• BMB Reports
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• 제38권1호
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• pp.24-27
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• 2005

The role of 3' exonuclease excision in DNA polymerization was evaluated for primer extension using inert allele specific primers with exonuclease-digestible ddNMP at their 3' termini. Efficient primer extension was observed in amplicons where the inert allele specific primers and their corresponding templates were mismatched. However, no primer-extended products were yielded by matched amplicons with inert primers. As a control, polymerase without proofreading activity failed to yield primer extended products from inert primers regardless of whether the primers and templates were matched or mismatched. These data indicated that activation was undertaken for the inert allele specific primers through mismatch proofreading. Complementary to our previously developed SNP-operated on/off switch, in which DNA polymerization only occurs in matched amplicon, this new mutation detection assay mediated by $exo^+$ DNA polymerases has immediate applications in SNP analysis independently or in combination of the two assays.

• BMB Reports
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• 제31권6호
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• pp.585-589
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• 1998

The herpes simplex virus type-1 (HSV-1)-encoded DNA polymerase consists of two subunits, the products of the UL30 and UL42 genes. UL30 and UL42 were coexpressed in Sf9 cells infected with recombinant baculoviruses carrying the two genes. The UL30 and UL42 gene products remained tightly associated throughout the purification, which led to a near homogeneous heterodimer composed of the DNA polymerase and UL42 protein. The DNA polymerase-UL42 protein heterodimer, purified from the recombinant baculovirus-infected Sf9 cells, showed the same high degree of processivity of deoxynucleotide polymerization as the enzyme purified from the HSV-1 infected primate cells. Like the latter, it contained a 3'-5' exonuclease activity that specifically hydrolyzes an incorrectly matched nucleotide at the 3' terminus of a primer, thereby contributing to the fidelity of DNA replication.

• 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.

• 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.

• BMB Reports
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• 제33권2호
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• pp.126-132
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• 2000

The hepadnaviruses replicate their nucleic acid through a reverse transcription step. The MBP-fused HBV polymerase was expressed in E. coli and purified by using amylase affinity column chromatography. The purified protein represented DNA-dependent DNA polymerase activity. In this report, the MBP-HBV polymerase was shown to lack 3'$\rightarrow$5' exonuclease activity, like other retroviral RTs. The ratio of the insertion efficiency for the wrong versus right vase pairs indicates the misinsertion frequency (f). The nucleotide insertion fidelity (1/f), observed with the MBP-HBV polymerase and HIV-1 RT, was between 60 and 54,000, and between 50 and 73,000, respectively, showing that they are in close range. A relatively efficient nucleotide incorporation by the MBP-HBV polymerase was observed with a specificity of three groups: (1) A : T, T : A>C : G, G : C (matched pairs), (2) A : C, C : A>G: T, T : G (purine-pyrimidine and pyrimidine-purine mispairs), and (3) C : C, A : A, G : G, T : T>T : C, C : T>A : G, G : A (purine-purine or pyrimidine-pyrimidine mispairs), and their order is (1)>(2)>(3). The data from the nucleotide insertion fidelity by the MBP-HBV polymerase suggest that the HBV polymerase may be as error-prone as HIV-1 RT.

• 미생물학회지
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• 제31권2호
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• pp.141-147
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• 1993

백색 부후균인 plaurotus ostreatus 의 4가지의 균주로부터 각각 10.2 kb 와 7.2 kb (NFFA 2), 두 종류의 10.2 kb (NFFA 4001) 11.2 kb (NFFA 4501), 10.2kb 와 11.2 kb(KFCC 11635) 크기의 미토콘드리아 플라스미드들을 분리해 내었다. NFFA 2 의 변종인 NFFA 2m1 과 NFFA 2m2 에서는 이들 플라스미드가 관찰되지 않았다. 분별 원심분리에 의해 얻은 미토콘드리아에서 핵산을 추출하여 agarose gel 에서 전기영동시키면 플라스미드가 관찰되지 않았으나 proteinase K 를 처리하고 핵산을 추출하여 전기영동한 결과 이들 플라스미드가 관찰되었는에, 이는 플라스미드상에 단백질인 결합되어 있음을 시사한다. Proteinase K 를 처리한 플라스미드 DNA 와 exonuclease 를 반응시킨 결과, 이들 플라스미드들은 5'말단에 단백질이 결합된 성형 이중가닥 DNA의 구조를 가진 것을 확인하였다. 각 플라스미드들의 상호관계를 조사하기 위하여 Southern hybridization 을 수행한 결과 최소 3가지 종류의 플라스미드들로 분류할 수 있었다. 이중 한 그룹 (group I) 은 모든 P. ostreatus 균주들에서 공통적으로 발견되었다. Pleurotus 속의 5가지 다른 종(P. cornucopiae, P. florida, P. pulmonarius, P. sajor-cuja, P. spodoleucus) 의 균주들로부터 미토콘드리아 플라스미드들을 분리하였다. 이들은 한균주당 1-4 개 까지의 플라스미드를 가지며, 플라스미드의 크기는 7.2 kb-14kb 범위에 있었다. P. ostreatus 의 NFFA 2 의 10.2 kb(group I) 플라스미드와 hybridization 을 수행한 결과 P. cornucopiae ASI 2011을 제외한 다른 모든 균주들이 유사한 염기서열의 플라스미드를 갖고 있음을 알았다.

• BMB Reports
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• 제37권3호
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• pp.269-274
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• 2004

DNA polymerases without the 3' exonuclease function ($exo^-$ pol) have been widely used in sequencing and SNP genotyping. As a major player that expedited the coming of the postgenomic era, $exo^-$ polymerases worked remarkably well in the Human Genome Sequencing Project. However, it has become a challenge for this class of polymerases to efficiently screen the large number of SNPs that are found in the human genome. For more than three decades it has been recognized that polymerase fidelity varied according to the presence of proofreading activity that is mediated by its internal 3' exonuclease. Polymerases with proofreading function are famous for their high fidelity in DNA replication both in vivo and in vitro, but this well-known class of polymerases has been almost completely neglected in genetic analysis in the postgenomic era. We speculate that $exo^+$ polymerases may exhibit higher nucleotide identification ability when compared to $exo^-$ polymerases for an in vitro genetic analysis. With the application of $exo^+$ polymerases in SNP assays, a novel mechanism for the maintenance of DNA replication, the on/off switch, was discovered. Two new SNP assays have been developed to carry out genome-wide genotyping, taking advantage of the enzymatic properties of $exo^+$ polymerases. Furthermore, the on/off switch mechanism embodies a powerful nucleotide identification ability, which can be used to discriminate the bases that are upstream of the 3' terminus, and thus defines a new concept in de novo sequencing technology. Application of $exo^+$ polymerases to genetic analysis, and especially SNP assays, will greatly accelerate the pace to personalized medicine.

• Asian Pacific Journal of Cancer Prevention
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• 제15권6호
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• pp.2571-2576
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• 2014

Background: Published studies on the association between the exonuclease 1 (EXO1) Glu589Lys polymorphism and cancer susceptibility have yielded conflicting results. Thus, a meta-analysis of published studies was performed to assess the possible association. Materials and Methods: All eligible case-control studies published up to January 2013 on the association between the EXO1 Glu589Lys polymorphism and cancer susceptibility were identified by searching PubMed, Web of Science, Science Direct and hand search. Either fixed-effect or random-effect models were used to calculate pooled odds ratios (ORs) with 95% confidence intervals (CIs) using the Comprehensive Meta-Analysis software version 2.2. Results: A total of 4,391 cancer cases and 4,339 controls from 10 studies were included. Overall, no significant association between the EXO1 Glu589Lys polymorphism and cancer susceptibility was observed in either genetic model. However; in subgroup analyses by cancer type, a significant association between EXO1 Glu589Lys and lung cancer risk was found (Lys vs Glu: OR=1.23, 95%CI=1.07-1.41, $p_{heterogeneity}$=0.05). Further, subgroup analysis by ethnicity indicated that there was a statistically increased cancer risk in Asians (Lys vs Glu: OR=1.42, 95%CI=1.30-1.55, $p_{heterogeneity}$=0.07; Lys/Lys vs Glu/Glu: OR=1.93, 95%CI=1.20-3.12, $p_{heterogeneity}$=0.01; Lys/Lys+Glu/Lys vs Glu/Glu: OR=1.52, 95%CI=1.37-1.68, $p_{heterogeneity}$=0.42; Lys/Lys vs Glu/Lys+Glu/Glu: OR=1.68, 95%CI=1.07-2.65, $p_{heterogeneity}$=0.02). However, significant association was absent in Caucasians. Conclusions: This meta-analysis suggests, for the first time, that the EXO1 Glu589Lys polymorphism is not associated with overall cancer susceptibility, although marginal associations were found for lung cancer and Asian subgroups. Additional well-designed studies with larger sample size focusing on different ethnicities and cancer types are needed to confirm these findings.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1994년도 춘계학술대회 and 제3회 신약개발 연구발표회
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• pp.192-192
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• 1994

본 논문에서는 cytochrome P450 LA1 유전자의 5'-upsteam 조절부위의 클로닝을 실시하였다. pUC19 vector에 연결시킨 3.4 Kb 크기의 Pstl DNA조각을 Sst1, Nco1 제한 효소로 자른 뒤, Exonuclease III 를 처리하여 약 200bp 씩의 차이를 갖는 여러 크기의 plasmid들을 얻었다. 이 plasmid 의 핵산서열을 알아보기 위해 dideoxy nucletide를 이용한 sequencing방법으로 그 핵산서열의 결정 실험을 시도하였다. 또한, 다환상 방향족 탄화수소 화합물에 반응성을 갖는 C57BL/6N 생쥐와 반응성을 갖지않는 DBA/2N 생쥐에 있어 phase II 대사 효소인UDP-glucuronosyltransferase 효소활성에 대한 3-methylcholanthrene의 영향을 알아보기 위해 C57BL/6N 생쥐와 DBA/2N 생쥐에 각각 다른 농도의 3-methylcholanthrene을 처리하거나 각기 다른 시간에 3-methylcholanthrene를 처리하였다. 그 결과 UDP-glucuronosyl-transferase의 mRNA가 3-methylcholanthrene양의 증가에 따라, 처치시간이 길어짐에 따라 증가되어지며 그 mRNA위 크기는 약 2.2Kb 정도임을 알았다. 이로부터 UDP-ghucuronosyltransferase 또한 cytochrome P45O와 함께 다환상 방향족 탄화수소 화합물 조절인자를 통한 조절을 받을 것이며 phase I phase II 약물 대사 효소가 조절상 밀접한 관련을 가짐을 예측할 수 있었다.

• BMB Reports
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• 제30권3호
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• pp.188-193
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• 1997

Membrane lipid peroxidation processes yield products that may react with DNA to cause mutations. Lipid hydroperoxides from linoleic acid in the presence of transition metal ions caused strand breaks in plasmid DNA. DNA damage induced by reactive aldehydes known to be produced by decomposition of lipid hydroperoxides, such as 4-hydroxynonenal or rnalondialdehyde, was repaired by endonucleases and exonuclease III which resulted in the increase of single strand breaks in DNA. Lipid hydroperoxides as well as malondialdehyde and 4-hydroxynonenal also caused mutations in the pUC18 lacZ' gene when measured as a loss of ${\alpha}-cornplementation$. In conclusion. the lipid peroxidation could be an important intermediary event in DNA damage and mutation by oxidative stress.