Unique substrate spectrum and PCR application of Nanoarchaeum equitans family B DNA polymerase.

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.

Protein trans-splicing and characterization of a split family B-type DNA polymerase from the hyperthermophilic archaeal parasite Nanoarchaeum equitans.

Nanoarchaeum equitans family B-type DNA polymerase (Neq DNA polymerase) is encoded by two separate genes, the large gene coding for the N-terminal part (Neq L) of Neq DNA polymerase and the small gene coding for the C-terminal part (Neq S), including a split mini-intein sequence. The two Neq DNA polymerase genes were cloned and expressed in Escherichia coli individually, together (for the Neq C), and as a genetically protein splicing-processed form (Neq P). The protein trans-spliced Neq C was obtained using the heating step at 80 degrees C after the co-expression of the two genes. The protein trans-splicing of the N-terminal and C-terminal parts of Neq DNA polymerase was examined in vitro using the purified Neq L and Neq S. The trans-splicing was influenced mainly by temperature, and occurred only at temperatures above 50 degrees C. The trans-splicing reaction was inhibited in the presence of zinc. Neq S has no catalytic activity and Neq L has lower 3'-->5' exonuclease activity; whereas Neq C and Neq P have polymerase and 3'-->5' exonuclease activities, indicating that both Neq L and Neq S are needed to form the active DNA polymerase that possesses higher proofreading activity. The genetically protein splicing-processed Neq P showed the same properties as the protein trans-spliced Neq C. Our results are the first evidence to show experimentally that natural protein trans-splicing occurs in an archaeal protein, a thermostable protein, and a family B-type DNA polymerase.

Cloning and expression of a DNA ligase from the hyperthermophilic archaeon Staphylothermus marinus and properties of the enzyme.

The gene encoding Staphylothermus marinus DNA ligase (Sma DNA ligase) was cloned and sequenced. The gene contains an open reading frame consisting of 1836bp, which encodes for 611 amino acid residues. Upon alignment of the entire amino acid sequence, Sma DNA ligase showed a high degree of sequence homology with the hyperthemophilic archaeal DNA ligases, 67% identity with Aeropyrum pernix K1, and 40% identity with both Pyrococcus abyssi and Thermococcus kodakarensis. An extremely high sequence identity was observed in the six conserved motifs indicative of DNA ligase. The Sma DNA ligase gene was expressed under the control of the T7lac promoter on the pET-22b(+) in Escherichia coli BL21-CodonPlus(DE3)-RIL. The expressed enzyme was then purified by heat treatment followed by ion exchange and metal affinity column chromatography. The enzyme was activated by both Mg(2+) and Mn(2+), and its activity was inhibited by Ca(2+) and Zn(2+). Sma DNA ligase can utilize both ATP and ADP as cofactors. The half-life of the enzyme at 100 degrees C was determined to be approximately 2.8h. The enzyme catalyzed cohesive-end intramolecular and intermolecular joining and blunt-end intermolecular joining in the presence of tricine-NaOH buffer and Mn(2+), using either ATP or ADP.

Mutational analyses of the thermostable NAD+-dependent DNA ligase from Thermus filiformis.

The crystal structure of NAD+-dependent DNA ligase from Thermus filiformis (Tfi) revealed that the protein comprised four structural domains. In order to investigate the biochemical activities of these domains, seven deletion mutants were constructed from the Tfi DNA ligase. The mutants Tfi-M1 (residues 1-581), Tfi-M2 (residues 1-448), Tfi-M3 (residues 1-403) and Tfi-M4 (residues 1-314) showed the same adenylation activity as that of wild-type. This result indicates that only the adenylation domain (domain 1) is essential for the formation of enzyme-AMP complex. It was found that the zinc finger and helix-hairpin-helix (HhH) motif domain (domain 3) and the oligomer binding (OB)-fold domain (domain 2) are important for the formation of enzyme-DNA complex. The mutant Tfi-M1 alone showed the activities for in vitro nick-closing and in vivo complementation in Escherichia coli as those of wild-type. These results indicate that the BRCT domain (domain 4) of Tfi DNA ligase is not essential for the enzyme activity. The enzymatic properties of Tfi-M1 mutant (deleted the BRCT domain) were slightly different from those of wild-type and the nick-closing activity of Tfi-M1 mutant was approximately 50% compared with that of wild-type.

Enhanced expression of the gene for beta-glycosidase of Thermus caldophilus GK24 and synthesis of galacto-oligosaccharides by the enzyme.

The gene (bglT) encoding Tca beta-glycosidase (Thermus caldophilus GK24 beta-glycosidase) was overexpressed under the control of the trp promoter on a high-copy-number plasmid, pTRPES, in Escherichia coli W3110. The purified Tca beta-glycosidase enzyme was used in a galactosyl-transfer reaction to synthesize galacto-oligosaccharides from lactose. The optimum temperature and pH for the enzyme to synthesize galacto-oligosaccharides from 30% (w/v) lactose were 80 degrees C and 6.0, respectively. The major product of the reaction was a trisaccharide. The thermostable Tca beta-glycosidase produced galacto-oligosaccharides efficiently during the hydrolysis of lactose.