• YAKHAK HOEJI
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• v.48 no.1
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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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• v.36 no.2
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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.

• YAKHAK HOEJI
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• v.42 no.1
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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.

• YAKHAK HOEJI
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• v.47 no.1
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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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• v.32 no.6
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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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• v.47 no.8
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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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• v.39 no.2
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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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• v.37 no.1
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• pp.56-63
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• 1994

Oligonucleotides for a conserved region of the coat protein gene of cucumber mosaic virus (CMV) and a hammerhead structure ribozyme against CMV RNA were synthesized using a DNA synthesizer. Both strands of oligonucleotides were annealed and restricted with BamHI/SacI, then cloned into a plasmid pBS SK (+). The cloned CMV substrate and ribozyme were sequenced to verify correct constructions. In vitro transcriptions were carried out by using T7 RNA polymerase with BssHII or SspI digests of $1\;{\mu}g$ of substrate and ribozyme clones. The size of substrate RNA was 176 nucleotides (nt) containing 50 nt of CMV RNA sequence, 6 nt of XbaI restriction site and 120 nt of vector-derived sequence in the case of BssHII digest. The size of ribozyme RNA was 164 nt containing 40 nt of ribozyme RNA sequence and same sequences of substrate. Substrate RNA was efficiently cleaved into two fragments (96 nt and 80 nt) by ribozyme RNA. This endonucleolytic cleavage occurred more efficiently at $55^{\circ}C$ than $37^{\circ}C$. SspI digest-derived substrate RNA (2234 nt) was also cleaved into two fragments by the same ribozyme. SspI digest-derived ribozyme RNA (2222 nt) cleaved the above substrate to two fragments. In vitro-tested ribozyme construct is being cloned into a plant transformation vector to develop virus-resistant plants.

• Applied Biological Chemistry
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• v.38 no.6
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• pp.522-527
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• 1995

To develop an antiviral agent for the rice black-streaked dwarf virus (RBSDV), a hammerhead type ribozyme, which has a potential target site on the genome segment 3, was designed. Oligonucleotides for the ribozyme and its substrate were synthesized, annealed, and cloned into a plasmid pBluescript II KS(+). Ribozyme and substrate RNAs were then synthesized by in vitro transcription with $T_3$ RNA polymerase, obtaining RNAs in expected size, 193 and 182 nucleotides, respectively. The substrate RNA was efficiently cleaved into two fragments when incubated with the ribozyme at $55^{\circ}C$, while the cleavage was not detected at $37^{\circ}C$. In addition, the segment 3 RNA of RBSDV was also cleaved into two fragments by the same ribozyme at $55^{\circ}C$. Taken together, our results demonstrated that the hammerhead ribozyme has an in vitro endonucleolytic activity and may be used as an antiviral agent in transgenic plants.