaeon Staphylothermus marinus. Enzyme Microb. Technol. 40, 1475–1483. Song, J.G., Kil, E.J., Cho, S.S., Kim, I.H., Kwon, S.T., 2010. An amino acid residue in the middle of the fingers subdomain is involved in Neq DNA polymerase processivity: enhanced processivity of engineered Neq DNA polymerase and its PCR application. Protein Eng. Des. Sel. 23, 835–842. Takagi, M., Nishioka, M., Kakihara, H., Kitabayashi, M., Inoue, H., Kawakami, B., Oka, M., Imanaka, T., 1997. Characterization of DNA polymerase from Pyrococcus sp. strain KOD1 and its application to PCR. Appl. Environ. Micobiol. 63, 4504–4510. Von Hippel, P.H., Fairfield, F.R., Dolejsi, M.K., 1994. On the processivity of polymerases. Ann. N. Y. Acad. Sci. 726, 118–131. Wang, Y., Dennis, E.P., Li, M., John, C.S., Michael, F., Peter, B.V.H., 2004. A novel strategy to engineer DNA polymerase for enhanced processivity and improved performance in vitro. Nucleic Acids Res. 32, 1197–1207. Zheng, R., Matsui, E., Shen, Y., Musti, K.V., Feng, Y., Darnis, S., Kawarabayasi, Y., Kikuchi, H., Harata, K., Matsui, I., 2001. The novel function of a short region K253 XRXXXD259 conserved in the exonuclease domain of hyperthermostable DNA polymerase I from Pyrococcus horikoshii. Extremophiles 5, 111–117.
