• 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.

• 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.35 no.2
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• pp.194-198
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• 2002

Eukaryotic replication protein A (RPA) is a single-stranded(ss) DNA binding protein with multiple functions in DNA replication, repair, and genetic recombination. The 70-kDa subunit of eukaryotic RPA contains a conserved four cysteine-type zinc-finger motif that has been implicated in the regulation of DNA replication and repair. Recently, we described a novel function for the zinc-finger motif in the regulation of human RPA's ssDNA binding activity through reduction-oxidation (redox). Here, we show that yeast RPA's ssDNA binding activity is regulated by redox potential through its RPA32 and/or RPA14 subunits. Yeast RPA requires a reducing agent, such as dithiothreitol (DTT), for its ssDNA binding activity. Also, under non-reducing conditions, its DNA binding activity decreases 20 fold. In contrast, the RPA 70 subunit does not require DTT for its DNA binding activity and is not affected by the redox condition. These results suggest that all three subunits are required for the regulation of RPA's DNA binding activity through redox potential.

• BMB Reports
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• v.44 no.5
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• pp.341-346
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• 2011

The single-stranded DNA binding activity of the Escherichia coli RecA protein is crucial for homologous recombination to occur. This and other biochemical activities of ssDNA binding proteins may be affected by various factors. In this study, we analyzed the effect of $CaCl_2$, NaCl and $NH_4NO_3$ salts in combination with the pH and nucleotide cofactor effect on the ssDNA-binding activity of RecA. The studies revealed that, in addition to the inhibitory effect, these salts exert also a stimulatory effect on RecA. These effects occur only under very strict conditions, and the presence or absence and the type of nucleotide cofactor play here a major role. It was observed that in contrast to ATP, ATP${\gamma}$S prevented the inhibitory effect of NaCl and $NH_4NO_3$, even at very high salt concentration. These results indicate that ATP${\gamma}$S most likely stabilizes the structure of RecA required for DNA binding, making it resistant to high salt concentrations.

• 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.

• 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 Chemical Society
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• v.46 no.2
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• pp.151-156
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• 2002

Fucoidan is a kind of polysaccharides in brown seaweeds. For the past years have been extensively studied due to their numerous biological activities : anticancer, anticoagulant, antithrombotic, anti-inflammatory and antiviral. In this study, we h ave extracted fucoidan from the Korean brown seaweeds and examined it's anticancer activities for employed SV40 DNA replication assay, RPA-ssDNA binding assay of replication protein A(RPA: known as human single-stranded DNA-binding protein essential for DNA rep-lication) and MCF7 cell growth inhibition assay. In addition to, we found that chemically sulfated fucoidan'santicancer activity is more higher than natural and desulfated fucoidan. It seem that fucoidan's sulfate group affect on DNA replication, cause of decrease RPA's DNA binding activity. These results suggests that sulfated fucoidan from Korean brown seaweeds have anticancer activity.

• Journal of the Korean Magnetic Resonance Society
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• v.15 no.1
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• pp.69-79
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• 2011

HP0827 has two RNP motif which is a very common protein domain involved in recognition of a wide range of ssRNA/DNA.We acquired 3D NMR spectra of HP0827 which shows well dispersed and homogeneous signals which allows us to assign 98% of all $^1H_N$, $^{15}N$, $^{13}C_{\alpha}$, $^{13}C_{\beta}$ and $^{13}C$=O resonances and 90% of all sidechain resonances. The sequence-specific backbone resonance assignment of HP0827 can be used to gain deeper insights into the nucleic acids binding specificity of HP0827 in the future study. Here, we report secondary structure prediction of HP0827 derived from NMR data. Additionally, ssRNA/DNA binding assay studies was also conducted. This study might provide a clue for exact function of HP0827 based on structure and sequence.

• BMB Reports
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• v.34 no.5
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• pp.428-433
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• 2001

A defect in uvsC of Aspergillus nidulans caused high methyl methansulfonate (MMS)-sensitivity, hyporecombination, and a lack of UV induced mutation. The uvsC gene of Aspergillus nidulans shares a sequence similarity with the RAD51 gene of Saccharomyces cerevisiae. In this study, in vitro and in vivo tests were conducted in order to determine whether or not the UVSC protein had functional similarities to RAD51, the recombination enzyme in yeast. The purified recombinant UVSC protein, following expression in Escherichia coli, showed binding activity to single-stranded DNA (ssDNA), when both ATP and magnesium are present. In addition, ATPase activity was also demonstrated and its activity was stimulated in the presence of ssDNA. The UVSC protein that was expressed under the ADH promoter in S. cerevisiae suppressed in part the sensitivity to MMS of the rad51 null mutant. Similarly, when the uvsC cDNA was expressed from the nmt promoter, the MMS sensitivity of the rhp51 null mutant of Schizosaccharomyces pombe was partially complemented. These results indicate that the A. nidulans UVSC protein is a functional homologue of the RAD51 protein.

• Korean Journal of Microbiology
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• v.43 no.4
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• pp.250-255
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• 2007

The RecA protein of Deinococcus radiodurans is essential for the extreme radiation resistance of this organism. The central steps involved in recombinational DNA repair require DNA-dependent ATP hydrolysis by recA protein. Key feature of RecA protein-mediated activities is the interactions with ssDNA and dsDNA. The ssDNA is the site where RecA protein filament formation nucleates and where initiation of DNA strand exchange takes place. The effect of sequence heterogeneity of ssDNA was examined in this experiment. The rate of homopolymeric synthetic ssDNA-dependent ATP hydrolysis was constant or nearly so over a broader range of pHs. For poly(dT)-dependent ATP or dATP hydrolysis, rates were generally faster, with a broader optimum between pH 7.0 and 8.0. Activities of RecA protein were affected by the ionic environment. The ATPase activity was shown to have different sensitivity to anionic species. The presence of glutamate seemed to slimulate the hydrolytic activity. Dr RecA protein was shown to require $Mg^{2+}$ ion greater than 2 mM for binding to etheno ssDNA and the binding stoichiometry of 3 nucleotide for RecA protein monomer.