• 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.

• Journal of the Korean Magnetic Resonance Society
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• v.20 no.3
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• pp.66-70
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• 2016

The replication protein A (RPA), is a heterotrimer with 70, 32 and 14 kDa subunits and plays a crucial role in DNA replication, recombination, and repair. The largest subunit, RPA70, binds to single-stranded DNA (ssDNA) and mediates interactions 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 temperatures, to understand the temperature dependency of ssDNA binding affinity of RPA70A. Essential residues for ssDNA binding were conserved while less essential parts were changed with the temperature. Our results provide valuable insights into the molecular mechanism of the ssDNA binding of human RPA.

• Korean Journal of Microbiology
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• v.55 no.3
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• pp.191-198
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• 2019

H-NS binds to promoter DNA and works as a general transcription silencer. DicA protein, by binding to the promoter DNA of dicA, activates dicA expression and at the same time inhibits expression of dicF and dicB, thus, exerting cell division control in Escherichia coli. H-NS complexed with a nucleoid protein Cnu was known to be involved in dicA expression. However, the exact nature of H-NS binding to dicA promoter DNA and the consequences of H-NS binding in expression of dicA is not clear. In this study, we explored the DNA binding activity of H-NS on the promoter DNA of dicA and found that H-NS binding occurs exclusively to the dicA promoter DNA. We never observed, however, H-NS binding at the vicinity of the dicA promoter. Temperature dependent oligomerization of H-NS was observed during DNA binding and the Cnu protein enhances the oligomerization process of H-NS binding. In vivo measurement of dicA expression in an hns deleted strain showed that dicA expression increased. These results demonstrated that H-NS binds specifically to dicA promoter DNA and functions as a transcription silencer.

• Proceedings of the Korean Vacuum Society Conference
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• 2016.02a
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• pp.140.1-140.1
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• 2016

We have measured DNA translocation through a nanocapillary functionalized with probe DNA. These DNA-functionalized nanocapillaries selectively facilitate the translocation of target ssDNAs that are complementary to the probe ssDNAs. In addition, translocation of the complementary target ssDNA exhibits two tendencies to translocation speed, such as fast and slow translocation, whereas that of non-complementary target ssDNA yields only one tendency, fast translocation. These observations suggest that the complementary and non-complementary target ssDNAs may be discriminated due to different interaction strengths between target and probe ssDNAs. The temperature dependence measurements of DNA translocation show that slow translocation events are ascribed to the complementary interaction between probe and target ssDNA. This confirms that their dwell time is dependent on the base-pair binding strength. These results demonstrate that mere single-base different target DNA can be selectively detectable by using the probe DNA-functionalized nanocapillaries.

• Journal of the Korean Magnetic Resonance Society
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• v.18 no.2
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• pp.52-57
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• 2014

The TCP domain is a DNA-binding domain present in plant transcription factors and plays important roles in various biological functions. The hydrogen exchange rate constants of the imino protons were determined for the three DNA duplexes containing the DNA-binding sites for the TCP11, TCP15, and TCP20 transcription factors using NMR spectroscopy. The M11 duplex displays unique hydrogen exchange property of the five base pairs in the first binding site (5'-GTGGG-3'). However, the M15 and M20 duplexes lead to clear changes in thermal stabilities of these five base pairs. The unique dynamic features of the five base pairs in the first binding site might play crucial roles in the sequence-specific DNA binding of the class I TCP transcription factors.

• Journal of Microbiology and Biotechnology
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• v.17 no.8
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• pp.1353-1360
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• 2007

Three csp-like genes were identified in the Corynebacterium glutamicum genome and designated cspA, cspB, and cspA2. The genes cspA and cspA2 encode proteins, comprising of 67 amino acid residues, respectively. They share 83% identity with each other. Identity of those proteins with Escherichia coli Csp proteins was near 50%. The cspB gene encodes a protein composed of 127 amino acids, which has 40% and 35% sequence identity with CspA and CspA2, respectively, especially at its N-terminal region. Analysis of the gene expression profiles was done using transcriptional cat fusion, which identified not only active expression of the three genes at the physiological growth temperature of $30^{\circ}C$ but also growth phase-dependent expression with the highest activity at late log phase. The promoters of cspA and cspA2 were more active than that of cspB. The expression of the two genes increased by 30% after a temperature downshift to $15^{\circ}C$, and such stimulation was more evident in the late growth phase. In addition, the cspA gene appeared to show DNA-binding activity in vivo, and the activity increased at lower temperatures. Interestingly, the presence of cspA in multicopy hindered the growth of the host C. glutamicum cells at $20^{\circ}C$, but not at $30^{\circ}C$. Altogether, these data suggest that cspA, cspB, and cspA2 perform functions related to cold shock as well as normal cellular physiology. Moreover, CspA and its ortholog CspA2 may perform additional functions as a transcriptional regulator.