• Journal of Microbiology and Biotechnology
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• v.8 no.6
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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.

• Korean Journal of Microbiology
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• v.29 no.4
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• pp.209-214
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• 1991

The PRD1 DNA polymerase is a small multi-functional enzyme containing conserved amino acid sequences shared by family B DNA polymerases. Thus the PRD1 DNA polymerase provides an useful model system with which to study structure-functional relationships of DNA polymerase molecules. In order to investigate the functional and structural roles of the highly conserved amino acid sequences, we have introduced three mutations into a conserved amino acid of the PRD1 DNA polymerase. Genetic complememtation study indicated that each mutation inactivated DNA polymerase catalytic activity.

• Korean Journal of Agricultural Science
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• v.41 no.3
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• pp.245-249
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• 2014

Among dozens of DNA polymerases cloned from thermophilic bacteria, Taq DNA polymerase from Thermus aquaticus has been most frequently used in polymerase chain reaction (PCR) that is being applied to gene cloning, DNA sequencing, gene expression analysis, and detection of infectious and genetic diseases. Since native Taq DNA polymerase is expressed at low level in T. aquaticus, recombinant Escherichia coli system was used to produce Taq DNA polymerase in a large amount. Taq DNA polymerase was expressed as a soluble form under the control of tac promoter in E. coli, and purified by heat treatment and ion exchange chromatographies. The purified Taq DNA polymerase was nearly homogeneous and exhibited a similar DNA amplification activity with a commercial Taq DNA polymerase.

• BMB Reports
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• v.31 no.5
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• pp.498-502
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• 1998

HBV polymerase shares several regions of amino acid homology with other DNA-directed and RNA-directed polymerases. The amino acid residues $Asp^{429}$, $Gly^{518}$, $Asp^{551}$, $Lys^{585}$, and $Gly^{641}$ in the conserved motifs A, B', C, D, and E in the polymerase domain of HBV polymerase were mutated to alanine or histidine by in vitro site-directed mutagenesis. Those mutants were overexpressed, purified, and analyzed against DNA-dependent DNA polymerase activity and affinity for DNA binding. All those mutants did not show DNA-dependent DNA polymerase activities indicating that those five amino acid residues are all critical in DNA polymerase activity. South-Western analysis shows that amino acid residues $ASp^{429}$ and $ASp^{551}$ are essential to DNA binding, and $Gly^{318}$ and $Gly^{585}$ also affect DNA binding to a certain extent.

• The Microorganisms and Industry
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• v.16 no.3
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• pp.6-14
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• 1990

PRDI DNA polymerase is the smallest member of the family B DNA polymerase (Jung et al., 1987). This DNA polyerase is specified by bacteriophage PRDI which infects a wide variety of gram-negative bacteria(Mindich and Bamford, 1988). Because PRDI is highly amenable to genetic and biochemical manipulation, it is a convenient model system with which to study structure-function relationships of DNA polymerase molecules. To determine the functional roles of the highly conserved regions of the family B DNA polymerases, we have initiated site-directed mutagenesis with PRD1 DNA polymerase, and our results show that mutations at the conserved regions within PRD1 DNA polymerase inactivate polymerase complementing activity and catalytic activity.

• Journal of Life Science
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• v.3 no.4
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• pp.194-208
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• 1993

DNA 복제시 중추적 단백질은 DNA 합성을 수행하는 DNA polymerase이다. 따라서 DNA polymerase의 구조 및 기능에 대한 연구는 DNA polymerase의 중합반응에 대한 기작을 비롯하여 교정 및 수선기능에 대한 정보를 얻게 함으로써 복잡한 DNA 복제 기적을 이해하는 첩경이 된다. Bacteriophage T7의 Gene 5 단백질은 T7 DNA polymerase로 Richardson group에 의해 처음으로 발견되었으며, E. coli의 12 KDa thioredoxin과 tight complex를 형성한다. T7 DNA polymerase의 클로닝은 분자생물학의 새로운 장을 열어준 중요한 의미를 지닌다 . 본 연구에서는 T7 DNA polymerase의 구조적, 기능적 특성을 파악하고 DNA 염기서열 분석에의 응용 및 DNA 염기서열 결정을 위한 새로운 전략 및 최근연구 동향에 대해 기술하였다.

• Korean Journal of Microbiology
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• v.30 no.2
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• pp.141-148
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• 1992

In order to overexpress bacteriophage PRD1 DNA polymerase in E. coli cells, the 2 kb HaeII fragment was isolated from phage genomic DNA. This fragment was then cloned into pEMBL/sup ex/ 3-expression vector. A specific 57bp deletion was performed by using uracil containing ss DNA and oligonucleotide spanning each region to remove an unwanted non-coding region. After this deletion, the PRD1 DNA polymerase gene is totally under the control of the vector promoter and SD sequence. Upon heat induction, a protein with an apparent size of 68 kdal was overexpressed as an active PRD1 DNA polymerase. The expression of PRD1 DNA polymerase was about 1% of total E. coli protein.

• Journal of Microbiology and Biotechnology
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• v.14 no.5
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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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• v.8 no.5
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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.

• BMB Reports
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• v.29 no.1
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• pp.38-44
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• 1996

Taq DNA polymerase from Thermus aquaticus has been shown to be very useful in the polymerase chain reaction method, which is being used for amplifying DNA. Not only does Taq DNA polymerase have high commercial value commercial value for the polymerase chain reaction application, but it is also important in studying DNA replication, because it is apparently an homologue to E. coli DNA polymerase I, which has long been used for DNA replication study (Lawyer et ai., 1993). The crystal structure determination of Taq DNA polymerase was initiated. An X-ray diffraction pattern breaks down a crystal structure into discrete sine waves in a Fourier series. The original shape of a crystal object in terms of electron density may be represented as the sum of those sine waves with varying amplitudes and phases in three dimensions. The molecular replacement method was initially employed to provide phase information for the structure of Taq DNA polymerase. The rotation search using the program MERLOT resulted in a solution peak with 5.4 r.m.s. PC-refinement of the X-PLOR program verified the result and also optimized the orientation angles. Next, the translation search using the X-PLOR program resulted in a unique solution peak with 7.35 r.m.s. In addition, the translation search indicated $P3_121$ to be the true space group out of two possible ones. The phase information from the molecular replacement was useful in the MIR phasing experiment.