• 생명과학회지
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• 제8권4호
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• pp.400-409
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• 1998

Formation of adduct was studied in benzo(a)pyrene(BP)- and doxorubicin(Dx)-treated human NC-37 cells and isolated nuclei. Major adducts formed were determined by fluorescence absorption spectrophotometery and DNA-lin-ked protein assay. When isolated nuclei were exposed to carcinogens BP and DMBA, and anticancer drugs m-AMSA, ellipticine and Dx, varying degrees of adduct formation occured between DNA-protein complex and these drugs. When the mixture was centrifuged 1.7 M sucrose solution, binding BP and DMBA appeared to be similar between the sediment and the supernatant. When the sediment was centrifuged again with 0.35% polymin-P, the amount of BP bound was 2-fold greater in the protein(1077$\pm$55cpm) than in DNA fraction (470$\pm$20cpm), whereas that of DMBA was 1.6-fold greater in the DNA than in protein fraction. In the case of m-AMSA, ellipticine and Dx, the amount of binding was slightly greater in supernatant than in sediment in centrifugation with 1.7 M sucrose, and more than 3 times greater in the DNA- than in protein- fraction in centrifugation with 0.35% polymin P. DNA fractions which associated with a subset of nonhistone chromosomal protein were isolated from NC-37 cells exposed to $^{3}$H-BP and $^{14}$C-Dx. They were separated into two distince components DNA-S and DNA-P by centrifugation with 2M Nacl chromatin extraction. The results indicated that the amount of $^{3}$H-BP bound was 6.0-fold greater in DNA-P as compared with DNA-S, while that of $^{14}$C-Dx binding appreaed to be 6.2-fold greater in DNA-S than in DNA-P fraction. When $^{3}$H-BP binding wasdetermined in the presence of cold Dx, the amount of binding was reduced only in the DNA-P fraction, indicating that the interaction between DNA and protein is decreased. Gene expression by these drugs, BP treated cells were increased to compare with nomal cells but reduced by treatment with BP-Dx. These results suggest that the protein moiety which tightly bound to DNA-P fraction may play an important role in the regulation of gene expression.

• Bulletin of the Korean Chemical Society
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• 제17권4호
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• pp.358-362
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• 1996

The ionic strength dependent binding mode of 9-aminoacridine (9AA), a well-known DNA intercalator, to DNA is studied by flow linear dichroism, circular dichroism, fluorescence techniques and equilibrium dialysis. The DNA-bound 9AA exhibits spectral properties corresponding to the intercalative binding mode disregarding the salt concentrations; the angle between the long-axis transition moment of the 9AA molecule and DNA helix axis is calculated to be about 65°, indicating a significant deviation from the classical intercalation. At low salt concentrations, however, upwards bending curve in Stern-Volmer plot is observed (where 9AA is a fluorophore and DNA a quencher), indicating the coexistence of both static and dynamic quenching mechanisms or the existence of an additional binding site.

• BMB Reports
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• 제34권1호
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• pp.1-7
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• 2001

Structural and physical properties of type wild type and various selected mutants of the vnd/NK-2 homeodomain, the protein product of the homeobox, and the implication in genetic disease are reviewed. The structure, dynamics and thermodynamics have been Investigated by NMR and by calorimetry. The interactions responsible for the nucleotide sequence-specific binding of the homeodomain to its consensus DNA binding site have been identified. There is a strong correlation between significant structural alterations within the homeodomain or its DNA complex and the appearance of genetic disease. Mutations in positions known to be important in genetic disease have been examined carefully For example, mutation of position 52 of vnd/NK-2 results in a significant structural modification and mutation of position 54 alters the DNA binding specificity and amity The $^{15}N$ relaxation behavior and heteronuclear Overhauser effect data was used to characterize and describe the protein backbone dynamics. These studies were carried out on the wild type and the double mutant proteins both in the free and in the DNA bound states. Finally, the thermodynamic properties associated with DNA binding are described for the vnd/NK-2 homeodomain. These thermodynamic measurements reinforce the hypothesis that water structure around a protein and around DNA significantly contribute to the protein-DNA binding behavior. The results, taken together, demonstrate that structure and dynamic studies of proteins combined with thermodynamic measurements provide a significantly more complete picture of the solution behavior than the individual studies.

• BMB Reports
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• 제44권5호
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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.

• 한국환경성돌연변이발암원학회지
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• 제18권2호
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• pp.77-82
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• 1998

In order to compare the suppressive effect of galangin on the genotoxicity by N-methyl-N-nitrosourea (MNU) or benzo[a]pyrene B(a)P, in vivo micronycleus test using mouse peripheral blood and in vitro sister chromatid exchange(SCE) test using mouse spleen lymphocytes were performed. MNU or B(a)P-induced micronucleated reticulocytes in vivo was decreased by the simultaneous treatment of galangin. MNU or B(a)P-induced SCEs in vitro was also decreased by the simultaneous treatment of galangin. On the other hand, the determinations of [$^3$H]MNU-induced total DNA binding and methylated DNA were performed to find out the mechanism of action. [$^3$H]MNU-induced total DNA binding was inhibited by the treatment of galangin in calf thymus DNA. HPLC analysis of DNA hydrolysates showed that galangin caused a decrease of 7-methyl guanine and $O^{6}$-methyl guanine in calf thymus DNA. To elucidate the action mechanism of galangin against B(a)P, alteration of B(a)P metabolism was studied. Galangin inhibited B(a)P metabolism in the presence of S-9 mix and decreased B(a)P-DNA binding in calf thymus DNA with S-9 mix.

• 생명과학회지
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• 제7권3호
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• pp.167-171
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• 1997

번역개시 및 인산화의 조절에 관여하는 RNA와 단백질의 결합 및 인식기작을 연구하기 위해서[$\alpha$^{-32}$P] UMP-labeled HIV Rev-responsive element(RRE) RNA를 이용한 affinity screening에 이해서 Hela ZA-PII cDNA library로부터 이중선RNA결합단백질의 cDNA (RBFII)를 분리하였다. RBFII의 cDNA에 대한 염기서열을 결정하였으며 기존에 연구된 바 있는 RBFII(RBF 또는 TRBP로 보고되었으며 본 연구에서는 RBFII와 구분하기 위해 RBFI으로 명명)과 대부분의 경우 공통적인 ORF를 지니는 것으로 나타났다. 그러나 5’말단에서는 공통적인 ORF 가 RBFI의 경우 21개의 아미노산을 의미하는 63 nt가 Lac-Z의 N-말단에 연결된데 비해서 특이한 46개 아미노기를 의미하는 138nt가 존재함이 밝혀졌다. 5’-말단에 처음 나타나는 ATG 및 부근의 염기서열을 분석해 볼 때 양 cDNA는 5’말단이 완전하지 않은 것으로 사료된다.

• 한국자기공명학회논문지
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• 제21권3호
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• pp.90-95
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• 2017

The Z-DNA domain of human ADAR1 ($Z{\alpha}_{ADAR1}$) produces B-Z junction DNA through preferential binding to the CG-repeat segment and destabilizing the neighboring AT-rich region. However, this study could not answer the question of how many base-pairs in AT-rich region are destabilized by binding of $Z{\alpha}_{ADAR1}$. Thus, we have performed NMR experiments of $Z{\alpha}_{ADAR1}$ to the longer DNA duplex containing an 8-base-paired (8-bp) CG-repeat segment and a 12-bp AT-rich region. This study revealed that $Z{\alpha}_{ADAR1}$ preferentially binds to the CG-repeat segment rather than AT-rich region in a long DNA and then destabilizes at least 6 base-pairs in the neighboring AT-rich region for efficient B-Z transition of the CG-repeat segment.

• 약학회지
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• 제49권5호
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• pp.428-436
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• 2005

DNA topoisomerase I(TOP1) helps the control of DNA replication, transcription and recombination by assist­ing breaking and rejoining of DNA double strand. Camptothecin (CPT) and its derivative, topotecan, are known to inhibit TOP1 by intercalating into TOP1-DNA complex. Recently various non-CPT intercalators are synthesized for a new class of TOP1 inhibitors. In this study, six compounds isolated from Poria cocos were investigated for their interaction with TOP1­DNA complex using the flexible docking program, FlexiDock. The binding modes were analyzed and compared with the TOP1 inhibition activities. The compounds that showed potent activity were intercalated between the + 1/-1 base pairs of DNA, located near the active site phosphotyrosine723 and formed hydrogen bonds with active site residues. On the other hand, compounds with no activity were not docked at all. The binding modes were well correlated with the inhibition activity, suggesting the possibility that potent inhibitors can be designed from the information presented by the docking study.

• 한국임상수의학회지
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• 제21권3호
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• pp.253-258
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• 2004

DNA double-strand break (DSB) is a serious treat for the cells including mutations, chromosome rearrangements, and even cell death if not repaired or misrepaired. Ku heterodimer regulatory DNA binding subunits (Ku70/Ku80) bound to double strand DNA breaks are able to interact with 470-kDa DNA-dependent protein kinase catalytic subunit (DNA-PKcs), and the interaction is essential for DNA-dependent protein kinase (DNA-PK) activity. The Ku80 mutants were designed to bind Ku70 but not DNA end binding activity and the peptides were treated in breast cancer cells for co-therapy strategy to see whether the targeted inhibition of DNA-dependent protein kinase (DNA-PK) activity sensitized breast cancer cells to ionizing irradiation or chemotherapy drug to develop a treatment of breast tumors by targeting proteins involved in damage-signaling pathway and/or DNA repair. We designed domains of Ku80 mutants, 26 residues of amino acids (HN-26) as a control peptide or 38 (HNI-38) residues of amino acids which contain domains of the membrane-translocation hydrophobic signal sequence and the nuclear localization sequence, but HNI-38 has additional twelve residues of peptide inhibitor region. We observed that the synthesized peptide (HNI-38) prevented DNA-PKcs from binding to Ku70/Ku80, resulting in inactivation of DNA-PK complex activity in breast cancer cells (MDA-465 and MDA-468). Consequently, the peptide treated cells exhibited poor to no DNA repair, and became highly sensitive to irradiation or chemotherapy drugs. The growth of breast cancer cells was also inhibited. These results demonstrate the possibility of synthetic peptide to apply breast cancer therapy to induce apoptosis of cancer cells.

• BMB Reports
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• 제42권11호
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• pp.764-768
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• 2009

The tandem ubiquitin-interacting motif (UIM) domain located at the N-terminus of Receptor Associated Protein 80 (RAP80) plays a crucial role in ionizing radiation (IR)-induced DNA damage response. RAP80 translocates to sites of IR-induced DNA damage through interaction of its UIM domain with ubiquitinated H2A and Lys63-linked polyubiquitin chains. The exact mechanism, however, through which RAP80 associates with Lys63-linked polyubiquitin chains is not clear. Here, we show by in vitro GST-pull down assays that modifying the linker region between the tandem ubiquitin binding domains of RAP80 changes the binding affinity for Lys63-linked polyubiquitin chains and affects translocation to sites of DNA breaks. Based on these findings, we suggest that the length of the linker region between the tandem ubiquitin binding domains of RAP80 may be a key factor in the binding of RAP80 with Lys63-linked polyubiquitin chains as well as in the translocation of RAP80 to DNA break sites.