• Molecules and Cells
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• v.27 no.1
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• pp.47-54
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• 2009

A cDNA clone for a transcript preferentially expressed during an early phase of flooding was isolated from Nicotiana tabacum. Nucleotide sequencing of the cDNA clone identified an open reading frame that has high homology to the previously reported glycine-rich RNA-binding proteins. The open reading frame consists of 157 amino acids with an N-terminal RNA-recognition motif and a C-terminal glycine-rich domain, and thus the cDNA clone was designated as Nicotiana tabaccum glycine-rich RNA-binding protein-1 (NtGRP1). Expression of NtGRP1 was upregulated under flooding stress and also increased, but at much lower levels, under conditions of cold, drought, heat, high salt content, and abscisic acid treatment. RNA homopolymer-binding assay showed that NtGRP1 binds to all the RNA homopolymers tested with a higher affinity to poly r(G) and poly r(A) than to poly r(U) and poly r(C). Nucleic acid-binding assays showed that NtGRP1 binds to ssDNA, dsDNA, and mRNA. NtGRP1 suppressed expression of the fire luciferase gene in vitro, and the suppression of luciferase gene expression could be rescued by addition of oligonucleotides. Collectively, the data suggest NtGRP1 as a negative modulator of gene expression by binding to DNA or RNA in bulk that could be advantageous for plants in a stress condition like flooding.

• Molecules and Cells
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• v.19 no.1
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• pp.54-59
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• 2005

Deoxyribonuclease I (DNase I) is a divalent cation dependent endonuclease and thought to be a significant barrier to effective gene delivery. The only known DNase I-specific inhibitor is monomeric actin which acts by forming a 1:1 complex with DNase I. Its use, however, is restricted because of tendency to polymerize under certain conditions. We screened two random phage peptide libraries of complexity $10^8$ and $10^9$ for DNase I binders as candidates for DNase I inhibitors. A number of DNase I-binding peptide sequences were identified. When these peptides were expressed as fusion proteins with Escherichia coli maltose binding protein, they inhibited the actin-DNase I interaction ($IC_{50}=0.1-0.7{\mu}M$) and DNA degradation by DNase I ($IC_{50}=0.8-8{\mu}M$). Plasmid protection activity in the presence of DNase I was also observed with the fusion proteins. These peptides have the potential to be a useful adjuvant for gene therapy using naked DNA.

• Journal of the Korean Magnetic Resonance Society
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• v.25 no.3
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• pp.39-44
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• 2021

The CRISPR-Cas system provides adaptive immunity for bacteria and archaea against invading phages and foreign plasmids. In the Class 1 CRISPR-Cas system, multi-subunit Cas proteins assemble with crRNA to bind to DNA targets. To disarm the bacterial defense system, bacteriophages evolved anti-CRISPR (Acr) proteins that actively inhibit the host CRISPR-Cas function. Here we report the backbone resonance assignments of AcrIF7 protein that inhibits the type I-F CRISPR-Cas system of Pseudomonas aeruginosa using triple-resonance nuclear magnetic resonance spectroscopy. We employed various computational methods to predict the structure and binding interface of AcrIF7, and assessed the model with experimental data. AcrIF7 binds to Cas8f protein via flexible loop regions to inhibit target DNA binding, suggesting that conformational heterogeneity is important for the Cas-Acr interaction.

• BMB Reports
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• v.42 no.1
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• pp.53-58
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• 2009

The methyl-directed mismatch repair (MMR) mechanism has been extensively studied in vitro and in vivo, but one of the difficulties in determining the biological relationships between the MMR-related proteins is the tendency of MutL to self-aggregate. The properties of a stable MutL homologue were investigated using a thermostable MutL (TmL) from Thermotoga maritima MSB8 and whose size exclusion chromatographic and crosslinking analyses were compatible with a dimeric form of TmL. TmL underwent conformational changes in the presence of nucleotides and single-stranded DNA (ssDNA) with ATP binding not requiring ssDNA binding activity of TmL, while ADPnP-stimulated TmL showed a high ssDNA binding affinity. Finally, TmL interacted with the T. maritima MutS (TmS), increasing the affinity of TmS to mismatched DNA base pairs and suggesting that the role of TmL in the formation of a mismatched DNA-TmS complex may be a pivotal observation for the study of the initial MMR system.

• Journal of Life Science
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• v.14 no.5
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• pp.732-740
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• 2004

MCM proteins are essential for eukaryotic DNA replication, playing roles in the initiation and elongation of DNA replication. MCM proteins expression is much higher in malignant tissues than normal tissues. Several reports have indicated the usefulness of MCM proteins as markers of cancer cells in histopathological diagnosis. However, the cause of enhanced expression of MCM proteins in cancer cells remain to be clarified. The purpose of this study is to examine the relative transcriptional activities of human mcm gene promoters in cancer and normal cells. The minimal promoter region required for transcription of a luciferase reporter gene was contained an E-box and one E2F site. In addition, luciferase activities from mcm7 and mcm2 promoter/luciferase gene reporter constructs were significantly increased in cancer cells at 8 times compared with normal cells. E-box and E2F binding site in the promoter of mcm genes are responsible for different mechanism of transcription regulation on the cellular environment.

• BMB Reports
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• v.41 no.8
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• pp.575-580
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• 2008

Thymocyte-specific transcriptional regulatory systems can be used to better understand the relationship between transcription and V(D)J recombination during early T cell development. In this study, we generated transgenic mice expressing the transactivator Gal4-VP16 or the Gal4 DNA binding domain (Gal4-DBD) under the control of the lck proximal promoter, which is only active in immature thymocytes. From these studies Gal4-VP16 and Gal4-DBD expression was shown to significantly alter thymic cellularity and differentiation without significantly changing the $CD3^+$ thymocyte distribution. Furthermore, the presence of Gal4-VP16 or Gal4-DBD in the transgenic thymocytes retarded the mobility of the Gal4 DNA binding motif as determined by a gel mobility shift assay, suggesting that the developmental alteration did not affect the functional property of the transgenic proteins. These results indicated that lck promoter-driven Gal4-VP16 or Gal4-DBD expression did not affect $CD3^+$ mature thymocytes, thus this system can be applied to study transcriptional regulation of transresponder genes in bigenic mouse model thymocytes.

• Journal of Photoscience
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• v.3 no.2
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• pp.85-90
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• 1996

The binding ability of leaf nuclear extracts to the lighbresponsive elements (LREs) of cab promoters of Arabidopsis thaliana has been investigated. The cab promoters were fragmented with restr ction endonucleases into LRE that were identified by Mitra et al. [Plant Mol. Biol. 12, 169179 ( 1989)] and other small fragments. After end labeling with Klenow fragment, the fragments were assayed for binding with the leaf nuclear proteins that were prepared by solubilizing the purified nuclei with 0.5 M ammonium sulfate. The binding ability was assayed by mobility shift assay. To perform successful mobility shift assay, several factors affecting the interaction of protein with DNA were optimized before performing the assay. The LREs had several retardation bands. However, the other promoter fragments from the transcription start site to the far upstream region of the promoters had also retardation bands. No particular relationships could be found between the retardation band distributions and the loci of LRE. It is likely that the light-regulation of cab gene expression may be controlled by the multiple interactions of the regulatory protein factors with DNA motifs.

• BMB Reports
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• v.44 no.10
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• pp.680-685
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• 2011

The AT-hook motif is a small DNA-binding protein motif that has been found in the high mobility group of non-histone chromosomal proteins. The Arabidopsis genome contains 29 genes encoding the AT-hook motif DNA-binding protein (AHL). Recent studies of Arabidopsis genes (AtAHLs) have revealed that they might play diverse functional roles during plant growth and development. In this report, we mined 20 AHL genes (OsAHLs) from the rice genome database using AtAHL genes as queries and characterized their molecular features. A phylogenetic tree revealed that OsAHL proteins can be classified into 2 evolutionary clades. Tissue expression pattern analysis revealed that all of the OsAHL genes might be functionally expressed genes with 3 distinct expression patterns. Nuclear localization analysis using transgenic Arabidopsis showed that several OsAHL proteins are exclusively localized in the nucleus, indicating that they may act as architectural transcription factors to regulate expression of their target genes during plant growth and development.

• Animal cells and systems
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• v.2 no.4
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• pp.539-543
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• 1998

Hrp1, of Schizosaccharomyces pombe, is a new member of the SW12/SNF2 protein family that contains a chromodomain and a DNA binding domain as well as ATPase/7 helicase domains. This configuration suggests that Hrp1 could be a homolog of mouse CHD1, which is thought to function in altering the chromatin structure to facilitate gene expression. To understand the enzymatic nature of Hrp1 we purified the 6-Histidine-tagged Hrp1 protein (6$\times$His-Hrp1) to homogeneity from a S. pombe Hrp1-overexpressing strain and hen examined its biochemical properties. We demonstrate that the purified 6$\times$His-Hrp1 protein exhibited a DNA-binding activity with a moderate preference to the (A＋T)-rich tract in double-stranded NA via a minor groove interaction. However, we failed to detect any intrinsic DNA helicase activity from the purified Hrp1 like other SW12/SNF2 proteins. These observations suggest that the DNA binding activities of Hrp1 may be involved in the remodeling of the chromatin structure with DNA-dependent ATPase. We propose that Hrp1 may function in heterochromatins as other proteins with a chromo- or ATPase/helicase domain and play an important role in the determination of chromatin architecture.

• Journal of Web Engineering
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• v.14 no.15
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• pp.3206-3218
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• 2022

Helicobacter pylori (H. pylori) is a Gram-negative bacterium that colonizes the gastric mucosa and triggers various stomach diseases. H. pylori induces reactive oxygen species (ROS) production and DNA damage. The heterodimeric Ku70/Ku80 protein plays an essential role in the repair of DNA double-strand breaks (DSB). Oxidative stress stimulate apoptosis and DNA damage that can be repaired by Ku70/80. However, excessive reactive oxygen species (ROS) can cause Ku protein degradation, resulting in DNA fragmentation and apoptosis. α-lipoic acid (α-LA), which is found in organ meats such as liver and heart, spinach, broccoli, and potatoes, quenches free radicals, chelates metal ions, and reduces intracellular DNA damage induced by oxidative stress. Here, we investigated whether H. pylori decreases Ku70/80 and induces apoptosis, and whether α-LA inhibits changes induced by H. pylori. We analyzed ROS, DNA damage markers (γ-H2AX, DNA fragmentation), levels of Ku70/80, Ku-DNA binding activity, Ku80 ubiquitination, apoptosis indices (Bcl-2, Bax, apoptosis-inducing factor (AIF), and caspase-3), and viability in a human gastric epithelial adenocarcinoma cell line (AGS). H. pylori increased ROS, DNA damage markers, Ku80 ubiquitination, and consequently induced apoptosis. It also decreased nuclear Ku70/80 levels and Ku-DNA-binding activity; increased Bax expression, caspase-3 cleavage, and truncated AIF; but decreased Bcl-2 expression. These H. pylori-induced alterations were inhibited by α-LA. The antioxidant N-acetylcysteine and proteasome inhibitor MG-132 suppressed H. pylori-induced cell death and decreased nuclear Ku70/80 levels. The results show that oxidative stress induced Ku70/80 degradation via the ubiquitin-proteasome system, leading to its nuclear loss and apoptosis in H. pylori-infected cells. In conclusion, α-LA inhibited apoptosis induced by H. pylori by reducing ROS levels and suppressing the loss of Ku70/80 proteins in AGS cells.