Characterization of a family B DNA polymerase from Thermococcus barophilus Ch5 and its application for long and accurate PCR.

The family B DNA polymerase gene from the euryarchaeon Thermococcus barophilus Ch5 (Tba5) contains an open reading frame of 6198 base pairs that encodes 2065 amino acid residues. The gene is split by three inteins that must be spliced out to form the mature DNA polymerase. A Tba5 DNA polymerase gene without inteins (genetically intein-spliced) was expressed under the control of the pET-28b(+)T7lac promoter in E. coli Rosetta 2(DE3)pLysS cells. The molecular mass of the purified Tba5 DNA polymerase was about 90kDa consistent with the 90,470Da molecular mass calculated based on the 776 amino acid sequence. The optimal pH for Tba5 DNA polymerase activity was 7.5 and the optimal temperature was 70-75°C. The enzyme possessed 3'→5' exonuclease activity and was activated by magnesium ions. PCR amplification using Tba5 DNA polymerase enables high-yield for 1- to 6-kb target DNA products, while 8- to 10-kb target DNA products were amplified at low or inefficient levels. To simultaneously improve product yield and amplification fidelity, Tba5 plus DNA polymerase mixtures were constituted with various amounts of Tba5 DNA polymerase mixed with Taq DNA polymerase. The Tba5 plus DNA polymerase mixtures robustly amplified up to 25-kb λ DNA fragments. In addition, the PCR error rate of Tba5 plus3 and Tba5 plus4 mixtures were much lower than those of wild-type Tba5 DNA polymerase, Pfu DNA polymerase, Taq DNA polymerase, and Pfu plus DNA polymerase.

Improved PCR performance and fidelity of double mutant Neq A523R/N540R DNA polymerase.

We previously reported that Neq A523R DNA polymerase is more efficient in PCR than wild-type Neq DNA polymerase, and amplifies products more rapidly. Neq A523R DNA polymerase also amplifies templates more rapidly than Pfu DNA polymerase, but has a lower fidelity than Pfu DNA polymerase. To improve product yield and the fidelity of amplification simultaneously, we constructed and characterized the double mutant Neq A523R/N540R. The yield of PCR products was greater for Neq A523R/N540R DNA polymerase than wild-type and other mutant DNA polymerases, and the Neq double mutant catalyzed amplification of a 12-kb PCR product from a lambda template with an extension time of 3 min. The PCR error rate of Neq A523R/N540R DNA polymerase (6.3×10(-5)) was roughly similar to that of Pfu DNA polymerase (4.8×10(-5)), but much lower than those of wild-type Neq DNA polymerase (57.2×10(-5)), Neq A523R DNA polymerase (13.1×10(-5)), and Neq N540R DNA polymerase (37.7×10(-5)). These results indicated that A523R and N540R mutations of Neq DNA polymerase had synergistic effects on its fidelity.

Enhanced PCR efficiency of high-fidelity DNA polymerase from Thermococcus waiotapuensis.

Twa DNA polymerase from hyperthermophilic archaeon Thermococcus waiotapuensis has exceedingly high fidelity among family B DNA polymerases. However, Twa DNA polymerase has significant shortcomings in terms of a low extension rate and poor processivity. To resolve these weaknesses, we focused on two amino acid residues (N565 and H633) in the palm and thumb subdomains of the Twa DNA polymerase. These two residues were replaced by site-directed mutagenesis and the enzymatic properties of the mutants were analyzed. Here, Twa H633R DNA polymerase showed significantly improved polymerase function compared to wild-type Twa DNA polymerase in terms of processivity (2-fold), extension rate (1.5-fold) and PCR efficiency. Kinetic analysis using DNA as a template revealed that the kcat value of the Twa H633R mutant was similar to that of wild-type, but the Km of the Twa H633R mutant was about 1.6-fold lower than that of the wild-type. These results showed that the Arg residue substitution at H633 located in the thumb subdomain has a positive effect on processivity, extension rate and PCR efficiency, suggesting that the Twa H633R mutant allows a conformational change for easy access of the primer-template to the binding site of the polymerase domain.

Mutations in the palm subdomain of Twa DNA polymerase to enhance PCR efficiency and its function analysis.

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.

Characterization of a family B DNA polymerase from the hyperthermophilic crenarchaeon Ignicoccus hospitalis KIN4/I and its application to PCR.

A family B DNA polymerase gene from the hyperthermophilic crenarchaeon Ignicoccus hospitalis KIN4/I was highly expressed under the control of T7lac promoter of pET-28ARG in Escherichia coli BL21-CodonPlus(DE3)-RIL cells. The produced I. hospitalis (Iho) DNA polymerase was purified by heat treatment followed by HisTrap™ HP column and HiTrap™ SP column chromatographies. The molecular mass of the purified Iho DNA polymerase was 88 kDa as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimal pH for Iho DNA polymerase activity was 7.0 and the optimal temperature was 70 °C. Iho DNA polymerase was strongly activated by the presence of magnesium ion at an optimum concentration of 3 mM. The optimal concentration of KCl for Iho DNA polymerase activity was 60 mM. The half-life of the enzyme at 94 °C was about 2 h. The optimal conditions for polymerase chain reaction (PCR) were determined. Iho DNA polymerase possesses 3'→5' exonuclease activity, and the fidelity of the Iho DNA polymerase was similar to that of Pfu and Vent DNA polymerases. However, Iho DNA polymerase provided more enhanced efficiency of PCR amplification than Pfu and Vent DNA polymerases. Iho DNA polymerase could successfully amplify a 2-kb λ DNA target with a 10-s extension time and could amplify a DNA fragment up to 8 kb λ DNA.

Broad nucleotide cofactor specificity of DNA ligase from the hyperthermophilic crenarchaeon Hyperthermus butylicus and its evolutionary significance.

The nucleotide cofactor specificity of the DNA ligase from the hyperthermophilic crenarchaeon Hyperthermus butylicus (Hbu) was studied to investigate the evolutionary relationship of DNA ligases. The Hbu DNA ligase gene was expressed under control of the T7lac promoter of pTARG in Escherichia coli BL21-CodonPlus(DE3)-RIL. The expressed enzyme was purified using the IMPACT™-CN system (intein-mediated purification with an affinity chitin-binding tag) and cation-ion (Arg-tag) chromatography. The optimal temperature for Hbu DNA ligase activity was 75 °C, and the optimal pH was 8.0 in Tris-HCl. The activity was highly dependent on MgCl2 or MnCl2 with maximal activity above 5 mM MgCl2 and 2 mM MnCl2. Notably, Hbu DNA ligase can use ADP and GTP in addition to ATP. The broad nucleotide cofactor specificity of Hbu DNA ligase might exemplify an undifferentiated ancestral stage in the evolution of DNA ligases. This study provides new evidence for possible evolutionary relationships among DNA ligases.

Characterization and PCR application of a new high-fidelity DNA polymerase from Thermococcus waiotapuensis.

The family B DNA polymerase gene from the euryarchaeon Thermococcus waiotapuensis (Twa) contains an open reading frame of 4404 bases that encodes 1467 amino acid residues. The gene is split by two intein-coding sequences that forms a continuous open reading frame with the three polymerase exteins. Twa DNA polymerase genes with (whole gene) and without (genetically intein-spliced) inteins were expressed in Escherichia coli Rosetta(DE3)pLysS. The inteins of the expressed whole gene were easily spliced during purification. The molecular mass of the purified Twa DNA polymerase was about 90 kDa, as estimated by SDS-PAGE. The optimal pH for Twa DNA polymerase activity was 6.0 and the optimal temperature was 75 °C. The enzyme was activated by magnesium ions. The half-life of the enzyme at 99 °C was about 4 h. The optimal buffer for PCR with Twa DNA polymerase was 50 mM Tris-HCl (pH 8.2), 2.0 mM MgCl2, 30 mM KCl, 2.0 mM (NH4)2SO4, 0.01% Triton X-100, and 0.005% BSA. The PCR fidelity of Twa DNA polymerase was higher than Pfu, KOD and Vent DNA polymerases. A ratio of 15:1 Taq:Twa DNA polymerase efficiently facilitated long-range PCR.

Characterization and PCR applications of dUTPase from the hyperthermophilic euryarchaeon Thermococcus pacificus.

We cloned and sequenced the gene encoding Thermococcus pacificus dUTPase (Tpa dUTPase). The Tpa dUTPase gene consists of 471 bp and encodes a 156-amino acid protein. The deduced amino acid sequence of Tpa dUTPase has high sequence similarity with other archaeal dUTPases. The Tpa dUTPase had an 18-kDa major protein band consistent with the 17,801 Da molecular mass calculated based on the amino acid sequence. The specific activity of Tpa dUTPase on dUTP at 85 °C was 90,909 U/mg. For Tpa dUTPase activity, we determined an optimum pH of 8.5 and temperature of 85 °C. Magnesium ions strongly induced activity, with an optimum concentration of 0.75 mM. The half-life of the enzyme at 94 °C was about 7 h. The specific activity of the Tpa dUTPase on dUTP was about 10-20-fold higher than that of Tpa dUTPase on dCTP. Tpa dUTPase enhanced the PCR amplification efficiency of long targets when Pfu and Vent DNA polymerases were used.

Improved PCR performance using mutant Tpa-S DNA polymerases from the hyperthermophilic archaeon Thermococcus pacificus.

We previously reported that Tpa-S DNA polymerase (constructed via fusion of the Sso7d DNA binding protein to the C-terminus of Thermococcus pacificus (Tpa) DNA polymerase) is more efficient in long and rapid PCR than wild-type Tpa, Taq, or Pfu DNA polymerases. However, Tpa-S DNA polymerase had a low yield of PCR products compared with commercialized Taq or Pfu DNA polymerases. To improve the yield of PCR products, mutant Tpa-S DNA polymerases were created via site-directed mutagenesis. In this study, we have targeted the N213 residue in the Exo II motif and the K501 residue in the Pol III motif. The mutant Tpa-S DNA polymerases showed enhanced PCR yields compared to that of the Tpa-S DNA polymerase. Specifically, the double mutant Tpa-S N213D/K501R DNA polymerase had an approximately three-fold increase in the yield of 8-10kb PCR products over that of the Tpa-S DNA polymerase, and catalyzed amplification of a 12kb PCR product using a lambda template with an extension time of 30s. Even though the mutation is in the Exo II motif, the error rate of the double mutant Tpa-S N213D/K501R (2.79×10(-5)) was nearly the same as that seen in the Pfu DNA polymerase (2.70×10(-5)).

Improved thermostability and PCR efficiency of Thermococcus celericrescens DNA polymerase via site-directed mutagenesis.

The Thermococcus celericrescens (Tcel) DNA polymerase gene, which contains a 2328-bp open reading frame that encodes 775 amino acid residues, was expressed in the Escherichia coli strain Rosetta(DE3)pLysS. The expressed enzyme was purified through heat treatment, HisTrap™ HP column chromatography and then HiTrap™ SP HP column chromatography. Tcel DNA polymerase has poor thermostability and PCR efficiency compared to those of other family B DNA polymerases. To improve thermostability and PCR efficiency, mutant Tcel DNA polymerases were created via site-directed mutagenesis. Specifically, we targeted the A752 residue for enhanced thermostability and the N213 residue for improved PCR efficiency. The mutant Tcel DNA polymerases all showed enhanced PCR efficiency and thermostability compared to those of the wild-type Tcel DNA polymerase. Specifically, the double mutant TcelA752K/N213D DNA polymerase had an approximately three-fold increase in thermostability over that of the wild-type enzyme and amplified a long 10-kb PCR product in an extension time of 2min. However, there was a small change in the 3'→5' exonuclease activity compared with that of the wild-type Tcel DNA polymerase, even though the mutation is in the ExoII motif. The double mutant TcelA752K/N213D DNA polymerase had a 2.6-fold lower error rate compared to that of Taq DNA polymerase. It seems that the double mutant TcelA752K/N213D DNA polymerase can be used in LA (long and accurate) PCR.

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.

Neq DNA polymerase is the first archaeal family B DNA polymerase reported to lack uracil recognition function and successfully utilize deaminated bases. We have focused on two amino acid residues (Y515, A523) in the fingers subdomain of Neq DNA polymerase, which were predicted to be located in the middle of the fingers subdomain, based on amino acid sequence alignment of the Neq DNA polymerase with structurally determined archaeal DNA polymerases. Those two residues were replaced by site-directed mutagenesis, and the enzymatic properties of the mutants were analyzed. Here, we show that the A523 residue located in the middle of the fingers subdomain affects the processivity of Neq DNA polymerase. Mutational analysis has allowed us to enhance the protein function as well as understand the function of the residues. One mutant protein, Neq A523R DNA polymerase, exhibited a roughly 3-fold enhanced processivity and extension rate compared to wild type, enabling more efficient PCR. In the presence of uracil, Neq A523R DNA polymerase outperformed Taq DNA polymerase with enhanced specificity and sensitivity. These results suggest that Neq A523R DNA polymerase could be most effectively utilized in real-time PCR using uracil-DNA glycosylase without the risk of carry-over contamination.

Biochemical properties and PCR performance of a family B DNA polymerase from hyperthermophilic Euryarchaeon Thermococcus peptonophilus.

The Thermococcus peptonophilus (Tpe) DNA polymerase gene was expressed under the control of the T7lac promoter on pET-22b(+) in Escherichia coli BL21-CodonPlus(DE3)-RIL in order to fully elucidate its biochemical properties and evaluate its feasibility in polymerase chain reaction (PCR) application. The expressed enzyme was then purified by heat treatment followed by two steps of column chromatography after which optimum pH and temperature of the enzyme were evaluated to be 7.0 and 75 degrees C, respectively. The optimal buffer for PCR with Tpe DNA polymerase consisted of 50 mM Tris-HCl (pH 8.0), 2 mM MgCl(2), 80 mM KCl, and 0.02% Triton X-100. Tpe DNA polymerase revealed a 3.6-fold higher fidelity (3.37 x 10(-6)) than Taq DNA polymerase (12.13 x 10(-6)) and performed significantly more efficiently in PCR amplification than both Taq and Pfu DNA polymerases. Ratios of 31:1 of Taq to Tpe DNA polymerases allowed PCR amplification of targets up to 15 kb in length with a 2.2-fold higher fidelity than Taq DNA polymerase. The results of the PCR experiments indicate that Tpe DNA polymerase may provide a higher fidelity DNA amplification in a shorter reaction time.

Clinical utility of chimerism status assessed by lineage-specific short tandem repeat analysis: experience from four cases of allogeneic stem cell transplantation.

Chimerism testing permits early prediction and documentation of successful engraftment, and also facilitates detection of impending graft rejection. In this study, we serially monitored chimerism status by short tandem repeat-based PCR in nucleated cells (NC), T cells and natural killer (NK) cells after myeloablative allogeneic stem cell transplantation (SCT). Four patients with myeloid malignancies showed discrepant chimerism results among those three fractions. Three patients had mixed chimerism (MC) of donor/host T cells at a time point around the onset of chronic graft-versus-host disease (GVHD). In two patients with disease relapse, MC of NK cells preceded a morphological relapse or NK cells showed a higher percentage of patient cells compared to NC. Therefore, our study shows that chimerism analysis in lineage-specific cells might be useful in predicting clinical outcome after allogeneic SCT in certain patients.