Characteristics of DNA polymerase I from an extreme thermophile, Thermus scotoductus strain K1

Abstract Several native and engineered heat‐stable DNA polymerases from a variety of sources are used as powerful tools in different molecular techniques, including polymerase chain reaction, medical diagnostics, DNA sequencing, biological diversity assessments, and in vitro mutagenesis. The DNA polymerase from the extreme thermophile, Thermus scotoductus strain K1, (TsK1) was expressed in Escherichia coli, purified, and characterized. This enzyme belongs to a distinct phylogenetic clade, different from the commonly used DNA polymerase I enzymes, including those from Thermus aquaticus and Thermus thermophilus. The enzyme demonstrated an optimal temperature and pH value of 72–74°C and 9.0, respectively, and could efficiently amplify 2.5 kb DNA products. TsK1 DNA polymerase did not require additional K+ ions but it did need Mg2+ at 3–5 mM for optimal activity. It was stable for at least 1 h at 80°C, and its half‐life at 88 and 95°C was 30 and 15 min, respectively. Analysis of the mutation frequency in the amplified products demonstrated that the base insertion fidelity for this enzyme was significantly better than that of Taq DNA polymerase. These results suggest that TsK1 DNA polymerase could be useful in various molecular applications, including high‐temperature DNA polymerization.

be used to create unique reagents (Aschenbrenner & Marx, 2017;Coulther et al., 2019;Reha-Krantz et al., 2014). This means that the search for novel DNA polymerases has been a major focus for the last couple of decades. A-type polymerases from the genus Thermus are the most frequently used in molecular biology and include the commonly used Taq DNA polymerase from T. aquaticus. Several polymerases with similarities to Taq have been mined from other Thermus species, including Tfi from T. filiformis, Tfl from T. flavus, Tbr from T. brockianus, Tca from T. caldophilus, and Tth from T. thermophilus. Slight amino acid sequence differences between polymerase enzymes can result in dramatic changes to their biochemical characteristics, suggesting that it is possible to mine for novel polymerases with improved functionality (Gibbs et al., 2009). Other A-type polymerases have been isolated from Thermotoga spp., including Tma polymerase from T. maritima and Tne from T. neapolitana (Spibida et al., 2017). Despite the number of available enzymes, the growing applications of molecular biology mean that there is still a demand for novel enzymes, and this is where the field of applied science might facilitate continued improvements.
Here, the expression, purification, and characterization of a recombinant DNA polymerase I from Thermus scotoductus strain K1 (TsK1 DNA polymerase) originating from a geothermal spring in Karvachar, Nagorno-Karabakh (Saghatelyan et al., 2015) is described.

| Source of enzyme
The enzyme was from T. scotoductus strain K1, which was isolated from a geothermal spring located in Karvachar, Nagorno-Karabakh.
The draft genome sequence of T. scotoductus K1 was deposited in the DBJ/EMBL/GenBank database under the RefSeq assembly accession no. GCF_001294665.1 (Saghatelyan et al., 2015). A phylogenetic tree depicting the evolutionary distance between TsK1 DNA polymerase (Accession no. MW080815) and other Thermus spp. polymerases was constructed based on the JTT matrix model using the maximum likelihood method (Jones et al., 1992) in MEGA X software (Kumar et al., 2018). The polI codons were optimized (GenScript) to maximize expression in E. coli while maintaining the original amino acid sequence. The codon-optimized gene was then synthesized by GenScript (https://www.gensc ript.com/).

| Cloning
A pUC57-Mini plasmid harboring the codon-optimized polI sequence, 2512 bp, was used to facilitate the downstream cloning experiments completed using the FX (fragment exchange) system (Geertsma & Dutzler, 2011). The expression vector p7xC3H (6999 bp) (Addgene, LGC Standards), which contained a T7 promoter, a C-terminal 3C protease cleavage site, and a C-terminal 10× His tag, was used as the expression vector. Expression constructs were identified and maintained using kanamycin resistance conferred by the vector.

| Protein expression and purification
1 L of 2×YT (yeast extract and tryptone) broth supplemented with 50 μg/ml kanamycin was used to culture E. coli BL21 (DE3) harboring the C-10 recombinant expression plasmid at 37°C until an OD 600 of 0.4 was obtained. Expression was induced by adding isopropyl β-D-1-thiogalactopyranoside to 0.4 mM and further cultivation at 20 °C for another 16 h (at 200 rpm). Cell harvesting was performed by centrifugation followed by resuspension in buffer R (20 mM NaCl, 50 mM Tris-HCl pH 7.5, 8% glycerol). The cells were then disrupted by lysozyme and freezing-thawing, which was followed by sonication. The lysate was then heated at 70 °C for 20 min to precipitate most of the heat-labile host proteins, and cell wall and insoluble debris were removed by further centrifugation at 7482 g, 4°C for 30 min. The supernatant was filtered using Whatman filters and applied to a HisTalon gravity column (Clontech Laboratories, Inc.), preequilibrated with the same buffer. The column was further washed with buffer R containing 10 mM imidazole. Elution was then performed using buffer R containing 50 mM imidazole. Purification efficiency was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) , and the fractions containing the highest concentrations of the target protein were merged. Gel-filtration on a PD-10 column (GE Healthcare) was used to remove the excess of imidazole. The purified fractions were then dialyzed against storage buffer (20 mM Tris-HCl, 40 mM KCl, 0.1 mM DTT, 0.1 mM EDTA, 0.5% Tween-20, 0.5% Nonidet P40, and 50% glycerol) (Gibbs et al., 2009) and stored at −20°C for further investigation. The final protein concentrations were measured using a Qubit Assay kit (Life Technologies).

| Activity assay
Polymerase activity was evaluated using a method described previously (Choi et al., 2006) with minor modifications. Briefly, the standard reaction mixture (50 μL) contained 20 mM Tris-HCl (pH 7.5); 40 mM KCl; 2 mM MgCl 2 ; 100 mM dCTP, dATP, and dGTP each; 10 mM dTTP; 0.5 μCi [ 3 H] thymidine 5'-triphosphate (2.59-3.33 TBq/mmol; PerkinElmer); 1.25 μg activated calf thymus DNA; and 0.5 μL TsK1 DNA polymerase solution. The mixture was incubated at 70°C for 10 min, and the reaction was terminated by placing the mixture on ice and adding 0.5 M EDTA. Subsequently, an aliquot was applied onto a DE81 filter-paper disc (23 mm; Whatman), which was then dried on a heat block, washed with 0.5 M sodium phosphate (pH 7.0) buffer for 10 min and in 70% (v/v) ethanol for 5 min, and dried. The radioactivity incorporated in the dried filter-paper disc was then measured using a Tri-Carb 2900 TR Liquid Scintillation Analyser (PerkinElmer).
To determine the optimal temperature, the reaction mix containing the optimal buffer was incubated at various temperatures (45-80°C) for 10 min and then subjected to the above-described activity assay.
The influence of K + ions on polymerase activity was determined by adding various concentrations (from 0 to 200 mM) of KCl to the basic reaction mixture and the dependence of the polymerase on divalent cations (Mg 2+ , Mn 2+ ) was determined using various concentrations (from 0 to 20 and from 0 to 10 mM, respectively) of MgCl 2 and MnCl 2 .
Various dilutions of the enzyme solution were used in each reaction under optimal pH and temperature conditions to determine the specific activity of TsK1 DNA polymerase. One unit of TsK1 DNA polymerase was defined as the amount of enzyme needed to convert 10 pmol of [ 3 H] TTP into an acid-insoluble product at 72°C in 10 min.
To investigate the thermostability properties of this enzyme, we subjected the purified enzyme, without any additional stabilizers, to 75, 80, 88, and 95°C for up to 1 h. Aliquots of the enzyme were removed at 5, 15, 30, 45, and 60 min and quenched on ice. The residual activity of these samples was then determined in the optimal reaction mix as described above.
All measurements were carried out in triplicate.

| Fidelity assay
The fidelity assays were performed using the blue-white screening method described previously ) with some modifications. Briefly, primers pUC19_F (5′-gcatgaAAGCTTGCAT-GCCTGCAGGTCGAC-3′) and pUC19_R (5′-gcatgaCATATGCGGT-GTGAAATACCGCAC-3′), which incorporate a HindIII and NdeI site, respectively (underlined), were used to amplify a 265 bp fragment of the lacZα gene using pUC19 as a template. The primers were designed manually using the Primer3 online tool (https://www.ncbi. nlm.nih.gov/tools/ prime r-blast/), and PCR was performed using TsK1 DNA polymerase, OneTaq (NEB), Fusion (Thermo Fisher), and Taq (Sigma) DNA polymerases in their optimal buffers and assayed under their optimal reaction conditions. The PCR products were then digested with HindIII and NdeI, purified, and ligated to the 2421 bp HindIII/NdeI fragment from pUC19. Each ligation mixture was then used to transform chemically competent E. coli TOP10 cells. Transformed cells were plated on LB agar plates supplemented with 100 μg/ml carbenicillin, 40 μg/ml X-gal, and 0.3 mM IPTG. Pale blue and white colonies were formed by cells containing mutant plasmids, while blue colonies were formed by cells containing wild-type plasmids. The percentage of white and pale blue colonies was then calculated.
Various commercially available and manually designed (using Primer3 online tool (https://www.ncbi.nlm.nih.gov/tools/ prime r-blast/)) primer sets were applied using both genomic and plasmid DNA as a template (Table 1)

| Optimal conditions for TsK1 DNA polymerase activity
We evaluated TsK1 DNA polymerase activity between 45 and 80°C and determined that the optimal temperature range for this enzyme was 68-75°C, and the highest activity was observed at 72°C ( Figure 3a). An evaluation of the pH requirements for this enzyme revealed that it was most active between pH 8.5 and 9.2 in Tris-HCl buffer, demonstrating the highest activity at pH 9.0 (Figure 3b). To study the effects of divalent cations on TsK1 DNA polymerase activity, we evaluated various concentrations of MgCl 2 and MnCl 2 and confirmed that the enzyme was highly dependent on Mg 2+ ions. We identified that the Mg 2+ ion concentration of 3-5 mM was optimal for the activity of this enzyme, whereas a concentration of >8 mM led to decreased activity. The presence of Mn 2+ ions had almost no influence on polymerase activity (Figure 3d).

| PCR by TsK1 DNA polymerase
The electrophoretogram of the PCR products amplified by using the TsK1 DNA polymerase is shown in Figure 5. Lanes 2 (500 bp) and 3 (1.5 kb) represent amplicons from bacterial genomic DNA, and lanes 1 (265 bp), 4 (1920 bp), and 5 (2.5 kb) represent products amplified from plasmid templates. The high intensities of the bands suggest that TsK1 DNA polymerase can amplify products of up to 2.5 kb with high efficiency.

| DISCUSS ION
The expression, purification, and characterization of a DNA polymerase I from T. scotoductus K1 are described in the current study. The amino acid sequence comparison showed that this enzyme shares a high degree of similarity to DNA polymerase from T. scotoductus SA-01. This finding was expected, as according to earlier evaluations of similarity (Saghatelyan et al., 2015). TsK1 DNA polymerase is relatively different from T. aquaticus DNA polymerase, which might explain the differences in the behavior of this enzyme as discussed below.
The FX cloning strategy used to clone TsK1 DNA polymerase was based on the use of type IIS restriction enzymes, which digest DNA at an exact distance from their asymmetric recognition sites.
The resulting overhang is defined only based on its distance from the recognition site and not by its sequence. Following optimization of buffering conditions, TsK1 DNA polymerase demonstrated the highest activity at pH 9.0 in Tris-HCl buffer, which is different from that of Taq that prefers a less alkaline pH (Chien et al., 1976;Lawyer et al., 1993). Polymerase I from T. caldophilus GK24 requires an optimum pH of 8.7 (Park et al., 1993).
TsK1 DNA polymerase activity was rather independent of KCl at lower concentrations, although high concentrations of this salt did inhibit its enzymatic activity. Reportedly, KCl at concentrations above 100 mM inhibits Taq polymerase as well (Chien et al., 1976).
Although TsK1 DNA polymerase does not appear to be dependent on KCl when using calf thymus DNA as a template, the influence of KCl may differ with different enzymes and different templates (Lawyer et al., 1993).
Divalent cations are necessary for polymerization. Here, we demonstrated that the TsK1 DNA polymerase activity was dependent on Mg 2+ to some extent. This is in agreement with the literature, which suggests that Mg 2+ is a critical component for polymerases (Choi et al., 2006). For TsK1 DNA polymerase, the optimal concentration of Mg 2+ is 3-5 mM, in contrast to that for Taq (10 mM), recombinant Taq (2-4 mM), and Tca (12 mM) (Chien et al., 1976;Lawyer et al., 1993;Park et al., 1993).
The optimal temperature for TsK1 DNA polymerase was shown to be 72-74°C, which is comparable with other Taq-like polymerases (Lawyer et al., 1993;Park et al., 1993) and the lower polymerization activity described at 80°C could also be explained by the template (activated calf thymus DNA) denaturation at higher temperatures, as the TsK1 DNA polymerase was stable at 80°C.
Although the half-life of TsK1 DNA polymerase (without the use of additional stabilizers) at 95°C (15 min) is lower than that of Taq (Lawyer et al., 1993), it is close to another commercially available rTaq (20 min) polymerase (Gibbs et al., 2009) (Park et al., 1993).
Numerous dilations to the basic PCR approach have been reported and respective enzymes with various degrees of similarity to Taq polymerase have been mined from other Thermus spp. (Gibbs et al., 2009). Gibbs and colleagues compared six recombinant polymerases originating from different Thermus species, which were selected based on their degree of divergence, and demonstrated that all of these enzymes retained similar biochemical characteristics.
Moreover, some studies suggest that the other polymerases originating from Thermus spp. can be more efficient in certain cases of PCR in contrast to Taq. For instance, it has been reported, that the reverse transcriptase activity of a recombinant DNA polymerase from T. thermophilus was 100-fold higher than the RT activity of T. aquaticus DNA pol, although these two strains are closely related (Gibbs et al., 2009 KCl and 3 mM MgCl 2 was shown to be optimal for the amplification of 2.5 kb products using various templates ( Figure 5).
Our results suggest that TsK1 DNA polymerase is twice as accurate as Taq (Gibbs et al., 2009).
Even with these limitations, we can still confidently assert that TsK1 DNA polymerase is at least 2 times more accurate than Taq but further investigations are needed to confirm this finding.

CO N FLI C T O F I NTE R E S T
None declared.

E TH I C S S TATEM ENT
None required.

DATA AVA I L A B I L I T Y S TAT E M E N T
The nucleotide sequence of the synthetic codon-optimized polI gene encoding the TsK1DNA