• Molecules and Cells
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• v.40 no.8
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• pp.533-541
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• 2017

Engineered DNA-binding domains provide a powerful technology for numerous biomedical studies due to their ability to recognize specific DNA sequences. Zinc fingers (ZF) are one of the most common DNA-binding domains and have been extensively studied for a variety of applications, such as gene regulation, genome engineering and diagnostics. Another novel DNA-binding domain known as a transcriptional activator-like effector (TALE) has been more recently discovered, which has a previously undescribed DNA-binding mode. Due to their modular architecture and flexibility, TALEs have been rapidly developed into artificial gene targeting reagents. Here, we describe the methods used to design these DNA-binding proteins and their key applications in biomedical research.

• Journal of the Korean Magnetic Resonance Society
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• v.20 no.3
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• pp.71-75
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• 2016

The replication protein A (RPA) plays a crucial role in DNA replication, recombination, and repair. RPA consists of 70, 32 and 14 kDa subunits and has high single-stranded DNA (ssDNA) binding affinity. The largest subunit, RPA70, mainly contributes to bind to ssDNA as well as interact with many cellular and viral proteins. In this study, we performed nuclear magnetic resonance experiments on the complex of the DNA binding domain A of human RPA70 (RPA70A) with ssDNA, d(CCCCC), at various pH, to understand the effect of pH on the ssDNA binding of RPA70A. The chemical shift perturbations of binding residues were most significant at pH 6.5 and they reduced with pH increment. This study provides valuable insights into the molecular mechanism of the ssDNA binding of human RPA.

• Journal of the Korean Magnetic Resonance Society
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• v.22 no.1
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• pp.18-25
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• 2018

Replication protein A (RPA) is an essential single-stranded DNA binding protein in DNA processing. It is known that N terminal domain of RPA70 (RPA70N) recruits various protein partners including damage-response proteins such as p53, ATRIP, Rad9, and MRE11. Although the common binding residues of RPA70N were revealed, dynamic properties of the protein are not studied yet. In this study, we measured $^{15}N$ relaxation parameters ($T_1,\;T_2$ and heteronuclear NOE) of human RPA70N and analyzed them using model-free analysis. Our data showed that the two loops near the binding site experience fast time scale motion while the binding site does not. It suggests that the protein binding surface of RPA70N is mostly rigid for minimizing entropy cost of binding and the loops can experience conformational changes.

• Journal of Microbiology and Biotechnology
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• v.15 no.5
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• pp.1022-1027
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• 2005

Based on plasmid display technology by the complexes of fusion protein and the encoding plasmid DNA, an in vitro selection method for high affinity DNA-binding protein was developed and experimentally demonstrated. The GAL4 DNA-binding domain (GAL4 DBD) was selected as a model DNA-binding protein, and enhanced green fluorescent protein (EGFP) was used as an expression reporter for the selection of target proteins. Error prone PCR was conducted to construct a mutant library of the model. Based on the affinity decrease with increased salt concentration, mutants of GAL4 DBD having high affinity were selected from the mutant protein library of protein-encoding plasmid complex by this method. Two mutants of (Lys33Glu, Arg123Lys, Ile127Lys) and (Ser47Pro, Ser85Pro) having high affinity were obtained from the first generation mutants. This method can be used for rapid in vitro selection of high affinity DNA-binding proteins, and has high potential for the screening of high affinity DNA-binding proteins in a sequence-specific manner.

• Korean Journal of Microbiology
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• v.37 no.4
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• pp.245-252
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• 2001

Erm proteins, MLS (macrolide-lincosamide-streptogramin B) resistance factor proteins, show high degree of amino acid sequence homology and comprise of a group of structurally homologous N-methyltransferases. On the basis of the recently determined structures of ErmC` and ErmAM, ErmSF was divided into two domains, N-terminal end catalytic domain and C-terminal end substrate binding domain and attempted to overexpress catalytic domain in E. coli using various pET expression systems. Three DNA fragments were used to express the catalytic domain: DNA fragment 1 encoding Met 1 through Glu 186, DNA fragment 2 encoding Arg 60 to Glu 186 and DNA fragment 3 encoding Arg 60 through Arg 240. Among the pET expression vectors used, pET 19b successfully expressed the DNA fragment 3 and pET23b succeeded in expression of DNA fragment 1 and 2. But the overexpressed catalytic domains existed as inclusion body, a insoluble aggregate. To assist the soluble expression of ErmSF catalytic domains, Coexpression of chaperone GroESL or Thioredoxin and lowering the incubation temperature to $22^{\circ}C$ were attempted, as did in the soluble expression of the whole ErmSF protein. Both strategies did not seem to be helpful. Solubilization with guanidine-HCl and renaturation with gradual removal of denaturant and partial digestion of overexpressed whole ErmSF protein (expressed to the level of 126 mg/ι culture as a soluble protein) with proteinase K, nonspecific proteinase are under way.

• Journal of the Korean Magnetic Resonance Society
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• v.20 no.4
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• pp.138-142
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• 2016

Replication Protein A (RPA) is the eukaryotic single-stranded DNA binding protein. It involves in DNA replication, repair, and damage response. Among three subunits, RPA70 has a protein-protein binding domain (RPA70N) at the N-terminal. It has known that the domain recruits several damage response proteins to the damaged site. Also, it is suggested that there are more candidates that interact with RPA70N. Even though several studies performed on the structural aspects of RPA70N and its ligand binding, the backbone assignments of RPA70N is not available in public. In this study, we present the backbone assignments of RPA70N.

• Proceedings of the Microbiological Society of Korea Conference
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• 2002.10a
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• pp.135-139
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• 2002

Yeast cells respond to condition of amino acid starvation by synthesizing GCN4, a typical eukaryotic transcriptional activator, which regulates the expression of many amino acids biosynthetic genes. By introducing point mutations in the DNA binding domain of GCN4, mutants with normal DNA binding activity but defective in transcriptional activity were isolated to identify unknown proteins that could suppress the mutant phenotype under an amino acid depletion condition. As a result, SSB(Stress-Seventy B) subfamily proteins were identified as suppressors of mutant GCN4. SSB proteins were known as a member of yeast hsp70 family that probably aids passage of nascent chain through ribosomes. Among them, the mechanism of suppression by SSB2 on the defective GCN4 mutant strains is under investigation. Gcn4p directly interacts with Ssb2p through the basic DNA binding domain of GCN4. It suggests the possibility that physical interaction might induce the transcriptional activation of Gcn4p.

• Journal of the Korean Magnetic Resonance Society
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• v.24 no.4
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• pp.117-122
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• 2020

Transcription factors are proteins that bind specific sites or elements in regulatory regions of DNA, known as promoters or enhancers, where they control the transcription or expression of target genes. MEIS1 protein is a DNA-binding domain present in human transcription factors and plays important roles in various biological functions. The hydrogen exchange rate constants of the imino protons were determined for the wild-type containing the consensus DNA-binding site for the MEIS1 and those of the mutant DNA duplexes using NMR spectroscopy. The G2A-, A3G- and C4T-mutant DNA duplexes lead to clear changes in thermal stabilities of these four consensus base pairs. These unique dynamic features of the four base pairs in the consensus 5'-TGAC-3' sequence might play crucial roles in the effective DNA binding of the MEIS1 protein.

• Korean Journal of Microbiology
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• v.55 no.3
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• pp.181-190
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• 2019

In Escherichia coli, DicA protein is involved in cell division control. DicA protein is known to bind DNA better at $25^{\circ}C$ than at $37^{\circ}C$. However, the molecular cause of the temperature dependent binding is not clear. In this study, we investigated how DicA binds DNA and why its DNA binding activity depends on temperature. An unique in vivo DNA binding assay developed in this laboratory showed that unlike the homologous proteins such as RovA or SlyA, DicA uses its N-terminal domain for DNA binding. The in vivo DNA binding assay of DicA also demonstrated that the temperature-dependent DNA binding activity does not come from Cnu or H-NS that is known to bind DNA better at $25^{\circ}C$ than at $37^{\circ}C$. Electrophoretic Mobility Shift Assay (EMSA), when performed with purified DicA protein, did not show temperature-dependent DicA binding activity. However when EMSA was performed with crude protein from WT E. coli cells, temperature-dependent DicA binding activity was observed, suggesting that there is a factor(s) that confers temperature DNA binding activity of DicA in vivo.

• Molecules and Cells
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• v.44 no.3
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• pp.179-185
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• 2021

Vancomycin response regulator (VncR) is a pneumococcal response regulator of the VncRS two-component signal transduction system (TCS) of Streptococcus pneumoniae. VncRS regulates bacterial autolysis and vancomycin resistance. VncR contains two different functional domains, the N-terminal receiver domain and C-terminal effector domain. Here, we investigated VncR C-terminal DNA binding domain (VncRc) structure using a crystallization approach. Crystallization was performed using the micro-batch method. The crystals diffracted to a 1.964 Å resolution and belonged to space group P212121. The crystal unit-cell parameters were a = 25.71 Å, b = 52.97 Å, and c = 60.61 Å. The structure of VncRc had a helix-turn-helix motif highly similar to the response regulator PhoB of Escherichia coli. In isothermal titration calorimetry and size exclusion chromatography results, VncR formed a complex with VncS, a sensor histidine kinase of pneumococcal TCS. Determination of VncR structure will provide insight into the mechanism by how VncR binds to target genes.