• 한국동물생명공학회지
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• 제38권3호
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• pp.99-108
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• 2023

Background: Efficient gene editing technology is needed for successful knock-in. Homologous recombination (HR) is a major double-strand break repair pathway that can be utilized for accurately inserting foreign genes into the genome. HR occurs during the S/G2 phase, and the DNA mismatch repair (MMR) pathway is inextricably linked to HR to maintain HR fidelity. This study was conducted to investigate the effect of inhibiting MMR-related genes using CdCl₂, an MMR-related gene inhibitor, on HR efficiency in HC11 cells. Methods: The mRNA and protein expression levels of MMR-related genes (Msh2, Msh3, Msh6, Mlh1, Pms2), the HR-related gene Rad51, and the NHEJ-related gene DNA Ligase IV were assessed in HC11 cells treated with 10 μM of CdCl₂ for 48 hours. In addition, HC11 cells were transfected with a CRISPR/sgRNA expression vector and a knock-in vector targeting Exon3 of the mouse-beta casein locus, and treated with 10 μM cadmium for 48 hours. The knock-in efficiency was monitored through PCR. Results: The treatment of HC11 cells with a high-dose of CdCl₂ decreased the mRNA expression of the HR-related gene Rad51 in HC11 cells. In addition, the inhibition of MMR-related genes through CdCl₂ treatment did not lead to an increase in knock-in efficiency. Conclusions: The inhibition of MMR-related gene expression through high-dose CdCl₂ treatment reduces the expression of the HR-related gene Rad51, which is active during recombination. Therefore, it was determined that CdCl₂ is an inappropriate compound for improving HR efficiency.

• Asian Pacific Journal of Cancer Prevention
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• 제14권11호
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• pp.6727-6731
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• 2013

Homologous recombination (HR) repair has a crucial role to play in the prevention of chromosomal instability, and it is clear that defects in some HR repair genes are associated with many cancers. To evaluate the potential effect of some HR repair gene polymorphisms with differentiated thyroid carcinoma (DTC), we assessed Rad51 (135G>C), Rad52 (2259C>T), XRCC2 (R188H) and XRCC3 (T241M) polymorphisms in Iranian DTC patients and cancer-free controls. In addition, haplotype analysis and gene combination assessment were carried out. Genotyping of Rad51 (135G>C), Rad52 (2259C>T) and XRCC3 (T241M) polymorphisms was determined by PCR-RFLP and PCR-HRM analysis was carried out to evaluate XRCC2 (R188H). Separately, Rad51, Rad52 and XRCC2 polymorphisms were not shown to be more significant in patients when compared to controls in crude, sex-adjusted and age-adjusted form. However, results indicated a significant difference in XRCC3 genotypes for patients when compared to controls (p value: 0.035). The GCTG haplotype demonstrated a significant difference (p value: 0.047). When compared to the wild type, the combined variant form of Rad52/XRCC2/XRCC3 revealed an elevated risk of DTC (p value: 0.007). It is recommended that Rad52 2259C>T, XRCC2 R188H and XRCC3 T241M polymorphisms should be simultaneously considered as contributing to a polygenic risk of differentiated thyroid carcinoma.

• Journal of Plant Biotechnology
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• 제42권3호
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• pp.154-160
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• 2015

The plant breeding technology was developed with genetic engineering. Many researchers and breeders have turned from traditional breeding to molecular breeding. Genetically modified organisms (GMO) were developed via molecular breeding technology. Currently, molecular breeding technologies facilitate efficient plant breeding without introducing foreign genes, in virtue by of gene editing technology. Gene targeting (GT) via homologous recombination (HR) is one of the best gene editing methods available to modify specific DNA sequences in genomes. GT utilizes DNA repair pathways. Thus, DNA repair systems are controlled to enhance HR processing. Engineered sequence specific endonucleases were applied to improve GT efficiency. Engineered sequence specific endonucleases like the zinc finger nuclease (ZFN), TAL effector nuclease (TALEN), and CRISPR-Cas9 create DNA double-strand breaks (DSB) that can stimulate HR at a target site. RecQl4, Exo1 and Rad51 are effectors that enhance DSB repair via the HR pathway. This review focuses on recent developments in engineered sequence specific endonucleases and ways to improve the efficiency of GT via HR effectors in plants.

• 한국동물생명공학회지
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• 제36권3호
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• pp.121-128
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• 2021

Increasing the efficiency of HR (homologous recombination) is important for a successful knock-in. Rad51 is mainly involved in homologous recombination and is associated with strand invasion. The HR-related mismatch repair system maintains HR fidelity by heteroduplex rejection and repair. Therefore, the purpose of this study is to control Rad51, which plays a critical role in HR, through UV-induced DNA damage. It is also to confirm the effect on the expression of MMR related genes (Msh2, Msh3, Msh6, Mlh1, Pms2) and HR-related genes closely related to HR through treatment with the MMR inhibitor CdCl₂. The mRNA expression of Rad51 gene was confirmed in both HC11 cells and mouse testes, but the mRNA expression of Dmc1 gene was confirmed only in mouse testes. The protein expression of Rad51 and Dmc1 gene increased in UV-irradiated HC11 cells. After 72 hours of treatment with 1 ㎛ of CdCl₂, the mRNA expression level of Msh3, Pms2, and Rad51 decreased, but the mRNA expression level of Msh6 and Mlh1 increased in HC11 cells. There was no significant difference in Msh2 mRNA expression between CdCl₂ untreated-group and the 72 hours treated group. In conclusion, HR-related gene (Rad51) was increased by UV-induced DNA damage. Treatment of the MMR inhibitor CdCl₂ in HC11 cells decreased the mRNA expression of Rad51.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제35권1호
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• pp.1-6
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• 2009

DNA double-strand breaks (DSBs) occur commonly in the all living and in cycling cells. They constitute one of the most severe form of DNA damage, because they affect both strand of DNA. DSBs result in cell death or a genetic alterations including deletion, loss of heterozygosity, translocation, and chromosome loss. DSBs arise from endogenous sources like metabolic products and reactive oxygen, and also exogenous factors like ionizing radiation. Defective DNA DSBs can lead to toxicity and large scale sequence rearrangement that can cause cancer and promote premature aging. There are two major pathways for their repair: homologous recombination(HR) and non-homologous end-joining(NHEJ). The HR pathway is a known "error-free" repair mechanism, in which a homologous sister chromatid serves as a template. NHEJ, on the other hand, is a "error-prone" pathway, in which the two termini of the broken DNA molecule are used to form compatible ends that are directly ligated. This review aims to provide a fundamental understanding of how HR and NHEJ pathways operate, cause genome instability, and what kind of genes during the pathways are associated with head and neck cancer.

• 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.

• BMB Reports
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• 제56권2호
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• pp.102-107
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• 2023

Genome editing using CRISPR-associated technology is widely used to modify the genomes rapidly and efficiently on specific DNA double-strand breaks (DSBs) induced by Cas9 endonuclease. However, despite swift advance in Cas9 engineering, structural basis of Cas9-recognition and cleavage complex remains unclear. Proper assembly of this complex correlates to effective Cas9 activity, leading to high efficacy of genome editing events. Here, we develop a CRISPR/Cas9-RAD51 plasmid constitutively expressing RAD51, which can bind to single-stranded DNA for DSB repair. We show that the efficiency of CRISPR-mediated genome editing can be significantly improved by expressing RAD51, responsible for DSB repair via homologous recombination (HR), in both gene knock-out and knock-in processes. In cells with CRISPR/Cas9-RAD51 plasmid, expression of the target genes (cohesin SMC3 and GAPDH) was reduced by more than 1.9-fold compared to the CRISPR/Cas9 plasmid for knock-out of genes. Furthermore, CRISPR/Cas9-RAD51 enhanced the knock-in efficiency of DsRed donor DNA. Thus, the CRISPR/Cas9-RAD51 system is useful for applications requiring precise and efficient genome edits not accessible to HR-deficient cell genome editing and for developing CRISPR/Cas9-mediated knockout technology.

• Animal Bioscience
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• 제35권1호
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• pp.126-137
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• 2022

Objective: Efficient gene editing technology is critical for successful knock-in in domestic animals. RAD51 recombinase (RAD51) gene plays an important role in strand invasion during homologous recombination (HR) in mammals, and is regulated by checkpoint kinase 1 (CHK1) and CHK2 genes, which are upstream elements of RAD51 recombinase (RAD51). In addition, mismatch repair (MMR) system is inextricably linked to HR-related pathways and regulates HR via heteroduplex rejection. Thus, the aim of this study was to investigate whether clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9)-mediated knock-in efficiency of human lactoferrin (hLF) knock-in vector in the bovine β-casein gene locus can be increased by suppressing DNA MMR-related genes (MSH2, MSH3, MSH6, MLH1, and PMS2) and overexpressing DNA double-strand break (DSB) repair-related genes (RAD51, CHK1, CHK2). Methods: Bovine mammary epithelial (MAC-T) cells were transfected with a knock-in vector, RAD51, CHK1, or CHK2 overexpression vector and CRISPR/sgRNA expression vector to target the bovine β-casein gene locus, followed by treatment of the cells with CdCl₂ for 24 hours. After 3 days of CdCl₂ treatment, the knock-in efficiency was confirmed by polymerase chain reaction (PCR). The mRNA expression levels of DNA MMR-related and DNA DSB repair-related genes were assessed by quantitative real-time PCR (RT-qPCR). Results: Treatment with CdCl₂ decreased the mRNA expression of RAD51 and MMRrelated genes but did not increase the knock-in efficiency in MAC-T cells. Also, the overexpression of DNA DSB repair-related genes in MAC-T cells did not significantly affect the mRNA expression of MMR-related genes and failed to increase the knock-in efficiency. Conclusion: Treatment with CdCl₂ inhibited the mRNA levels of RAD51 and DNA MMR-related genes in MAC-T cells. However, the function of MMR pathway in relation to HR may differ in various cell types or species.

• Journal of Animal Science and Technology
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• 제63권5호
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• pp.984-997
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• 2021

This study sought to evaluate DNA damage and repair in porcine postovulatory aged oocytes. The DNA damage response, which was assessed by H2A.X expression, increased in porcine aged oocytes over time. However, the aged oocytes exhibited a significant decrease in the expression of RAD51, which reflects the DNA damage repair capacity. Further experiments suggested that the DNA repair ability was suppressed by the downregulation of genes involved in the homologous recombination (HR) and nonhomologous end-joining (NHEJ) pathways. The expression levels of the cell cycle checkpoint genes, CHEK1 and CHEK2, were upregulated in porcine aged oocytes in response to induced DNA damage. Immunofluorescence results revealed that the expression level of H3K79me2 was significantly lower in porcine aged oocytes than in control oocytes. In addition, embryo quality was significantly reduced in aged oocytes, as assessed by measuring the cell proliferation capacity. Our results provide evidence that DNA damage is increased and the DNA repair ability is suppressed in porcine aged oocytes. These findings increase our understanding of the events that occur during postovulatory oocyte aging.

• Asian Pacific Journal of Cancer Prevention
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• 제15권23호
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• pp.10225-10229
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• 2015

Genetic polymorphisms in homologous recombination repair genes cause an abnormal development of cancerous cells. In the present study we evaluated the possibility of breast cancer association with single nucleotide polymorphisms of RAD51, XRCC2 and XRCC3 genes. Polymorphisms selected in this study were RAD51 135G/C, XRCC2 Arg188His; and XRCC3 Thr241Met. Each polymorphism was genotyped using Polymerase chain reaction-restriction fragment length polymorphism in study cohort of 306 females (156 breast cancer patients and 150 controls). We observed that heterozygous variant genotype (GC) of RAD51 135 G/C polymorphism was associated with a significantly (OR=2.70; 95%CI (0.63-1.79); p<0.03) increased risk of breast cancer. In case of the XRCC3 gene we observed that frequency of heterozygous (OR=2.88; 95%CI (1.02-8.14); p<0.02) and homozygous (OR=1.46; 95%CI (0.89-2.40); p<0.04) genotype of Thr241Met polymorphism were significantly higher in breast cancer patients. For the Arg188His polymorphism of XRCC2, ~2fold increase in breast cancer risk (OR=1.6, 95%CI = 0.73-3.50) was associated with GA genotype with a p value for trend of 0.03. Our results suggest that the 135G/C polymorphism of the RAD51, Thr241Met polymorphism of XRCC3 and Arg188His polymorphism of XRCC2 can be independent markers of breast cancer risk in Pakistan.