• Journal of Microbiology and Biotechnology
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• 제14권5호
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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.

• Bulletin of the Korean Chemical Society
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• 제30권5호
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• pp.1031-1034
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• 2009

Fluorescence of quinolones including norfloxacin, ciprofloxacin and S- and R-ofloxacin is quenched upon association with single and double-stranded DNA (ss- and ds-DNA). The ratios of fluorescence intensity in the presence of DNA to its absent were plotted with respect to the DNA concentration to construct the Stern-Volmer plot. The slope of the Stern-Volmer plot become larger as the temperature is lowered, ensuring that the fluorescence quenching is static process, i.e., the fluorescence is quenched by formation of the non-fluorescent complex between quinolone and DNA. In the static quenching mechanism, the quenching constant which is equivalent to the slope of the Stern-Volmer plot, is considered as the equilibrium constant for the association of quinolones and DNA. From the temperature-dependent equilibrium constant, ${\Delta}H^0\;and\;{\Delta}S^0$ was obtained using the van’t Hoff relation. In general, association of the quinolone with ds- as well as ss-DNA is energetically favorable (an exothermic) process while the entropy change was unfavorable. Due to the steric effect of the substituents, the effect of the quinolone ring is smaller on the ss-DNA compared to ds-DNA.

• The Korean Journal of Physiology and Pharmacology
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• 제27권5호
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• pp.427-436
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• 2023

Mitotic arrest deficient 2 like 2 (Mad2L2, also known as Mad2B), the human homologue of the yeast Rev7 protein, is a regulatory subunit of DNA polymerase ζ that shares high sequence homology with Mad2, the mitotic checkpoint protein. Previously, we demonstrated the involvement of Mad2B in the cisplatin-induced DNA damage response. In this study, we extend our findings to show that Mad2B is recruited to sites of DNA damage in human cancer cells in response to cisplatin treatment. We found that in undamaged cells, Mad2B exists in a complex with Polζ-Rev1 and the APC/C subunit Cdc27. Following cisplatin-induced DNA damage, we observed an increase in the recruitment of Mad2B and Cdc20 (the activators of the APC/C), to the complex. The involvement of Mad2B-Cdc20-APC/C during DNA damage has not been reported before and suggests that the APC/C is activated following cisplatin-induced DNA damage. Using an in vitro ubiquitination assay, our data confirmed Mad2B-dependent activation of APC/C in cisplatin-treated cells. Mad2B may act as an accelerator for APC/C activation during DNA damage response. Our data strongly suggest a role for Mad2B-APC/C-Cdc20 in the ubiquitination of proteins involved in the DNA damage response.

• Rapid Communication in Photoscience
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• 제3권4호
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• pp.67-69
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• 2014

Bis(2-thienyl)-diketopyrrolopyrrole having two $Zn^{II}$-cylcens (DPPCy) was synthesized. DPP-aggregates were constructed by self-organization of DPPCy and $dT_n$-DNAs. In the presence of L-ascorbic acid as an electron sacrifice reagent, the DPP aggregates immobilized on a gold electrode exhibit good anodic photocurrent responses as well as cathodic photocurrent responses in the presence of methyl viologen. The anodic photocurrent responses depend on the DNA lengths because of the formation of uniform DPP-aggregates corresponding to the DNA lengths. The present results show that photocurrent responses of the DPP-aggregates can be controlled by DNA lengths and electron sacrifice reagents.

• Journal of Microbiology and Biotechnology
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• 제12권6호
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• pp.1013-1016
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• 2002

Cyclo(L-prolyl-L-phenylalanyl) [cyclo(pro-phe)] was isolated from Streptomyces sp. AMLK-335 and found to inhibit DNA topoisomerase I activity. In a DNA relaxation assay using supercoiled pBR322 DNA, cyclo(pro-phe) inhibited the DNA topoisomerase activity more strongly than camptothecin, a known topoisomerase inhibitor. However, at a concentration of $10{\mu}M$, cyclo(pro-phe) produced a lower degree of DNA relaxation than camptothecin, therefore, the inhibition of topoisomerase I activity by cyclo(pro-phe) was also found to be dose dependent. Accordingly, the current results suggest that cyclo(pro-phe) may be a novel inhibitor of topoisomerase I.

• Bulletin of the Korean Chemical Society
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• 제30권2호
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• pp.297-301
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• 2009

We have developed the yttrium oxide (YNP) or ytterbium oxide (YbNP) nanoparticle/polymer matrices for the size-dependent separation of DNA ranging from 100 bp to 9,000 bp. High separation efficiency (> $10^6$ plates/m) and the baseline resolution for various DNA standards (100 bp, 500 bp, and 1 kbp DNA ladder) were obtained in 10 min with these matrices. The effects of concentrations of both polyethylene oxide (PEO) and nanoparticles were investigated and the highest performance was obtained at 0.02% PEO with 0.02% YNP or YbNP. Similar sieving power for both YNP and YbNP matrices was observed probably due to the similar sizes of nanoparticles, resulting in the formation of comparable sieving networks for DNA separation. For the reduction of electrosmotic flow, either dynamic or permanent coating of the capillary inner wall was compared and it turned out that PEO was superior to polyvinylpyrrolidone (PVP) or polyacrylamide (PAA) for better separation efficiency.

• 미생물학회지
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• 제55권3호
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• pp.191-198
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• 2019

H-NS는 대장균에서 DNA 결합 단백질로 수많은 유전자의 발현에 영향을 주는 것으로 잘 알려져 있다. DicA 단백질은 dicF, dicB의 발현을 억제하여 대장균의 분열을 조절한다. dicA의 발현에 Cnu, H-NS의 관여 여부는 CnuK9E 돌연변이가 $37^{\circ}C$에서 dicA의 발현을 억제하여 대장균이 길게 자라는 현상을 일으키며 처음 알려졌다. 하지만 Cnu와 H-NS 두 단백질이 어떻게 dicA의 발현을 조절하는지에 대한 분자적인 기작 연구는 잘 되어있지 않다. 본 연구에서 H-NS가 dicA와 dicC 유전자의 프로모터 부근에 염기서열 특이적으로 결합하며, $37^{\circ}C$ 보다 $25^{\circ}C$에서 DNA 더 잘 결합하는 것을 확인하였다. 그리고 EMSA를 통해 Cnu는 H-NS의 DNA 결합의 oligomeric state를 변화시키는 방식으로 작용하는 것을 보여주었다. In vivo transcription assay와 real time PCR을 통해 H-NS가 제거된 대장균에서 dicA 프로모터 활성이 높아지고, 분열 초기 dicA의 발현이 조절 받지 못하고 증가하는 것으로 보아, H-NS는 dicA의 발현에 억제자로서 기능한다.

• Journal of Photoscience
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• 제9권2호
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• pp.229-232
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• 2002

There are two distinct UV-responsive signaling pathways in UV-irradiated mammalian cells, i.e., the DNA damage-dependent and -independent pathways. The former occurs in nucleus and results in growth arrest and apoptosis via post-translational modification of p53. The latter is initiated by oxidative stress and/or by damages in cell membrane or cytoplasm, which activate signaling cascade through intracellular molecules including mitogen activated protein kinases (MAPK). In normal human fibroblastic cells, all of MAPK family members, extracellular signal-related kinases (ERK), c-Jun N-terminal kinases (JNK) and p38, were rapidly phosphorylated following UV-irradiation. ERK phosphorylation was suppressed by an inhibitor of receptor tyrosine kinases (RTK). As ERK usually responds to mitogenic stimuli from RTK ligands, UV-induced ERK phosphorylation may be linked to the proliferation of survived cells. In contrast, phosphorylation of JNK and p38, as well as apoptosis, were modulated by the level of UV-generated oxidative stress Therefore, JNK and p38 may take part in oxidative stress-mediated apoptosis. Phosphorylation of p53 at Ser and Thr residues are essential for stabilization and activation of p53. Among several sites reported, we confirmed phosphorylation at Ser-15 and Ser-392 after UV-irradiation. Both of these were inhibited by a phosphoinositide 3-kinase inhibitor, presumably due to the shutdown of signals from DNA damage to p53. Phosphorylation at Ser-392 was also sensitive to an antioxidant and a p38 inhibitor, suggesting that Ser-392 of p53 is one of the possible points where DNA damage-dependent and -independent apoptic signals merge. Thus, MAPK pathway links UV-induced intracellular signals to the nuclear responses and modifies DNA damage-dependent cellular outcome, resulting in the determination of cell death.

• 한국식품영양학회지
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• 제27권5호
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• pp.845-850
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• 2014

In the present study, the effects of extracts from Korean plants on the DNA damage response in HaCaT cells exposed to ultraviolet B (UVB) were investigated. The activity of cells treated for 24 hr with ethanol extracts from Vaccinium spp. (VS), and Vitis vinifera L (VV) alone was similar to that of the non-treated control, but gradually decreased at concentrations above $200{\mu}g/mL$. However, when post-incubation of UVB-exposed cells was carried out for 24 hr in medium containing VS or VV extracts, the cell activity increased in a concentration-dependent manner compared with that in the normal growth medium. The cell viability of UVB-exposed cells also increased when post-incubated in medium containing VS or VV extracts, in a concentration-dependent manner. Nuclear fragmentation analysis showed that post-incubation with VS or VV extracts decreased the UVB-induced apoptosis by about 10 and 13%, respectively, of that in cells post-incubated in growth medium. After 24 hr of post-incubation in medium containing VS or VV extracts, the level of CPD and 8-OHdG decreased in time- and concentration-dependent manners. Overall these results suggest that VS and VV extracts assist the survival of UVB-exposed cells, in accordance with the respective decrease in the levels of UVB-induced DNA damage.

• 한국동물학회:학술대회논문집
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• 한국동물학회 1995년도 한국생물과학협회 학술발표대회
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• pp.323.1-323
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• 1995