• Title/Summary/Keyword: ssDNA binding

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Comparative Study of Nucletic Acid Binding of the Purified RBF Protein and Its Inhibition of PKR phosphorylation (RBF정제단백질의 핵산결합도 및 PKR효소의 인산화억제효과의 비교에 관한 연구)

  • 박희성;김인수
    • Journal of Life Science
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    • v.8 no.2
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    • pp.119-125
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    • 1998
  • Column-purified double-stranded RNA binding factor (RBF) protein was tested for its binding affinity for the different forms of nucleic acids structure such as single-stranded(ss) and double-stranded(ds)RNA and ss- and dsDNA. The RBF protein was incubated with each of these nucleic acid structures in separate reactions and its comparative binding affnity was visualized by SDS-polyacrylamide gel electrophoresis. The RBF protein bound to the dsRNA molecule to form a tight RNA:protein complex in agreement with previous studies, but not to the other nucleic acid molecules confirming its distinctive affinity for the dsRNA structure. In phosphorylation assay in vito, the purified RBF protein significantly inhibited the autophosphorylation of the PKR derived from not only human but mouse source in the presence of poly(I):poly(C). It is suggesting that PKR vs. RBF is similarly under a competitive interaction among different eukaryotic organisms during protein synthesis.

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Homologous Expression and T3SS-Dependent Secretion of TAP-Tagged Xo2276 in Xanthomonas oryzae pv. oryzae Induced by Rice Leaf Extract and Its Direct In Vitro Recognition of Putative Target DNA Sequence

  • Kim, Seunghwan;Nguyen, Thi-Dieu-Hanh;Lee, Joohee;Hong, Myoung-Ki;Pham, Tan-Viet;Ahn, Yeh-Jin;Lee, Byoung-Moo;Han, Ye Sun;Kim, Dong-Eun;Kim, Jeong-Gu;Kang, Lin-Woo
    • Journal of Microbiology and Biotechnology
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    • v.23 no.1
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    • pp.22-28
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    • 2013
  • Xo2276 is a putative transcription activator-like effector (TALE) in Xanthomonas oryzae pv. oryzae (Xoo). Xo2276 was expressed with a TAP-tag at the C-terminus in Xoo cells to enable quantitative analysis of protein expression and secretion. Nearly all TAP-tagged Xo2276 existed in an insoluble form; addition of rice leaf extracts from a Xoosusceptible rice cultivar, Milyang23, significantly stimulated secretion of TAP-tagged Xo2276 into the medium. In a T3SS-defective Xoo mutant strain, secretion of TAPtagged Xo2276 was blocked. Xo2276 is a Xoo ortholog of Xanthomonas campestris pv. vesicatoria (Xcv) AvrBs3 and contains a conserved DNA-binding domain (DBD), which includes 19.5 tandem repeats of 34 amino acids. Xo2276- DBD was expressed in E. coli and purified. Direct in vitro recognition of Xo2276-DBD on a putative target DNA sequence was confirmed using an electrophoretic mobility shift assay. This is the first study measuring the homologous expression and secretion of Xo2276 in vitro using rice leaf extract and its direct in vitro binding to the specific target DNA sequence.

Flooding Stress-Induced Glycine-Rich RNA-Binding Protein from Nicotiana tabacum

  • Lee, Mi-Ok;Kim, Keun Pill;Kim, Byung-gee;Hahn, Ji-Sook;Hong, Choo Bong
    • 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.

Comparison of structure, function and regulation of plant cold shock domain proteins to bacterial and animal cold shock domain proteins

  • Chaikam, Vijay;Karlson, Dale T.
    • BMB Reports
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    • v.43 no.1
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    • pp.1-8
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    • 2010
  • The cold shock domain (CSD) is among the most ancient and well conserved nucleic acid binding domains from bacteria to higher animals and plants. The CSD facilitates binding to RNA, ssDNA and dsDNA and most functions attributed to cold shock domain proteins are mediated by this nucleic acid binding activity. In prokaryotes, cold shock domain proteins only contain a single CSD and are termed cold shock proteins (Csps). In animal model systems, various auxiliary domains are present in addition to the CSD and are commonly named Y-box proteins. Similar to animal CSPs, plant CSPs contain auxiliary C-terminal domains in addition to their N-terminal CSD. Cold shock domain proteins have been shown to play important roles in development and stress adaptation in wide variety of organisms. In this review, the structure, function and regulation of plant CSPs are compared and contrasted to the characteristics of bacterial and animal CSPs.

Effect of Cobaltous Chloride on the Repair of UV-induced DNA Damage (UV에 의해 손상된 DNA 회복에 미치는 cobaltous chloride의 효과)

  • Kim, Kug-Chan;Kim, Yung-Jin;Lee, Kang-Suk
    • Journal of Radiation Protection and Research
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    • v.20 no.2
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    • pp.71-78
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    • 1995
  • To develop methods to reduce radiation risk and apply such knowledge to improvement of radiation protection, the effects of cobaltous chloride known as bioantimutagen on the function of E. coli RecA protein involved in the repair of DNA damage were examined. The results demonstrated two distinct effects of cobaltous chloride on the RecA protein function necessary for the strand exchange reaction. Cobaltous chloride enhanced the ability of RecA protein to displace SSB protein from single-stranded DNA and the duplex DNA-dependent ATPase activity. RecA protein was preferentially bound with UV-irradiated supercoiled DNA as compared with nonirradiated DNA The binding of RecA protein to UV-irradiated supercoiled DNA was enhanced in a dose-dependent manner. It is likely that studies on the factors affecting repair efficiency and the DNA repair proteins may provide information on the repair of ionizing radiation-induced DNA damage and the mechanism for DNA radioprotection.

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Biochemical Properties of the Minichromosomal Maintenance Complex after the Phosphorylation by Cdc7 Kinase

  • Lee, Joon-Kyu
    • Animal cells and systems
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    • v.10 no.1
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    • pp.1-6
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    • 2006
  • Previous studies showed that Cdc7 kinase of Schizosaccharomyces pombe phosphorylated the minichromosome maintenance (Mcm) complex efficiently in the presence of spMcm10 protein. The biochemical properties of the phosphorylated Mcm complexes were examined to understand the activation mechanism of the Mcm complex by Cdc7 kinase. The phosphorylation of Mcm complex in the presence of spMcm10 by Cdc7 kinase did not affect the stability of the Mcm complex containing all six subunits, and the changes in the sedimentation properties were not observed after the phosphorylation. The reconstitution of the Mcm complex using the purified proteins showed that the phosphorylation of Mcm2 proteins did not affect the interactions between Mcm proteins. The phosphorylation of the Mcm2-7 complex at the same condition also did not activate the other biochemical activities such as DNA helicase and single stranded (ss) DNA binding activities. On the other hand, spMcm10 protein that was used for the stimulation of Mcm phosphorylation showed single stranded DNA binding activity, and inhibited the DNA helicase activity of the Mcm4/6/7 complex. These inhibitory effects were reduced by the addition of Cdc7 kinase, suggesting that the phosphorylation by Cdc7 kinase decreased the interactions between spMcm10 and the Mcm complex. Taken together, these results suggested that the phosphorylation by Cdc7 kinase alone is not sufficient for the remodeling and the activation of the Mcm complex, and the additional factors or the phosphorylations might be required for the activation of the Mcm complex.

Termination Sites of fleplication Are Anchored to the Nuclear Matrix during S Phase in Mouse LPI-1 Cells (생쥐 LP1-1 세포에서 S phase 동안 nuclear matrix에 고정되어 있는 복제 끝점)

  • 이형호;이갑열
    • The Korean Journal of Zoology
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    • v.37 no.3
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    • pp.318-323
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    • 1994
  • The association of replication origins/termini with nuclear matrix during S phase was investigated by DNase digestion of halo structures in synchronized mouse LPI-1 cells. The binding of parental DNA to nuclear matrix was constant throughout S phase. When nuclear matrix was isolated from the cells pulse-labeled with 3H-thvmidine at various stases of S phase, total 3H-labels associated with nuclear matrix were specifically higher at So, Sa and Ss stages than other stases of S phase, suggesting that the newly synthesized DNAs at those stages are not excluded out of nuclear matrix. Similar patterns were obsenred from the pulse-chase experiments, in which cells were pulse-labeled at each stage of S phase and further incubated for 1 hr. These results suggest that the replication origins and termini are fixed at the nuclear matrix, and that the nuclear matrix binding fractions of DNA at 3C-pause may contain a large population of replication origins and termination sites.

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Inhibition of Platelet Aggregation by Anti-thyroglobulin Monoclonal Antibodies (Thyroglobulin에 대한 단일클론 항체의 혈소판응집 저해 작용)

  • Shon Yun Hee;Kim Cheorl Ho;Jeon Byung Hun;Nam Kyung Soo
    • Journal of Physiology & Pathology in Korean Medicine
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    • v.18 no.2
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    • pp.534-537
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    • 2004
  • We produced twelve monoclonal antibodies(mAbs) against thyroglobulin and characterized the bindig profiles. Among them, three mAbs(TN-1, TN-2 and TN-3) were further characterized their binding specificities. TN-2 had a potent lupus anticoagulant activity and potentiated the anticoagulant effect of venom phospholipase A₂. he anticoagulant mechanism of TN-2 was elongation of the partial thromboplastin time and binding to phosphatidylserine which may have a pivot role in blood coagulation. And TN-2 was cross-reacted with ss-DNA and ds-DNA and had a characteristic of autoantibody. These results suggest that TN-2 may provide a useful tool for studying the correlation between autoimmune thyroiditis and its therapeutic effect.

The Schizosaccharomyces pombe Proteins that Bind to the Human HnRNPA1 Winner RNA

  • Kim, Jeong-Kook
    • Journal of Microbiology
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    • v.35 no.4
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    • pp.327-333
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    • 1997
  • Although extensively characterized in human cells, no heterogeneous nuclear ribonucleoprotein(hnRNP) has been found in the fission yeast Schizosaccharomyces pombe which is amenable to genetic studies and more similar to mammals than Saccharomyces cerevisiae is in terms of RNA processing. As a first step to characterize hnRNPs from S. pombe, attempt was made to find human hnRNP A1 homologs from S. pombe. The RNA molecule (A1 winner) containing the consensus high-affinity hnRNP A1 binding site (UAGGGA/U) was synthesized in vitro and used in an ultraviolet(UV) light-induced protein-RNA cross-linking assay. A number of S, pombe proteins bound to the A1 winner RNA. An approximately 50-kDa protein(p50) cross-linked more efficiently to the A1 winner RNA than other proteins. The p50 protein did not cross-link to a nonspecific RNA, but rather to the A1-5’ SS RNA in which the consensus 5’ splice junction sites of S. pombe introns were abolished. This suggests that the p50 protein, however, did not bind to the single-stranded DNA to shich the human hnRNP A1 could bind and be eluted with 0.5M NaCl. Further analysis should reveal more features of this RNA-binding protein.

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