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 DNA fragments up to 10 kb with an approximately 2.2-fold-lower error rate (5.53 ؋ 10 ؊6 ) than Taq DNA polymerase (11.98 ؋ 10 ؊6 ). Uniquely, Neq DNA polymerase also amplified 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.DNA polymerase (EC 2.7.7.7) plays essential roles in cellular DNA replication and repair. In recent years, thermostable archaeal DNA polymerases have been identified in many species (6,7,11,19,20,34,35). On the basis of amino acid sequences, DNA polymerases can be classified into at least six distinct families (A, B, C, D, X, and Y) (27), and most archaeal DNA polymerases have been identified as members of family B, along with eukaryotic replicative DNA polymerases and Escherichia coli DNA polymerase II (6). Previously reported archaeal family B DNA polymerases from hyperthermophiles possess a 3Ј35Ј proofreading exonuclease activity and so are capable of carrying out PCR with higher fidelity than Thermus aquaticus (Taq) DNA polymerase. Another notable feature of archaeal family B DNA polymerases is the recognition of uracil in DNA templates, which causes DNA synthesis to stall (14,22). The archaeal family B DNA polymerases have a readahead function concerning uracil detection, based on an Nterminal pocket specific for uracil (12). In addition, archaeal family B DNA polymerases interact with another deaminated base, hypoxanthine (13).Archaea have been recognized as a third domain of living organisms, distinct from the Bacteria and Eukarya (38). From a phylogenetic perspective on the basis of rRNA sequences, Archaea have been classified into four phyla: Crenarchaeota, Euryarchaeota, Korarchaeota, and Nanoarchaeota (3, 17). Nanoarchaeum equitans, which belongs to Nanoarchaeota, is a nano-sized and hyperthermophilic anaerobe and is the firstknown obligate archaeal symbiont, which grows on the surface of a specific crenarchaeal host, Ignicoccus sp. strain KIN4/I (17, 18). The 490,885-bp N. equitans genome is one of the smallest microbial genomes and has been completely sequenced (37).N. equitans family B DNA polymerase (Neq DNA polymerase) is encoded by two open reading fra...
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.
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.
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.
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