• 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.

• Proceedings of the Botanical Society of Korea Conference
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• 1987.07a
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• pp.149-155
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• 1987

Growth and development of a higher plant, or any living organism for that matter, could be defined as an orderly expression of the genome in time and space in close interaction with the environment. During differentiation and development of a tissue or organ a group of genes must be selectively turned on or turned off mainly by trans-acting regulators. In this general concept of regulation of regulation of gene expression, a DNA molecule is recognized at a specific nucleotide sequence by DNA-binding factors. Molecular biology of the regulatory factors such as hormones, and their receptors, target DNA sequences and DNA-binding proteins are well advanced. What is not clearly understood is the molecular basis of the interactions between DNA and binding factors, expecially of the usages of the dyad symmetry of the target DNA sequences and the dimeric nature of the DNA-binding proteins. A unique 3-dimensional structure of DNA has been proposed that may play an important role in the orderly expression of the gene. A foldback intercoil (FBI) DNA configuration which was originally found by electron microscopy among mtDNA molecules from pearl millet has some unique features. The FBI configuration of DNA is believed to be formed when a flexible double helix folds back and interwines in the widened major grooves resulting in a four stranded, intercoil DNA whose thickness is the same as that of double stranded DNA. More recently, the FBI structure of DNA has been also induced in vitro by a novel enzyme which was purified from pearl millet mitochondria. It has been proposed that the FBI DNA could be utillized in intramolecular recombination which leads to inversion or deletion, and in intermolecular recombination which can lead to either site-specific recombination, genetic recombination via single strand invasion, or cross strand recombination. The structure and function of DNA in 3-dimensional aspect is emphasized for better understanding orderly expression of genes during growth and development.

• BMB Reports
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• v.38 no.6
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• pp.690-694
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• 2005

Transcription termination of the human mitochondrial genome requires specific binding to termination factor mTERF. In this study, mTERF was produced in E. coli and purified by two-step chromatography. mTERF-binding DNA sequences were isolated from a pool of randomized sequences by the repeated selection of bound sequences by gel-mobility shift assay and polymerase chain reaction. Sequencing and comparison of the 23 isolated clones revealed a 16-bp consensus sequence of 5'-GTG$\b{TGGC}$AGANCCNGG-3' in the light-strand (underlined residues were absolutely conserved), which nicely matched the genomic 13-bp terminator sequence 5'-$\b{TGGC}$AGAGCCCGG-3'. Moreover, mTERF binding assays of heteroduplex and single-stranded DNAs showed mTERF recognized the light strand in preference to the heavy strand. The preferential binding of mTERF with the light-strand may explain its distinct orientation-dependent termination activity.

• Journal of Life Science
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• v.6 no.3
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• pp.185-192
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• 1996

Bacteriophage T7 gene 2.5 protein, a single-stranded DNA binding protein, is required for T7 DNA replication, recombination, and repair. T7 gene 2.5 protein has two distinctive domains, DNA binding and C-terminal domain, directly involved in protein-protein interaction. Gene 2.5 protein participates in the DNA replication of Bacteriophage T7, which makes this protein essential for the T7 growth and DNA replication. What gene 2.5 protein makes important at T7 growth and DNA replication is its binding affinity to single-stranded DNA and the protein-protein important at T7 DNA replication proteins which are essential for the T7 DNA synthesis. We have constructed pGST2.5(WT) encoding the wild-type gene 2.5 protein and pGST2.5$\Delta $21C lacking C-terminal 21 amino acid residues. The purified GST-fusion proteins, GST2.5(WT) and GST2.5(WT)$\Delta$21C, were used for whether the carboxyl-terminal domain participates in the protein-protein interactions or not. GST2.5(WT) and GST2.5$\Delta$21C showed the difference in the protein-protein interaction. GST2.5(WT) interacted with T7 DNA polymerase and gene 4 protein, but GST2.5$\Delta$21C did not interact with either protein. Secondly, GST2.5(WT) interacts with gene 4 proteins (helicase/primase) but not GST2.5$\Delta$21C. these results proved the involvement of the carboxyl-terminal domain of gene 2.5 protein in the protein-protein interaction. We clearly conclude that carboxy-terminal domain of gene 2.5 protein is firmly involved in protein-protein interactions in T7 replication proteins.

• 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.

• BMB Reports
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• v.28 no.2
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• pp.143-148
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• 1995

In E. coli, chromosomal DNA associated with proteins is condensed into an organized structure known as nucleoid. Using a nitrocellulose filter binding assay to identify proteins forming nucleoid, a 21 kDa protein was purified from E. coli. The molecular weight of the purified protein was 21 kDa on SDS-polyactylamide gel electrophoresis and 24 kDa on gel permeation chromatography. A molecular weight of 21 kDa on SDS-polyacrylamide gel electrophoresis is unique among known proteins which are believed to be involved in the formation of nucleoid in E. coli. The 21 kDa protein nonspecifically binds to both double-stranded and single-stranded DNA. Sedimentation in a sucrose gradient revealed that the protein induced significant condensation of both supercoiled plasmid DNA and linear bacteriophage $\lambda$ DNA On the basis of quantitative Western-blot analysis, approximately 40,000 molecules of the protein were estimated to exist in an E. coli. The biochemical properties and cellular abundance of the 21 kDa protein suggest that this protein participates in the formation of nucleoid in E. coli.

• 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.

• BMB Reports
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• v.28 no.6
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• pp.484-489
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• 1995

The product of bacteriophage T7 gene 2.5 is a single-stranded DNA binding protein and plays an important role in T7 DNA replication, recombination, and repair. Genetic analysis of T7 phage defective in gene 2.5 shows that the gene 2.5 protein is essential for T7 DNA replication and growth (Kim and Richardson, 1993). The C-terminal truncated gene 2.5 protein ($GP2.5-{\Delta}21C$) cannot substitute for wild-type gene 2.5 protein in vivo; suggesting that the C-terminal domain of gene 2.5 protein is essential for protein-protein interactions (Kim and Richardson, 1994; J. Biol. Chem. 269, 5070-5078). Truncated gene 2.5 proteins lacking 19 residues ($GP2.5-{\Delta}19N$) and 39 residues ($GP2.5-{\Delta}39N$) from the amino-terminal domain were constructed by in vitro mutagenesis. $GP2.5-{\Delta}19N$ can support the growth of T7 phage lacking gene 2.5 while $GP2.5-{\Delta}39N$ cannot substitute for wild-type gene 2.5 protein in vivo; however, its ability to bind to single-stranded DNA is not affected. These results clearly demonstrate that the 20~39 amino-terminal region of gene 2.5 protein is required for T7 growth in vivo but may not be involved in DNA binding activity.

• 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 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.