• Journal of Microbiology and Biotechnology
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• 제8권6호
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• pp.669-675
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• 1998

Taq DNA polymerase exhibits a sizable drawback compared to the other thermophilic DNA polymerases in that it demonstrates lower proof-reading activity due to the deficiency of 3'-5'exonuclease activity. A study was undertaken to improve the 3'-5' exonuclease activity in the PCR of Taq DNA polymerase. The three-dimensional structural alignment of the polymerase and 3'-5' exonuclease domains from the pol I family DNA polymerases explains why Taq DNA polymerase has just a background level of 3'-5'exonuclease activity. A comparison indicated that the two polymerase domains are very similar in primary and tertiary conformations, even though Taq DNA polymerase carries a much shorter 3'-5'exonuclease domain than that of E. coli DNA polymerase I. Those two polymerase domains were interchanged between Taq DNA polymerase and E. coli DNA polymerase I. The 3'-5' exonuclease domain from E. coli DNA polymerase I was separated and pasted into the polymerase domain of Taq DNA polymerase I, which resulted in a functional fusion-Stoffel fragment. The 3'-5'exonuclease activity of the fusion-Stoffel fragment increased up to 48% of the value of the Klenow fragment, while that of Taq DNA polymerase remained at 6.0% of the Klenow fragment.

• Journal of Microbiology and Biotechnology
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• 제8권5호
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• pp.471-477
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• 1998

Taq DNA polymerase from Thermus aquaticus is very useful in the polymerase chain reaction. Taq DNA polymerase is classified in the pol I family, represented by E. coli DNA polymerase I. The three-dimensional structural alignment of 3'-5'exonuclease domains from the pol I family DNA polymerases explains why Taq DNA polymerase does not carry out proofreading in polymerase chain reactions. Three sequence motifs, Exo I, II, and III, must exist to carry out 3'-5'exonuclease activity for proof- reading by a 3'-5'exonuclease reaction, but these are abolished in Taq DNA polymerase. The key catalytic module in 3'-5'exonuclease is two metal ions chelated by four active-site carboxylic amino acids. Taq DNA polymerase was mutagenized to construct the catalytic module in the active site. The circular dichroism technique supported the formation of the catalytic module, and the radioactive assay showed that the 3'-5'exonuclease activity doubled in the mutant Taq DNA polymerase.

• Journal of Microbiology and Biotechnology
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• 제10권2호
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• pp.272-274
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• 2000

Temperate bacteriophage P2 has a nonessential gene called old(overcoming lysoginization defection). In the presence of old, the growth of the host (Escherichia coli) with recBC- genotype is ingibited, and another bacteriophage, lambda, cannot superinfect. The Old protein has been shown to possess an exonuclease actibity. Three mutant P2s(old 1, old 17, old 49) which did gene was coned into expression vectors to produce hexahistidine-tagged proteins. The proteins were affinity-purified and shown to lose its exonuclease activity on both double-stranded and single-stranded DNA substrates. Thus, it was concluded that the lambda exclusion was related to Old's exonuclease activity.

• BMB Reports
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• 제36권6호
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• pp.525-528
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• 2003

The role of 3' exonuclease excision in DNA polymerization was evaluated in primer extensions using 3' allele-specific primers that had exonuclease-digestible and exonuclease-resistant 3' termini. With exonuclease-digestible unmodified 3' mismatched primers, the exo+ polymerase yielded template-dependent products. Using exonuclease-resistant 3' mismatched primers, no primer-extended product resulted from exo+ polymerase. As a control, polymerase without proofreading activity yielded primer-dependent products from 3' mismatched primers. These data indicated that a successful removal of the mismatch is required for DNA polymerization from the 3' mismatched primers by exo+ polymerase. In addition to the well-known proofreading from this mismatch removal, the premature termination in DNA polymerization, due to the failure of the efficient removal of the mismatched nucleotides, worked as an off-switch in maintaining the high fidelity in DNA replication from exo+ polymerase.

• Journal of Microbiology and Biotechnology
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• 제9권4호
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• pp.381-385
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• 1999

Taq DNA polymerase from Thermus aquaticus has been shown to be very useful in a polymerase chain reaction. Taq DNA polymerase has a domain at the amino terminus (residues 1 to 290) that has 5'-3' exonuclease activity and a domain at the C-terminus that catalyzes the polymerase reaction. Taq DNA polymerase is classified into the Pol I family, which is represented by E. coli DNA polymerase I. The alignment of amino acid sequences for the 5'-3' exonuclease domains of the Pol I family DNA polymerases shows ten highly conserved carboxylic amino acids. Crystallographic studies suggested that six of the carboxylic amino acids are clustered within a 7 $\AA$ radius by chelating three metal ions in the active site. Those six carboxylic residues are mutagenized to alanines in order to better understand their function. All six carboxylic residues, Asp l8, Glu1l7, Asp1l9, Asp120, Asp142, and Aspl44, are crucial for catalysis of 5'-3' exonuclease.

• Journal of Microbiology and Biotechnology
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• 제14권5호
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• pp.1022-1030
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• 2004

The gene encoding Aquifex pyrophilus (Apy) DNA polymerase was cloned and sequenced. The Apy DNA polymerase gene consists of 1,725 bp coding for a protein with 574 amino acid residues. The deduced amino acid sequence of Apy DNA. polymerase showed a high sequence homology to Escherichia coli DNA polymerase I-like DNA polymerases. It was deduced by amino acid sequence alignment that Apy DNA polymerase, like the Klenow fragment, has only the two domains, the $3'{\rightarrow}5'$ exonuclease domain and the $5'{\rightarrow}3'$ polymerase domain, containing the characteristic motifs. The Apy DNA polymerase gene was expressed under the control of T7lac promoter on the expression vector pET-22b(+) in E. coli. The expressed enzyme was purified by heat treatment, and Cibacron blue 3GA and $UNO^{TM}$ Q column chromatographies. The optimum pH of the purified enzyme was 7.5, and the optimal concentrations of KCl and $Mg^{2+}$ were 20 mM and 3 mM, respectively. Apy DNA polymerase contained a double strand-dependent $3'{\rightarrow}5'$ proofreading exonuclease activity, but lacked any detectable $5'{\rightarrow}3'$ exonuclease activity, which is consistent with its amino acid sequence. The somewhat lower thermostability of Apy DNA polymerase than the growth temperature of A. pyrophilus was analyzed by the comparison of amino acid composition and pressure effect.

• Journal of Microbiology and Biotechnology
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• 제28권9호
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• pp.1467-1472
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• 2018

In this study, DNA-templated copper nanoclusters (DNA-CuNCs) were used to detect exonuclease III (Exo III) activity, which is important for the diagnosis and therapy of several diseases. The results of this study showed that Exo III was affected by the concentrations of magnesium ions and sodium ions, and its optimal conditions for cleavage were $5mM\;Mg^{2+}$ and less than $25mM\;Na^+$. With a blunt-end DNA, more than 98% of DNA was digested by Exo III. As expected, with two or four cytosines in the terminal position of a 4-base overhanging DNA such as 5'-GGCC-3' and 5'-CCCC-3', there was little cleavage by Exo III compared with a blunt-end DNA.

• BMB Reports
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• 제32권5호
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• pp.492-496
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• 1999

We have investigated the biochemical properties of the herpes simplex virus type 1 (HSV-1) DNA polymerase without the UL42 protein (Pol), purified from insect cells infected with a recombinant baculovirus containing the UL30 gene. BSA and DTT have inhibitory effects on dAMP incorporation. Pol showed a greater turnover rate of steady-state single nucleotide incorporation at 12 mM $MgCl_2$ than at 2 mM $MgCl_2$. However, it showed a greater processivity of DNA synthesis at lower $MgCl_2$ concentration (1 mM, 2 mM) than at a higher $MgCl_2$ concentration (12.5 mM). These results are consistent with a slow DNA dissociation at lower $MgCl_2$ concentrations. Pol does not incorporate a correct nucleotide into the primer with an incorrect nucleotide at the end; instead, it preferentially excises the incorrect nucleotide at the 3' end of the primer. Pol has DNA polymerase activity at pHs 6.5 and 7.5 but little at pHs 5.5, 8.5, and 9.5. It has exonuclease activity at pHs 6.5, 7.5, and 8.5 but little at pHs 4.5, 5.5, and 9.5. The finding that Pol has exonuclease activity but not DNA polymerase at pH 8.5 suggests that DNA binds to Pol, but deoxynucleotide binding or incorporation does not occur at pH 8.5.

• 한국미생물·생명공학회지
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• 제38권2호
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• pp.158-162
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• 2010

고온균 Thermus thermophilus HJ6 유래의 N-말단 결실 Tod polymerase($\Delta$Tod polymerase)는 온도 감수성 프로모터 (lambda pR and pL)를 포함하는 pJLA503 벡터를 이용하여 대장균에서 발현하였다. N-말단 250개 아미노산이 제거된 $\Delta$Tod polymerase는 5'$\rightarrow$3' exonuclease 활성은 없어지고 DNA 중합반응의 활성은 그대로 유지되었다. $\Delta$Tod polymerase는 $MgCl_2$의 존재 하에서 매우 효율적으로 역전사 반응과 PCR 반응을 수행하였다. 또한 $\Delta$Tod polymerase는 one-step RT-PCR 반응에서 Taq polymerase 보다 높은 cDNA 증폭 효율을 나타내었다.

• Journal of Microbiology and Biotechnology
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• 제15권6호
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• pp.1359-1367
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• 2005

The gene encoding Pyrobaculum arsenaticum DNA polymerase (Par DNA polymerase) was cloned and sequenced. The gene consists of 2,361 bp coding for a protein with 786 amino acid residues. The deduced amino acid sequence of Par DNA polymerase showed a high similarity to archaeal family B-type DNA polymerases (Group I), and contained all of the motifs conserved in the family B-type DNA polymerases for $3'{\rightarrow}5'$ exonuclease and polymerase activities. The Par DNA polymerase gene was expressed under the control of the T7lac promoter on the expression vector pET-22b(+) in Escherichia coli BL21-CodonPlus(DE3)-RP. The expressed enzyme was purified by heat treatment, and Cibacron blue 3GA and $Hirap^{TM}$ Heparin HP column chromatographies. The optimum pH of the purified enzyme was 7.5. The enzyme activity was activated by divalent cations, and was inhibited by EDTA and monovalent cations. The half-life of the enzyme at $95^{\circ}C$ was 6 h. Par DNA polymerase possessed associated $3'{\rightarrow}5'$ proofreading exonuclease activity, which is consistent with its deduced amino acid sequence. PCR experiment with Par DNA polymerase showed an amplified product, indicating that this enzyme might be useful in DNA amplification and PCR-based applications.