• BMB Reports
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• v.35 no.3
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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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• v.35 no.4
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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.

• Journal of Life Science
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• v.19 no.3
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• pp.305-310
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• 2009

Plasmid pGKV21 contains a 229-nucleotide (nt) single-strand DNA initiation (ssi) signal. Using asymmetric PCR, we prepared a small single-stranded (ss) DNA fragment of the ssi signal and, using the 229-nt ssDNA fragment, determined the requirements of RNA polymerase for priming and DNA-protein interaction. The ssi fragment prepared was able to generate primer RNAs with almost the same efficiency as the $M13{\Delta}lac182/229$ phage DNA. However, the cssi (complementary strand of the ssi signal) fragment could not synthesize primer RNAs. This result suggests that the 229-nt ssi signal functions in a strand specific manner. Gel retardation and DNase I footprinting demonstrated that the synthesized ssi fragment could interact with both E. coli RNA polymerase and SSB protein to synthesize primer RNA. In Escherichia coli [pWVAp], an addition of rifampicin resulted in an accumulation of ssDNA, indicating that the host-encoded RNA polymerase is involved in the conversion of ssDNA to double-stranded plasmid DNA.

• Biomedical Science Letters
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• v.15 no.1
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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.

• Biomedical Science Letters
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• v.11 no.2
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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.

• Microbiology and Biotechnology Letters
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• v.37 no.1
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• pp.17-23
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• 2009

The gene encoding Thermus thermophilus HJ6 DNA polymerase (Tod) was cloned and sequenced. The open reading frame (ORF) of the Tod gene was composed of 2,505 nucleotides and encoded a protein (843 amino acids) with a predicted molecular weight of 93,795 Da. The deduced amino acid sequence of Tod showed 98% and 86% identities to the Thermus thermophilus HB8 DNA pol and Thermus aquaticus DNA pol, respectively, The Tod gene was expressed under the control of the bacteriophage $\lambda$ promoters PR and PL on the expression vector pJLA503 in Escherichia coli strain BL21 (DE3) codon plus. The expressed enzyme was purified by heat treatment, $HiTrap^{TM}$ Q column, and $HiPrep^{TM}$ Sephacryl S-200 HR 26/60 column chromatographies. The optimal temperature and pH for DNA polymerase activity were found to be $75{\sim}80^{\circ}C$ and 9.0, respectively. The optimal concentrations of $Mg^{2+}$ and $Mn^{2+}$ were 2.5 mM and 1 mM, respectively. The enzyme activity was activated by divalent cations, and was inhibited by monovalent cations. The result of the PCR experiment with Tod DNA polymerase indicates that this enzyme might be useful in DNA amplification and PCR-based applications.

• Journal of Microbiology and Biotechnology
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• v.12 no.5
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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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• v.35 no.2
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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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• v.32 no.5
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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.

• Korean Journal of Microbiology
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• v.37 no.1
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• pp.37-41
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• 2001

Two linear plasmid-like DNAs, 10.2 kb and 7.2 kb were found in the mitochondria of P. ostreatus. They have covalently linked 5'-terminal proteins in both ends. Two continuous fragments of 4.7 kb and 2.3 kb from 7.2 kb DNA were cloned and sequenced. Two long open reading frames (ORF1; 2982 bp, 993 a.a and ORF2; 2703 bp, 900 a.a) and one short open reading frame(ORF3; 771 bp, 256 a.a) were found in the 7.2 kb plasmid. The putative ORF1 and ORF2 have conserved motifs of DNA polymerases and RNA polymerases, respectively, while the ORF3 has homologous regions with phosphatase from Plasmodium, and also with adhesine from Mycoplasma.