• BMB Reports
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• 제35권3호
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• pp.320-329
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• 2002

A gene, coined tay, for a thermostable DNA polymerase from the novel, extremely thermophilic bacterium Thermoanaerobacter yonseiensis was cloned and expressed in E. coli. Using a DNA polymerase homologous PCR product as a hybridization probe, tay was isolated and sequenced to consist of 2621 nucleotides that encode 872 amino acids. A database analysis showed that DNA polymerase, coined Tay, from T. yonseiensis shared a 39% to 47% identity in the amino acid sequence with those from other DNA polymerases. Tay was overexpressed in E. coli as a fusion protein with a poly-histidine tag at the C-terminus. It was purified by heat treatment, followed by a $Ni^{2+}$-chelate column. The molecular weight of purified Tay was approximately 97 kDa, as shown by SDS PAGE, and it showed high DNA polymerase activity and thermostability. However, it had no 3'$\rightarrow$5' exonuclease activity.

• Journal of Microbiology
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• 제35권4호
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• pp.264-270
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• 1997

The structure of plasmid mlp1, a linear 10.2kb mitochondrial plasmid of Pleurotus ostreatus NFF A2 was determined by restriction enzyme mapping and partial sequencing. The plasmid encodes at least two proteins; a putative RNA polymerase showing homology to yeast mitochondrial RNA polymerase and to viral-encoded RNA polymerases, and a putative DNA polymerase showing significant homology to the family B thpe DNA polymerases. It also contains terminal inverted repeat sequences at both ends which are longer than 274 bp. A 1.6 kb EcoRI restriction fragment of m1p1 containing the putative RNA polymerase gene did not hybridize to the nuclear or motochondrial genomes from P. ostreatus, suggesting that it may encode plasmidspecific RNA polymerase. The gene fragment also did not hybridize with the RNA polymerase gene (RPO41) from Saccaromyces cerevisiae. The relationship between genes in m1p1 and those in another linear plasmid pC1K1 of Claviceps purpurea was examined by DNA hybridization. The result indicates that the genes for DNA and RNA polymerases are not closely related with those in C. purpurea.

• 생명과학회지
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• 제19권3호
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• pp.305-310
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• 2009

플라스미드 pGKV21에는 229-nt single-strand DNA initiation (ssi) signal이 존재한다. Asymmetric PCR 기법으로 합성된 229-nt ssDNA 단편을 이용하여 실제로 RNA polymerase에 의한 priming ability와 protein interaction을 확인하였다. in vitro primer RNA 합성 실험 결과, 229-nt ssDNA 단편은 filamentous M13 phage의 주형 DNA에서와 비슷한 효율로 시발체 RNA를 합성하였으며, 이 반응은 strand-specific하게 이루어졌다. DNase I footprinting과 gel retardation 실험 결과, RNA polymerase와 SSB 단백질은 229-nt ssDNA 단편에 stable interaction을 하며, 시발체 RNA를 합성하였다. 또한, in vivo 조건 하에서 RNA polymerase의 저해제인 rifampicin을 처리하여 세포내에 ssDNA 중간체가 집적되는 정도를 비교하여 본 결과, 플라스미드 pGKV21은 rifampicin-sensitive RNA polymerase가 상보가닥 합성에 관여 함을 보여 주었다.

• 대한의생명과학회지
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• 제15권1호
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• pp.1-8
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• 2009

Human genomic DNA is continuously attacked by oxygen radicals originated from cellular metabolic processes and numerous environmental carcinogens. 2-deoxyribonolactone (dL) is a major type of oxidized abasic (AP) lesion implicated in DNA strand scission, mutagenesis, and formation of covalent DNA-protein crosslink (DPC) with DNA polymerase (Pol) ${\beta}$. We show here that human DNA polymerase (Pol)${\iota}$ and mitochondrial $Pol{\gamma}$ give rise to stable DNA-protein crosslink (DPC) formation that is specifically mediated by dL lesion. $Pol{\gamma}$ mediates DPC formation at the incised dL residue by its 5'-deoxyribose-5-phosphate (dRP) lyase activity, while $Pol{\gamma}$ cross links with dL thorough its intrinsic dRP lyase and AP lyase activities. Reactivity in forming dL-mediated DPC was significantly higher with $Pol{\gamma}$ than with $Pol{\iota}$. DPC formation by $Pol{\gamma}$, however, can be reduced by an accessory factor of $Pol{\gamma}$ holoenzyme that may attenuate deleterious effects of crosslink adducts on mitochondrial DNA. Comparative kinetic analysis of DPC formation showed that the rate of DPC formation with either $Pol{\iota}$ or $Pol{\gamma}$ was lower than that with $Pol{\beta}$. These results revealed that the activity of catalytic lyase in DNA polymerases determine the efficiency of DPC formation with dL damages. Irreversible crosslink formation of such DNA polymerases by dL lesions may result in a prolonged strand scission and a suicide of DNA repair proteins, both of which could pose a threat to the genetic and structural integrity of DNA.

• 대한의생명과학회지
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• 제11권2호
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• pp.103-108
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• 2005

2-Deoxyribonolactone (dL) arises as a major DNA damage induced by a variety of agents, involving free radical attack and oxidation of C1'-deoxyribose in DNA. We investigated whether dL lesions can be repaired in mammalian cells and the mechanisms underlying the role of DNA polymerase $\beta$ in processing of dL lesions. Pol $\beta$ appeared to be trapped by dL residues, resulting in stable DNA-protein cross-links. However, repair DNA synthesis at site-specific dL sites occurred effectively in cell-free extracts, but predominantly accompanied by long-patch base excision repair (BER) pathway. Reconstitution of long-patch BER demonstrated that FEN1 was capable of removing the displaced flap DNA containing a 5'-dL residue. Cellular repair of dL lesions was largely dependent on the DNA polymerase activity of Pol $\beta$. Our observations reveal repair mechanisms of dL and define how mammalian cells prevent cytotoxic effects of oxidative DNA lesions that may threaten the genetic integrity of DNA.

• 한국미생물·생명공학회지
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• 제37권1호
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• pp.17-23
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• 2009

PCR법을 이용하여 Thermus thermophilus HJ6 유래 DNA polymerase(Tod) 유전자를 클로닝하고 염기서열을 분석한 결과, ORF는 2,505개의 뉴클레오타이드로 구성되고 834개의 아미노산을 암호화하였다. 아마노산 서열을 바탕으로 상동성을 분석한 결과, Thermus thermophilus HB8 유래 DNA polymerase와 98%, Thermus aquaticus 유래 DNA polymorase와 86%의 identity를 나타내었다. 이 유전자를 박테리오파지 $\lambda$ 유래 온도감수성 프로모터(PR, PL)를 포함하는 pJLA503 벡터를 이용하여 대장균에서 발현하였다. 발현된 효소는 열처리, $HiTrap^{TM}$ Q column과 $HiPrep^{TM}$ Sephacryl S-200 HR 26/60 columun으로 정제하여 94 kDa의 단백질을 얻을 수 있었다. 정제된 효소의 DNA 중합 활성에 대한 최적온도는 $75{\sim}80^{\circ}C$이고 최적 pH가 9.0이었다. $Mg^{2+}$ and $Mn^{2+}$에 대한 최적 농도는 각각 2.5mM과 1mM이었고 효소활성은 2가 양이온의 존재 하에서는 활성화 되지만 1가양이온에서는 저 해되었다. Tod DNA 중합효소를 이용한 PCR 실험결과, Tod DNA 중합효소는 DNA 증폭 및 PCR 관련 기술에 응용 가능할 것으로 생각된다.

• Journal of Microbiology and Biotechnology
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• 제12권5호
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• pp.841-843
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• 2002

Pfu DNA polymerase from Pyrococcus furiosus was expressed in the E. coli periplasm, and the fully active polymerase was partially purified by applying osmotic shock, ammonium sulfate precipitation, and heat treatment. This method represents a new way of expressing and purifying functional Pfu DNA polymerase without the use of chromatography.

• BMB Reports
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• 제35권2호
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• pp.248-250
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• 2002

Discrimination between the amplification of mRNA and contaminating genomic DNA is a common problem when performing a reverse transcriptase-polymerase chain reaction (RT-PCR). Even after treatment of the samples with DNAse, it is possible that negative controls (samples in which no reverse transcriptase was added) will give positive results. This indicates that there was amplification of DNA, which was not generated during the reverse transcriptase step. The possibility exists that Taq DNA polymerase acts as a reverse transcriptase, generating cDNA from RNA during the PCR step. In order to test this hypothesis, we incubated samples with a DNAse-free RNAse after the cDNA synthesis. Comparison of the results that were obtained from these samples (incubated with or without DNAse-free RNAse) confirms that the reverse transcriptase activity of Taq DNA polymerase I is a possible source of false positive results when performing RT-PCR from intronless genes. Moreover, we describe here a simple and rapid method to overcome the false positive results that originate by this activity of Taq polymerase.

• 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.

• 미생물학회지
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• 제37권1호
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• pp.37-41
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• 2001

Pleurotus ostreatus에서는 10.2 kb와 7.2 kb의 미토콘드리아 선상 플라스미드 DNA가 존재함이 발견되었다. 이 플라스미드 DNA의 양쪽 5'말단에는 단백질이 공유 결합되어있다고 알려져 있다. 최근에 P.ostreatus의 10.2 kb 미토콘드리아 선상 플라스미드 DNA를 다른 곰팡이의 선상 플라스미드 DNA에 존재하는 open reading frame(ORF)와 비교 분석하여 Podospora anserina의 플라스미드 pAL2의 DNA polymerase 및 RNA polymerase 암호부위와 유사성이 있음이 확인되었다. 본 연구에서는 7.2 kb선상 DNA를 HindIII잘라서 얻은 2조각의 절편(4.7 kb, 2.3 kb)을 클로닝하고 염기 서열을 분석하였다. 클로닝된 7 kb 절편에는 3개의 ORF,즉 ORF1(2982 bp, 993 amino acids), ORF2(2703 bp, 900 amino acids), ORF3(771 bp, 256 amino acids)가 존재함을 확인하였다 또한, 이들의 ORF를 분석한 결과, DNA polymerase와 RNA polymerase 암호부위와 유사성 이 있음을 확인하였다.