• Molecules and Cells
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• 제46권4호
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• pp.200-205
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• 2023

DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a member of the phosphatidylinositol 3-kinase-related kinase family is a well-known player in repairing DNA double-strand break through non-homologous end joining pathway. This mechanism has allowed us to understand its critical role in T and B cell development through V(D)J recombination and class switch recombination, respectively. We have also learned that the defects in these mechanisms lead to the severely combined immunodeficiency (SCID). Here we highlight some of the latest evidence where DNA-PKcs has been shown to localize not only in the nucleus but also in the cytoplasm, phosphorylating various proteins involved in cellular metabolism and cytokine production. While it is an exciting time to unveil novel functions of DNA-PKcs, one should carefully choose experimental models to study DNA-PKcs as the experimental evidence has been shown to differ between cells of defective DNA-PKcs and those of DNA-PKcs knockout. Moreover, while there are several DNA-PK inhibitors currently being evaluated in the clinical trials in an attempt to increase the efficacy of radiotherapy or chemotherapy, multiple functions and subcellular localization of DNA-PKcs in various types of cells may further complicate the effects at the cellular and organismal level.

• Journal of Radiation Protection and Research
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• 제41권4호
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• pp.339-343
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• 2016

Background: Evaluation of deoxyribonucleic acid (DNA)-strand break is important to elucidate the biological effect of ionizing radiations. The conventional methods for DNA-strand break evaluation have been achieved by Agarose gel electrophoresis and others using an electrical property of DNAs. Such kinds of DNA-strand break evaluation systems can estimate DNA-strand break, according to a molecular weight of DNAs. However, the conventional method needs pretreatment of the sample and a relatively long period for analysis. They do not have enough sensitivity to detect the strand break products in the low-dose region. Materials and Methods: The sample is water, methanol and plasmid DNA solution. The plasmid DNA pUC118 was multiplied by using Escherichia coli JM109 competent cells. The resonance frequency and Q-value were measured by means of microwave dielectric absorption spectroscopy. When a sample is located at a center of the electric field, resonance curve of the frequency that existed as a standing wave is disturbed. As a result, the perturbation effect to perform a resonance with different frequency is adopted. Results and Discussion: The resonance frequency shifted to higher frequency with an increase in a concentration of methanol as the model of the biological material, and the Q-value decreased. The absorption peak in microwave power spectrum of the double-strand break plasmid DNA shifted from the non-damaged plasmid DNA. Moreover, the sharpness of absorption peak changed resulting in change in Q-value. We confirmed that a resonance frequency shifted to higher frequency with an increase in concentration of the plasmid DNA. Conclusion: We developed a new technique for an evaluation of DNA damage. In this paper, we report the evaluation method of DNA damage using microwave dielectric absorption spectroscopy.

• Radiation Oncology Journal
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• 제11권2호
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• pp.219-225
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• 1993

각 방사선량의 조사에 따른 사람 정상임파구, 마우스 정상임파구, 그리고 EL-4마우스 백혈병 세포의 DNA double strand breaks 발생율에 대하여 비교분석 하였던 바 다음과 같은 결과를 얻었다. LPS와 PHA를 첨가하여 각각 배양한 마우스 정상임파구의 DNA double strand breaks의발생율을 strand scission factor의 기울기로 비교평가해 본 바 LPS를 첨가배양한 군이 DNA DSB가 더 많이 형성되었다(P<0.005). 이것으로 볼 때 DNA double strand breaks발생에 있어서는 B cells이 T cells보다 더 민감하였다. 또한 EL-4 백혈병 세포는 정상 임파구보다 유의하게 DNA DSB가 더 많이 형성된 것이 관찰되었다(p<0.005). 한편 사람 정상임파구와 마우스 정상임파구 사이의 intrinsic radiosensitivity는 시험관내 실험에서 비교적 유사한 kinetics를 나타냈으나, 마우스 정상임파구가 사람 정상임파구보다 DNA DSB수율이 더 낮았다. 그래서 방사선을 조사하고 3.5시간이 지난후에 $10\%$ DNA DSB발생에 필요한 선량을 두 군간에 비교평가해 본 바 마우스 정상임파구의 선량이 더 높게 나타났다. 이상의 실험결과를 정확하게 설명하기는 어 렵지만 아마도 환경적인 요인과 유전적인 요인 때문인 것으로 사료되었다. 그리고 본 연구결과는 방사선조사에 따른 임상반응의 이해와 cytometric assessment의 방사선 생물학적 parameter로 이용될 수 있을 것으로 판단되었다.

• Journal of Microbiology and Biotechnology
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• 제30권3호
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• pp.469-475
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• 2020

During meiosis I, programmed DNA double-strand breaks (DSBs) occur to promote chromosome pairing and recombination between homologs. In Saccharomyces cerevisiae, Mec1 and Tel1, the orthologs of human ATR and ATM, respectively, regulate events upstream of the cell cycle checkpoint to initiate DNA repair. Tel1^ATM and Mec1^ATR are required for phosphorylating various meiotic proteins during recombination. This study aimed to investigate the role of Tel1^ATM and Mec1^ATR in meiotic prophase via physical analysis of recombination. Tel1^ATM cooperated with Mec1^ATR to mediate DSB-to-single end invasion transition, but negatively regulated DSB formation. Furthermore, Mec1^ATR was required for the formation of interhomolog joint molecules from early prophase, thus establishing a recombination partner choice. Moreover, Mec1^ATR specifically promoted crossover-fated DSB repair. Together, these results suggest that Tel1^ATM and Mec1^ATR function redundantly or independently in all post-DSB stages.

• Radiation Oncology Journal
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• 제13권2호
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• pp.113-120
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• 1995

Purpose : To investigate the effect of alkaloid fraction from Korean ginseng on radiation-induced DNA double strand breaks (dsb) formation and repair in murine lymphocytes Materials and Methods : We used the neutral filter elution technique to assay $^{60}Co\;{\gamma}$ ray-induced DNA double strand breaks formation and repair in C57BL/6 mouse spleen lymphocytes for evaluating the dose-response relationship in the presence of alkaloid fraction as a radioprotective agent. The lymphocytes were stimulated with Phytohemagglutinin (PHA, 2 u g/ml) to label $^3[H]-thymidine.$ Isotope-labelled lymphocytes in suspension were exposed to 100 Gy at $0^{\cdot}C$ in the alkaloid fraction-treated group and elution procedure was performed at PH 9.6. The extents of formation of radiation-induced DNA double strand breaks and repair were compared respectively via strand scission factor (SSF) and relative strand scission factor (RSSF). Results: Alkaloid fraction reduced the formation of double strand breaks with dose modification factor of 2 15, compared to control group Rejoining of DNA dsb appeared to take place via two components. The first fast component was completed within 20.4 minutes, but the second slow component was not completed until 220.2 minutes after irradiation. About $30\%$ of dsb formed by irradiation was ultimately unrejoined despite the administration of alkaloid fraction. The administration of alkaloid fraction had a great effect on the second slow component of repair; the half-time of fast component repair was not changed, but that of slow component was 621.8 minutes. Conclusion: Neutral filter elution assay Proved to be a very effective method to quantitate the extents of DNA dsb formation and its repair. By using this technique, we were able to evaluate the efficiency of alkaloid fraction from Korean ginseng as a valuable radioprotector. Alkaloid fraction can be used prophylactically to prevent or ameliorate the severe radiation damages in workers and neighbors around the atomic power plants. For more refined study, however, more advanced purification of alkaloid fraction wil be needed in the near future.

• Molecules and Cells
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• 제39권3호
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• pp.204-210
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• 2016

DNA damage responses are important for the maintenance of genome stability and the survival of organisms. Such responses are activated in the presence of DNA damage and lead to cell cycle arrest, apoptosis, and DNA repair. In Caenorhabditis elegans, double-strand breaks induced by DNA damaging agents have been detected indirectly by antibodies against DSB recognizing proteins. In this study we used a comet assay to detect DNA strand breaks and to measure the elimination of DNA strand breaks in mitotic germline nuclei of C. elegans. We found that C. elegans brc-1 mutants were more sensitive to ionizing radiation and camptothecin than the N2 wild-type strain and repaired DNA strand breaks less efficiently than N2. This study is the first demonstration of direct measurement of DNA strand breaks in mitotic germline nuclei of C. elegans. This newly developed assay can be applied to detect DNA strand breaks in different C. elegans mutants that are sensitive to DNA damaging agents.

• Asian Pacific Journal of Cancer Prevention
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• 제14권12호
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• pp.7091-7094
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• 2013

DNA repair capacity is crucial in maintaining cellular functions and homeostasis. However, it can be altered based on DNA sequence variations in DNA repair genes and this may lead to the development of many diseases including malignancies. Identification of genetic polymorphisms responsible for reduced DNA repair capacity is necessary for better prevention. Homologous recombination (HR), a major double strand break repair pathway, plays a critical role in maintaining the genome stability. The present study was performed to determine the frequency of the HR gene XRCC3 Exon 7 (C18067T, rs861539) polymorphisms in Saudi Arabian population in comparison with epidemiological studies by "MEDLINE" search to equate with global populations. The variant allelic (T) frequency of XRCC3 (C>T) was found to be 39%. Our results suggest that frequency of XRCC3 (C>T) DNA repair gene exhibits distinctive patterns compared with the Saudi Arabian population and this might be attributed to ethnic variation. The present findings may help in high-risk screening of humans exposed to environmental carcinogens and cancer predisposition in different ethnic groups.

• Journal of Microbiology and Biotechnology
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• 제25권7호
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• pp.1026-1035
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• 2015

Condensin is not only responsible for chromosome condensation, but is also involved in double-strand break (DSB) processing in the cell cycle. During meiosis, the condensin complex serves as a component of the meiotic chromosome axis, and mediates both proper assembly of the synaptonemal complex and DSB repair, in order to ensure proper homologous chromosome segregation. Here, we used the budding yeast Saccharomyces cerevisiae to show that condensin participates in a variety of chromosome organization processes and exhibits crucial molecular functions that contribute to meiotic recombination during meiotic prophase I. We demonstrate that Ycs4 is required for efficient DSB formation and establishing homolog bias at the early stage of meiotic prophase I, which allows efficient formation of interhomolog recombination products. In the Ycs4 meiosis-specific allele (ycs4S), interhomolog products were formed at substantial levels, but with the same reduction in crossovers and noncrossovers. We further show that, in prophase chromosomal events, ycs4S relieved the defects in the progression of recombination interactions induced as a result of the absence of Rec8. These results suggest that condensin is a crucial coordinator of the recombination process and chromosome organization during meiosis.

• Journal of electromagnetic engineering and science
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• 제15권3호
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• pp.134-141
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• 2015

The effect of a 60 Hz time-varying electric field was studied using a facing-electrode device (FED) and a coplanar-electrode device (CED) for further investigation of the genotoxicity of 60 Hz time-varying magnetic field (MF) from preceding research. Neither a single 30-minute exposure to the CED or to the FED had any obvious biological effects such as DNA double strand break (DSB) and apoptosis in cancerous SCC25, and HeLa cells, normal primary fibroblast IMR90 cells, while exposures of 60 Hz time-varying MF led to DNA damage with induced electric fields much smaller than those used in this experiment. Nor did repetitive exposures of three days or a continuous exposure of up to 144 hours with the CED induce any DNA damage or apoptosis in either HeLa or IMR90 cells. These results imply that the solitary electric field produced by time-varying MF is not a major cause of DSBs or apoptosis in cancer or normal cells.

• Molecules and Cells
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• 제42권7호
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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.