• 약학회지
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• 제48권1호
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• pp.13-19
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• 2004

A bacterial expression vector for the human foamy virus (HFV) integrase was constructed and expressed in Escherichia coli. By two-step purification using a nickel-chelated column and a SP-sepharose chromatography; the HFV into-grase protein of 43 kDa was purified to near homogeneity, and used to investigate biochemical characteristics of the enzymatic activities, such as endonucleolytic and disintegration activities. Oligonucleotide substrates were specifically and efficiently cleaved by the purifed HFV integrase in the presence of Mn $^{+2}$, but not in the presence of Mg $^{+2}$, indicating that the HFV integrase is not able to use Mg $^{+2}$ as a cofactor Endonucleolytic reaction was almost completed in 60 min at 37 $^{\circ}C$. In addition, the maximum enzymatic activities were observed at 5 mM Mn $^{+2}$ in the buffer of which pH was from 7.0 to 9.0. The endonucleolytic activities were dose-dependently blocked in the addition of baicalein or chicolic acid which is a well-known inhibitor of human immunodeficiency virus integrase.

• BMB Reports
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• 제36권2호
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• pp.201-206
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• 2003

Two lymphoid-specific proteins, RAG1 and RAG2, are required for the initiation of the V(D)J recombination in vitro. The V(D)J cleavage that is mediated by RAG proteins at the border between the coding and signal sequences results in the production of a hairpin at the coding end and a double-stranded break at the signal end. Two hairpin coding ends are re-opened, modified, and sealed; whereas, the signal ends are directly ligated. Here I report that only RAG1 can carry out a distinct endonucleolytic activity in vitro using an oligonucleotide substrate that is tethered by a short single-stranded DNA. The purified RAG1 protein alone formed a nick at the near position to the recombination signal sequence. This endonucleolytic activity was eliminated by immunoprecipitation using the RAG1-specific antibody, and required the 3'-hydroxy group. All of the RAG1 mutants that were incapable of the nick and hairpin formation in the V(D)J cleavage analysis also showed this new endonucleolytic activity. This suggests that the nicking activity that was observed might be functionally different from the nick formation in the V(D)J cleavage.

• 약학회지
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• 제42권1호
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• pp.46-52
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• 1998

Retroviral integrase is required for integration of viral DNA into the host cell chromosome. Human immunodeficiency virus type-1 integrase was partially purified as a part of a fusion protein linked to a maltose-binding protein and characterized in terms of an endonucleolytic activity. The concentration of the fusion protein purified through an amylose column was about 12mg/ml. Indicating that the solubility of the fusion protein is highly increased by the presence of a maltose-binding protein, considering that the integrase protein alone is poorly solubilized. The endonucleolytic activity of the fusion protein was detected at 0.1 to 1.OmM $Mn^{++}$ ion, but not at any concentrations tested of $Mn^{++}$ ion.

• 약학회지
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• 제47권1호
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• pp.46-51
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• 2003

Baicalein and baicalin are flavonoid compounds isolated from medicinal herb Scutellaria baicalensis Georgi (Labiatae) and have been known to possess antiviral activities. In the present study, we investigated the in vitro effects of baicalein and baicalin on the three distinctive enzymatic activities of the human immunodeficiency virus type-1 (HIV-1) integrase-endonucleolytic, integration, and disintegration activities. Both compounds inhibited the three enzymatic activities in a dose-dependent manner. The 50％ inhibitory concentrations of baicalein and baicalin for endonucleolytic activities of HIV-1 integrase were 4.4$\pm$3.3 and 25.9$\pm$4.0$\mu$M, respectively. In general, baicalein exhibited nearly 6- to 10-fold stronger inhibition than baicalin for the three enzymatic activities. These data demonstrate that baicalein or baicalin can be used as a leading compound to develop anti-AIDS chemotherapeutic agents targeting to the HIV-1 integrase.

• BMB Reports
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• 제32권6호
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• pp.599-604
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• 1999

A highly conserved amino acid, glutamic acid (Glu), present at position 152 in the catalytic domain of the human immunodeficiency virus type 1 (HIV-1) integrase (IN) protein has been known to be critical for enzymatic function since substitution of Glu 152 with other residues results in a complete loss of enzymatic activities. In order to better understand the role of Glu 152 as a conserved residue in enzymatic action, intragenic second site mutations have been introduced around residue 152 of a mutant IN (E152A), and their biochemical properties were analyzed in terms of enzymatic activities. Disintegration activities were found to be significantly restored in several second site mutant INs, while integration activities were only recovered weakly. However, endonucleolytic activities were not discovered in all the mutant INs. These findings indicate that the second site mutations can partially restore that catalytic structure of the active site disturbed by the E152A mutation and lead to the regaining of integration and disintegration activities. In addition, it is also suggested that endonucleolytic activity requires a more accurate structure of the catalytic site than that for the integration and disintegration activities.

• BMB Reports
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• 제47권8호
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• pp.417-423
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• 2014

MicroRNAs (miRNAs) are a large family of post-transcriptional regulators, which are 21-24 nt in length and play a role in a wide variety of biological processes in eukaryotes. The past few years have seen rapid progress in our understanding of miRNA biogenesis and the mechanism of action, which commonly entails a combination of target degradation and translational repression. The target degradation mediated by Argonaute-catalyzed endonucleolytic cleavage exerts a significant repressive effect on target mRNA expression, particularly during rapid developmental transitions. This review outlines the current understanding of the mechanistic aspects of this important process and discusses several different experimental approaches to identify miRNA cleavage targets.

• BMB Reports
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• 제39권2호
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• pp.140-144
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• 2006

A deoxyriboendonuclease has been purified to near homogeneity from a fast growing mycobacterium species, M. smegmatis and characterized to some extent. The size of enzyme is about 43 kDa as determined by a denaturing gel analysis. It shows optimum activity at $32^{\circ}C$ in Tris-HCl buffer (pH 7.2) containing 2.5 mM of $MgCl_2$. Both EDTA and $K^+$ but not $Na^+$ inhibit its activity. Evidences show that the enzyme is not a restriction endonuclease but catalyzes the endonucleolytic cleavage of both the double- as well as the single-strand DNA non-specifically. It has been shown that the cleavage by this enzyme generates DNA fragments carrying phosphate groups at 5' ends and hydroxyl group at the 3' ends, respectively. Analysis reveals that no endonuclease having size and property identical to our deoxyriboendonuclease had been purified from M. smegmatis before. The property of our enzymes closely matches with the deoxyriboendonucleases purified from diverse sources including bacteria.

• Applied Biological Chemistry
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• 제37권1호
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• pp.56-63
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• 1994

오이모자이크 바이러스(CMV)의 외피단백질 유전자의 일정한 염기서열을 보유하는 부분과 CMV RNA에 대항한 헤머헤드(hammerhead) 구조의 ribozyme을 만드는 올리고뉴클 레오타이드(oligonucleotide, nt)를 DNA 합성기를 이용하여 제조하였다. 올리고뉴클레오타이드의 양쪽가닥을 서로 합친 후 제한효소 BamHl과 SacI으로 처리하여 플라스미드 pBS SK(+)에 삽입하였고 CMV 기질과 ribozyme 클론의 염기서열을 결정하여 확인하였다. 기질과 ribozyme 클론 $1\;{\mu}g$ BssHII이나 SspI으로 처리한후 T7 RNA 합성효소를 이용하여 튜브내에서 전사반응을 실시하였다. 제한효소 BssHII를 처리한 경우 만들어진 기질 RNA의 크기는 176 nt 였는데 50 nt의 CMV RNA 염기, 6 nt의 Xbal 제한효소 염기, 120 nt의 벡터에서 비롯된 염기를 포함한다. Ribozyme RNA의 크기는 164 nt인데 38 nt의 ribozyme 염기부분과 그외는 기질의 것과 같은 염기를 포함한다. CMV 기질 RNA는 ribozyme RNA에 의하여 특이적으로 절단되어 96 nt와 80 nt 두개의 조각을 만들었다. 이러한 특이적 절반은$37^{\circ}C$ 보다 $55^{\circ}C$에서 더 빠르게 일어났다. SspI으로 처리한 경우 만들어진 기질 RNA(2234 nt)도 역시 위치 ribozyme에 의해 두조각으로 절반되었으며 SspI 처리 후 만들어진 ribozyme RNA(2222 nt)에 의해서 특이적으로 절단되었다.

• Applied Biological Chemistry
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• 제38권6호
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• pp.522-527
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• 1995

흑조위축병 바이러스(RBSDV)에 대한 antiviral agent를 개발하기위하여 RBSDV 게놈 조각 3번을 절단하는 망치머리형 구조의 라이보자임을 설계하였다. 라이보자임과 기질의 oligonucleotides는 DNA 합성기로 합성 후 서로 합치고, pBluescript KS(+)에 삽입하였다. 라이보자임과 기질 RNA는 $T_3$ RNA polymerase로 기내 전사하여 각각 193과 183 nucleotide의 RNA를 얻었다. 기질 RNA는 라이보자임 RNA에 의해 $55^{\circ}C$에서 2개의 조각으로 절단되었으나 $37^{\circ}C$에서는 절단되지 않았다. 또한 RBSDV의 3번 조각 RNA도 동일한 라이보자임에 의해 절단되었다. 이러한 결과로 합성된 헤머해드 라이보자임은 기내 절단 능력을 가지며 형질전환 식물체에서 antiviral agent로 이용될 수 있을 것이다.