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ABSTRACT: 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.
Extremophiles 04/2013; · 2.94 Impact Factor
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ABSTRACT: We cloned and sequenced the gene encoding Thermococcus pacificus dUTPase (Tpa dUTPase). The Tpa dUTPase gene consists of 471bp 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 17,801Da molecular mass calculated based on the amino acid sequence. The specific activity of Tpa dUTPase on dUTP at 85°C was 90,909U/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 0.75mM. The half-life of the enzyme at 94°C was about 7h. 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.
Enzyme and microbial technology. 12/2012; 51(6-7):342-7.
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ABSTRACT: A recombinant DNA ligase from Sulfophobococcus zilligii that shows multiple cofactor specificity (ATP, ADP and GTP) was expressed in Escherichia coli and purified under reducing conditions. Crystals were obtained by the microbatch crystallization method at 295 K in a drop containing 1 µl protein solution (10 mg ml(-1)) and an equal volume of mother liquor [0.1 M HEPES pH 7.5, 10%(w/v) polyethylene glycol 10 000]. A data set was collected to 2.9 Å resolution using synchrotron radiation. The crystals belonged to space group P1, with unit-cell parameters a=63.7, b=77.1, c=77.8 Å, α=83.4, β=82.4, γ=74.6°. Assuming the presence of two molecules in the unit cell, the solvent content was estimated to be about 53.4%.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 12/2010; 66(Pt 12):1583-5. · 0.51 Impact Factor
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ABSTRACT: DNA ligases are divided into two groups according to their cofactor requirement to form ligase-adenylate, ATP-dependent DNA ligases and NAD(+)-dependent DNA ligases. The conventional view that archaeal DNA ligases only utilize ATP has recently been disputed with discoveries of dual-specificity DNA ligases (ATP/ADP or ATP/NAD(+)) from the orders Desulfurococcales and Thermococcales. Here, we studied DNA ligase encoded by the hyperthermophilic crenarchaeon Sulfophobococcus zilligii. The ligase exhibited multiple cofactor specificity utilizing ADP and GTP in addition to ATP. The unusual cofactor specificity was confirmed via a DNA ligase nick-closing activity assay using a fluorescein/biotin-labelled oligonucleotide and a radiolabelled oligonucleotide. The exploitation of GTP as a catalytic energy source has not to date been reported in any known DNA ligase. This phenomenon may provide evolutionary evidence of the nucleotide cofactor utilization by DNA ligases. To bolster this hypothesis, we summarize and evaluate previous assertions. We contend that DNA ligase evolution likely started from crenarchaeotal DNA ligases and diverged to eukaryal DNA ligases and euryarchaeotal DNA ligases. Subsequently, the NAD(+)-utilizing property of some euryarchaeotal DNA ligases may have successfully differentiated to bacterial NAD(+)-dependent DNA ligases.
Environmental Microbiology 12/2008; 10(12):3212-24. · 5.84 Impact Factor
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ABSTRACT: The known archaeal family B DNA polymerases are unable to participate in the PCR in the presence of uracil. Here, we report on a novel archaeal family B DNA polymerase from Nanoarchaeum equitans that can successfully utilize deaminated bases such as uracil and hypoxanthine and on its application to PCR. N. equitans family B DNA polymerase (Neq DNA polymerase) produced lambda DNA fragments up to 10 kb with an approximately 2.2-fold-lower error rate (5.53 x 10(-6)) than Taq DNA polymerase (11.98 x 10(-6)). Uniquely, Neq DNA polymerase also amplified lambda DNA fragments using dUTP (in place of dTTP) or dITP (partially replaced with dGTP). To increase PCR efficiency, Taq and Neq DNA polymerases were mixed in different ratios; a ratio of 10:1 efficiently facilitated long PCR (20 kb). In the presence of dUTP, the PCR efficiency of the enzyme mixture was two- to threefold higher than that of either Taq and Neq DNA polymerase alone. These results suggest that Neq DNA polymerase and Neq plus DNA polymerase (a mixture of Taq and Neq DNA polymerases) are useful in DNA amplification and PCR-based applications, particularly in clinical diagnoses using uracil-DNA glycosylase.
Applied and environmental microbiology 10/2008; 74(21):6563-9. · 3.69 Impact Factor
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ABSTRACT: In this study, the gene encoding Bacillus sp. HJ171 uracil-DNA glycosylase (Bsp HJ171 UDG) was cloned and sequenced. The Bsp HJ171 UDG gene consists of a 738-bp DNA sequence, which encodes for a protein that is 245-amino-acid residues in length. The deduced amino acid sequence of the Bsp HJ171 UDG had a high sequence similarity with other bacterial UDGs. The molecular mass of the protein derived from this amino acid sequence was 27.218 kDa. The Bsp HJ171 UDG gene was expressed under the control of a T7lac promoter in the pTYB1 plasmid in Escherichia coli BL21 (DE3). The expressed enzyme was purified in one step using the Intein Mediated Purification with an Affinity Chitin-binding Tag purification system. The optimal temperature range, pH, NaCl concentration, and KCl concentration of the purified enzyme was 20-25 degrees C, 8.0, 25 and 25 mM, respectively. The half-life of the enzyme at 40 degrees C and 50 degrees C were approximately 131 and 45 s, respectively. These heat-labile characteristics enabled Bsp HJ171 UDG to control carry-over contamination in the polymerase chain reaction product (PCR) without losing the PCR product.
Applied Microbiology and Biotechnology 08/2008; 80(5):785-94. · 3.42 Impact Factor
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ABSTRACT: Owing to its selective uracil-excision property, uracil-DNA glycosylase (UDG) has been widely utilized in diagnostic PCR applications as an effective decontamination method. Since mesophilic UDGs in PCR has been shown to degrade not just contaminant DNA but also target amplicon, there has been an increase in demand for cold-active UDGs. We characterized UDG from Photobacterium aplysiae GMD509 (Pap GMD509 UDG) expressed in Escherichia coli BL21 (DE3). The optimal temperature range of the enzyme was 25–30 °C, which is considerably lower than any other reported UDG, and the half-life of the enzyme at 40 °C and 50 °C was approximately 77 s and 33 s, respectively. These results clearly demonstrate the fragility of this enzyme upon heating. In addition, we compared the carryover contamination control property of Pap GMD509 UDG with other commercialized UDGs. The results indicate that Pap GMD509 UDG is capable of degrading contaminant DNA without a preincubation step before the main PCR reaction. These attributes imply that the Pap GMD509 UDG is a highly adequate enzyme to prevent carryover contamination during PCR.
Enzyme and Microbial Technology.
