• BMB Reports
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• 제30권5호
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• pp.362-369
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• 1997

Multiple phosphorylation of the carboxy-terminal domain (CTD) of the largest subunit in RNA polymerase II (RNAP II) is thought to play an important role in the transcription cycle. The preinitiation complex in a partially purified complete transcription system suggested that RNA polymerase IIA containing unphosphorylated CTD is involved in complex assembly, whereas RNA polymerase IIO containing Ser and Thr phosphorylated CTD is not involved in preinitiation complex assembly. Recently a minimal transcription system was developed which requires chemically defined minimal components for its transcription: TBP, TFIIB, TFIIF, RNAP II and a supercoiled adenovirus-2 major late promoter (Ad-2 MLP). It would be using interesting to determine the consequence of CTD phosphorylation on preinitiation complex formation using the minimal transcription system. Contrary to the results from the partially purified complete transcription system, both RNA polymerase IIA and IIO are equally recruited in the preinitiation complex formation. The discrepancy may result from the two different assays used to determine complex formation, the use of chemically undefined complete and defined minimal transcription systems. This implicates that some factors in the complete transcription system are involved in the distinction between RNAP IIA and IIO in complex assembly. In addition multiple tyrosine phosphorylation of the CTD of RNAP II was prepared with c-Abl kinase and its recruiting ability in the preinitiation complex was examined. Compare with Ser and Thr phosphorylated RNAP IIO, Tyr phosphorylated RNAP IlOy forms a stable preinitiation complex in both the minimal and complete transcription systems. Based on these results, it seems that tyrosine phosphorylation of the CTD is important in the transcription cycle on the special subset of class-II promoter or has a different role in the transcription process.

• Journal of Microbiology and Biotechnology
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• 제18권8호
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• pp.1377-1385
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• 2008

The uracil-sensing domain in archaeal family B-type DNA polymerases recognizes pro-mutagenic uracils in the DNA template, leading to stalling of DNA polymerases. Here, we describe our new findings regarding the molecular, mechanism underpinning the stalling of polymerases. We observed that two successive deaminated bases were required to stall TNA1 and KOD1 DNA polymerases, whereas a single deaminated base was enough for stalling Pfu DNA polymerase, in spite of the virtually identical uracil-sensing domains. TNA1 and KOD1 DNA polymerases have a much higher extension rate than Pfu DNA polymerase; decreasing the extension rate resulted in stalling by TNA1 and KOD1 DNA polymerases at a single deaminated base. These results strongly suggest that these polymerases require two factors to stop DNA polymerization at a single deaminated base: the presence of the uracil-sensing domain and a relatively slow extension rate.

• Journal of Microbiology and Biotechnology
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• 제17권7호
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• pp.1090-1097
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• 2007

Genomic analysis of Thermococcus sp. NA1 revealed the presence of a 3,927-base-pair (bp) family B-type DNA polymerase gene, TNA1_pol. TNA1_pol, without its intein, was overexpressed in Escherichia coli, purified using metal affinity chromatography, and characterized. TNA1_pol activity was optimal at pH 7.5 and $75^{\circ}C$. TNA1_pol was highly thermostable, with a half-life of 3.5h at $100^{\circ}C$ and 12.5h at $95^{\circ}C$. Polymerase chain reaction parameters of TNA1_pol such as error-rate, processivity, and extension rate were measured in comparison with rTaq, Pfu, and KOD DNA polymerases. TNA1_pol averaged one incorrect bp every 4.45 kilobases (kb), and had a processivity of 150 nucleotides (nt) and an extension rate of 60 bases/s. Thus, TNA1_pol has a much faster elongation rate than Pfu DNA polymerase with 7-fold higher fidelity than that of rTaq.

• 한국동물학회지
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• 제26권3호
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• pp.171-179
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• 1983

MMS와 자외선에 의한 DNA의 절제회복과 단사절단에 미치는 poly(ADP-ribose) polymerase의 저해제인 3-aminobenzamide의 영향을 CHO 세포를 재로로 조사하여 다음과 같은 결과를 얻었다. 1. MMS에 의한 비주기성 DNA 합성률과 DNA 단사 절단률은 이 저해제에 의해 모두 증가하였다. 이는 poly (ADP-ribose) polymeraserk MMS에 의해 유발된 염기 절제회복의 incision step를 억제하는 결과라 생각된다. 2. 자외선에 의한 비주기성 DNA 합성률과 DNA단사 절단률은 이 저해제에 의해 모두 감소하였다. 이는 poly(ADP-ribose) polymerase가 자외선에 의해 유발된 nucleotide 절제회복의 incision step을 돕는 작용을 하는 결과로 생각된다. 3. MMS와 자외선을 복합처리한 실험군에서는, DNA 단사 절단률은 이 저해제에 의해 영향을 받지 않았으며, 비주기성 DNA 합성률은 자외선 단독 처리군의 수준으로 증가되었다. 이는, 이 저해제가 MMS와 자외선으로 유발된 절제회복의 incision step에는 독립적으로 작용하며, 그 이후의 단계에서, MMS에 의해 부분적으로 불활성화 되었던 pyrimidine dimers의 절제를 완전하게 해주는 것으로 해석된다.

• Asian Pacific Journal of Cancer Prevention
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• 제14권9호
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• pp.5489-5494
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• 2013

In the current study we aimed to show the common YMDD motif mutations in viral polymerase gene in chronic hepatitis B patients during lamivudine and adefovir therapy. Forty-one serum samples obtained from chronic hepatitis B patients (24 male, 17 female; age range: 34-68 years) were included in the study. HBV-DNA was extracted from the peripheral blood of the patients using an extraction kit (Invisorb, Instant Spin DNA/RNA Virus Mini Kit, Germany). A line probe assay and direct sequencing analyses (INNO-LIPA HBV DR v2; INNOGENETICS N.V, Ghent, Belgium) were applied to determine target mutations of the viral polymerase gene in positive HBV-DNA samples. A total of 41 mutations located in 21 different codons were detected in the current results. In 17 (41.5%) patients various point mutations were detected leading to lamivudin, adefovir and/or combined drug resistance. Wild polymerase gene profiles were detected in 24 (58.5%) HBV positive patients of the current cohort. Eight of the 17 samples (19.5%) having rtM204V/I/A missense transition and/or transversion point mutations and resistance to lamivudin. Six of the the mutated samples (14.6%) having rtL180M missense transversion mutation and resistance to combined adefovir and lamivudin. Three of the mutated samples (7.5%) having rtG215H by the double base substituation and resistance to adefovir. Three of the mutated samples (7.5%) having codon rtL181W due to the missense transversion point mutations and showed resistance to combined adefovir and lamivudin. Unreported novel point mutations were detected in the different codons of polymerase gene region in the current HBV positive cohort fromTurkish population. The current results provide evidence that rtL180M and rtM204V/I/A mutations of HBV-DNA may be associated with a poor antiviral response and HBV chronicity during conventional therapy in Turkish patients.

• 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.

• 생명과학회지
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• 제11권4호
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• pp.371-378
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• 2001

The effects of the RM 60 series on DNA replication systems were examined by using simian virus 40 (SV40) DNA replication system in vitro. The RM 60 series inhibited the DNA replication in the initiation step rather than in the elongation step. Polymerase$\alpha$-primase activity and toposiomerase I activity study were performed subsequently, the RM 60 seies increased the polymerase $\alpha$-primase activity in a low dose, but decreased the activity in a higher dose. The topoisomerase I activity was inhibited by the RM 60 series. This result suggests that the RM 60 series might inhibit some molecules which are required to establish replication forks during the initiation step of the DNA replication.

• Journal of Microbiology and Biotechnology
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• 제10권6호
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• pp.881-884
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• 2000

A polymerase chain reaction (PCR) was performed to rapidly identify Lactobacillus plantarum from type strains and kimchi samples. The PCR experiments were carried out using specific oligonucleotide primer sets based on the 16S rRNA gene sequences of L. plantarum. The expected DNA amplificate of 419 bp was obtained when either purified DNA or whole cells of L. plantarum strains reacted with LP primers, yet not with any of the other strains. The PCR product was confirmed by DNA sequencing. Accordingly, since the PCR method used is simple, specific, and rapid, it will be useful for monitoring and evaluation L. plantarum in the mixed microbial population found in kimchi.

• Journal of Microbiology
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• 제44권1호
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• pp.126-128
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• 2006

This study confirms that Taq DNA polymerase could be contaminated with the $blaTEM-1_a$ gene. It also proposes two different methods that could be used to overcome DNA contamination: (i) DNase I treatment prior to PCR amplification; and (ii) the use of a highly purified Taq DNA polymerase which was devoid of detectable contamination.

• Journal of Microbiology
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• 제40권4호
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• pp.327-330
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• 2002

The purpose of this research was to test multiplex polymerase chain reaction in investigating the microbiological quality of the risky surfaces in social living places of a military base where over 15 thousand people live together. In 22 samples of 99, there were no bacteria. Only four of the samples contained Shigella, and one of them contained Salmonella, but 77 of the samples contained thermotolerant coliform organisms. There was no statistically significant difference among the microbiological quality of different sites and different equipment surfaces (p>0.05).