• IMMUNE NETWORK
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• v.2 no.4
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• pp.208-216
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• 2002

Background: Reactive oxygen and nitrogen are produced by rheumatoid arthritis (RA) synovial tissue and can induce mutations in key genes. Normally, this process is prevented by a DNA mismatch repair (MMR) system that maintains sequence fidelity. Key members of the MMR system include MutS${\alpha}$ (comprised of hMSH2 and hMSH6), which can sense and repair single base mismatches and 8-oxoguanine, and MutS${\beta}$ (comprised of hMSH2 and hMSH3), which repairs longer insertion/deletion loops. Methods: To provide further evidence of DNA damage, we analyzed synovial tissues for microsatellite instability (MSI). MSI was examined by PCR on genomic DNA of paired synovial tissue and peripheral blood cells (PBC) of RA patients using specific primer sequences for 5 key microsatellites. Results: Surprisingly, abundant MSI was observed in RA synovium compared with osteoarthritis (OA) tissue. Western blot analysis of the same tissues for the expression of MMR proteins demonstrated decreased hMSH6 and increased hMSH3 in RA synovium. To evaluate potential mechanisms of MMR regulation in arthritis, fibroblast-like synoviocytes (FLS) were isolated from synovial tissues and incubated with the nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP). Western blot analysis demonstrated constitutive expression of hMSH2, 3 and 6 in RA and OA FLS. When FLS were cultured with SNAP, the RA synovial pattern of MMR expression was reproduced (high hMSH3, low hMSH6). Conclusion: Therefore, oxidative stress can relax the DNA MMR system in RA by suppressing hMSH6. Decreased hMSH6 can subsequently interfere with repair of single base mutations, which is the type observed in RA. We propose that oxidative stress not only creates DNA adducts that are potentially mutagenic, but also suppresses the mechanisms that limit the DNA damage.

• The Korean Journal of Zoology
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• v.19 no.4
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• pp.179-186
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• 1976

DNA repari synthesis and chromosome aberrations induced by various concentrations of alkylating agents (MMS, MNNG, MMC) in cultured human lymphocytes and HeLa $S_3$ cells were studied to determine the possibility of correlation between these two types of biological phenomena, and the results obtained were as follows: DNA repair synthesis was detected in MMC, MNNG and MMS treated HeLa $S_3$ cells at the concentrations of $3 \\times 10^{-7}M, 1 \\times 10^{-6}M, 5 \\times 10^{-4}M$, respectively. These results indicate that MMC is the most potent mutagen followed by MNNG, and MMS is the least potent among these three types of alkylating agents. MMC and MNNG did not show any significant increases of DNA repair synthesis as dose increased, while MMS did. Chromosome aberrations induced by MMC in human lymphocytes was increased as dose increased, but not chromosome exchanges. MNNG did not induce any significant amount of chromosome aberrations with doses, and exchanges were not observed in MNNG treated cells. MMS, however, induced both chromosome aberrations and exchanges, and their rates were increased as dose increased. These results suggest that DNA repair synthesis induced by these alklating agents may not be directly related to the production of chromosome aberrations and exchanges.

• Environmental Mutagens and Carcinogens
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• v.17 no.2
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• pp.71-77
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• 1997

The single cell gel electrophoressis(SCGE) assay, also known as the comet assay, is a rapid, simple, visual and sensitive technique for measuring and analysing DNA breakage in mammalian cells. The SCGE or comet assay is a promising test for the detection of DNA damage and repair in individnal cells. It has widespread potential applications in DNA damage and repair studies, genotoxicity testing and biomonitoring. In this microgel electrophoresis technique, cells are embedded in agarose gel on microscope slides, iysed and electrophoresed under alkaline conditions. Cells with increased DNA damage display increased migration of DNA from the nucleus towards the anode. The length of DNA migration indicates the amount of DNA breakage in the cell. The comet assay is also capable of identifying apoptotic cells which contain highly fragmented DNA. Here we review the development of the SCGE assay, existing protocols for the detection and analysis of comets, the relevant underlying principles determining the behaviour of DNA and the potential applications of the technique.

• Korean Journal of Microbiology
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• v.28 no.2
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• pp.109-113
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• 1990

The mutagenesis-enhancing plasmid pKM101 and its mutant pSL4 were introduced into Escherichia coli B/r strains possessing different DNA repair capacities ($phr^{-}, recA^{-}, uvrA^{-}, uvrB^{-}$) and determined the protection effect and mutagenecity for UV and MNNG. The mutability and protection effect of plasmid pKM101 and pSL4 were affected by different DNA repair capacity. The mutagenecity and resistance of two plasmids were increased against UV and MNNG, and plasmid pSL4 had a higher effect than pKM101. We suggest that the functional differences between pKM101 and pSL4 is due to the variety of mutator gene.

• BMB Reports
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• v.52 no.2
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• pp.151-156
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• 2019

RAD51 recombinase plays a critical role in homologous recombination and DNA damage repair. Here we showed that expression of RAD51 is frequently upregulated in lung cancer tumors compared with normal tissues and is associated with poor survival (hazard ratio (HR) = 2, P = 0.0009). Systematic investigation of lung cancer cell lines revealed higher expression of RAD51 in KRAS mutant (MT) cells compared to wildtype (WT) cells. We further showed that MT KRAS, but not WT KRAS, played a critical role in RAD51 overexpression via MYC. Moreover, our results revealed that KRAS MT cells are highly dependent on RAD51 for survival and depletion of RAD51 resulted in enhanced DNA double strand breaks, defective colony formation and cell death. Together, our results suggest that mutant KRAS promotes RAD51 expression to enhance DNA damage repair and lung cancer cell survival, suggesting that RAD51 may be an effective therapeutic target to overcome chemo/radioresistance in KRAS mutant cancers.

• Journal of Veterinary Clinics
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• v.21 no.3
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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.

• Molecules and Cells
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• v.42 no.7
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• pp.546-556
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• 2019

Protein phosphatase 4 (PP4) is a crucial protein complex that plays an important role in DNA damage response (DDR), including DNA repair, cell cycle arrest and apoptosis. Despite the significance of PP4, the mechanism by which PP4 is regulated remains to be elucidated. Here, we identified a novel PP4 inhibitor, protein phosphatase 4 inhibitory protein (PP4IP) and elucidated its cellular functions. PP4IP-knockout cells were generated using the CRISPR/Cas9 system, and the phosphorylation status of PP4 substrates (H2AX, KAP1, and RPA2) was analyzed. Then we investigated that how PP4IP affects the cellular functions of PP4 by immunoprecipitation, immunofluorescence, and DNA double-strand break (DSB) repair assays. PP4IP interacts with PP4 complex, which is affected by DNA damage and cell cycle progression and decreases the dephosphorylational activity of PP4. Both overexpression and depletion of PP4IP impairs DSB repairs and sensitizes cells to genotoxic stress, suggesting timely inhibition of PP4 to be indispensable for cells in responding to DNA damage. Our results identify a novel inhibitor of PP4 that inhibits PP4-mediated cellular functions and establish the physiological importance of this regulation. In addition, PP4IP might be developed as potential therapeutic reagents for targeting tumors particularly with high level of PP4C expression.

• Journal of Photoscience
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• v.10 no.2
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• pp.195-198
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• 2003

In the cellular response to DNA damaging agents, cells undergo cell cycle arrest or apoptosis against irrepairable DNA damage. RpS3 is known to function as UV DNA repair endonuclease III and ribosomal protein S3. In this study, we used normal and rpS3-overexpressed 293T cells to examine the role of rpS3 in response to DNA damaging agents. When 293T cells transfected with rpS3 were irradiated with UV, the pattern of cell cycle was dramatically changed in comparison with un-transfected 293T cells. We also found that the expression of rpS3 in normal cells was increased by treatment with DNA damaging agents. By means of Western and Northern blot analyses in rat tissues, we showed the expression pattern of rpS3 protein and its mRNA. These data suggest that DNA repair and cell cycle arrest are interrelated to each other through rpS3, and the increased expression of rpS3 seems to regulate the cell cycle arrest by DNA damaging agents.

• Molecules and Cells
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• v.26 no.1
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• pp.5-11
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• 2008

Stalled DNA replication forks activate specific DNA repair mechanism called post-replication repair (PRR) pathways that simply bypass DNA damage. The bypassing of DNA damage by PRR prevents prolonged stalling of DNA replication that could result in double strand breaks (DSBs). Proliferating cell nuclear antigen (PCNA) functions to initiate and choose different bypassing pathways of PRR. In yeast, DNA replication forks stalled by DNA damage induces monoubiquitination of PCNA at K164, which is catalyzed by Rad6/Rad18 complex. PCNA monoubiquitination triggers the replacement of replicative polymerase with special translesion synthesis (TLS) polymerases that are able to replicate past DNA lesions. The PCNA interaction motif and/or the ubiquitin binding motif in most TLS polymerases seem to be important for the regulation of TLS. The TLS pathway is usually error-prone because TLS polymerases have low fidelity and no proofreading activity. PCNA can also be further polyubiquitinated by Ubc13/ Mms2/Rad5 complex, which adds an ubiquitin chain onto monoubiquitinated K164 of PCNA. PCNA polyubiquitination directs a different PRR pathway known as error-free damage avoidance, which uses the newly synthesized sister chromatid as a template to bypass DNA damage presumably through template switching mechanism. Mammalian homologues of all of the yeast PRR proteins have been identified, thus PRR is well conserved throughout evolution. Mutations of some PRR genes are associated with a higher risk for cancers in mice and human patients, strongly supporting the importance of PRR as a tumor suppressor pathway.

• Korean Journal of Veterinary Research
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• v.43 no.2
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• pp.211-218
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• 2003

Human ovarian cancerous cells survive in a way that they trigger the nucleotide excision repair (NER) or double-strand DNA repair (dsDNA) repair mechanism to show resistance to anticancer drugs and activate many kinds of repair protein, thus removing damaged DNAs. Two experiments on the PA-1 human ovarian teratocareinoma cell line that hardly has any expression of epidermal growth factor receptor (EGFR) were conducted in the study; first, EGF-R was transfected and its receptor was obtained. The receptor was investigated in terms of its mutual relations with many kinds of protein concerning NER or dsDNA repair. Second, it was examined what kind impact cisplatin and adriamycin had on the effects of EGF-R over the PA-1 cell line lacking EGF-R. When being administered with cisplatin and adriamycin, Hey and Hey C2 cell lines showed a high level of resistance while PA-1 cell line a high level of sensitivity. Hey and Hey C2 cell lines that are resistant against anticancer drugs exhibited a high level of EGF-R expression while PA-1 cell line that is sensitive to them did a much lower level of the expression. When PA-1 cell line was transfected for the expression of DNA adduct and EGF-R, it showed a higher level of resistance compared to the control group. There was no difference in the expression of DNA repair proteins (DNA- dependent protein kinase, Ku70, and Ku80) between Hey and the PA-1 cell lines. The results indicate that the Hey cell line that is resistant against cisplatin and adriamycin works along the signaling system responding to the changes of EGF-R while the PA-1 cell line that is sensitive to both of them does to the lack of EGF-R.