• Molecules and Cells
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• 제41권2호
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• pp.127-133
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• 2018

Chromatin remodeling factors are involved in many cellular processes such as transcription, replication, and DNA damage response by regulating chromatin structure. As one of chromatin remodeling factors, remodeling and spacing factor 1 (RSF1) is recruited at double strand break (DSB) sites and regulates ataxia telangiectasia mutated (ATM) -dependent checkpoint pathway upon DNA damage for the efficient repair. RSF1 is overexpressed in a variety of cancers, but regulation of RSF1 levels remains largely unknown. Here, we showed that protein levels of RSF1 chromatin remodeler are temporally upregulated in response to different DNA damage agents without changing the RSF1 mRNA level. In the absence of SNF2h, a binding partner of RSF1, the RSF1 protein level was significantly diminished. Intriguingly, the level of RSF1-3SA mutant lacking ATM-mediated phosphorylation sites significantly increased, and upregulation of RSF1 levels under DNA damage was not observed in cells overexpressing ATM kinase. Furthermore, failure in the regulation of RSF1 level caused a significant reduction in DNA repair, whereas reconstitution of RSF1, but not of RSF1-3SA mutants, restored DSB repair. Our findings reveal that temporal regulation of RSF1 levels at its post-translational modification by SNF2h and ATM is essential for efficient DNA repair.

• 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.

• 한국발생생물학회지:발생과생식
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• 제20권2호
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• pp.141-147
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• 2016

Rad51 is a key component of homologous recombination (HR) to repair DNA double-strand breaks and it forms Rad51 recombinase filaments of broken single-stranded DNA to promote HR. In addition to its role in DNA repair and cell cycle progression, Rad51 contributes to the reprogramming process during the generation of induced pluripotent stem cells. In light of this, we performed reprogramming experiments to examine the effect of co-expression of Rad51 and four reprogramming factors, Oct4, Sox2, Klf4, and c-Myc, on the reprogramming efficiency. Co-expression of Rad51 significantly increased the numbers of alkaline phosphatase-positive colonies and embryonic stem cell-like colonies during the process of reprogramming. Co-expression ofRad51 significantly increased the expression of epithelial markers at an early stage of reprogramming compared with control cells. Phosphorylated histone H2AX (${\gamma}H2AX$), which initiates the DNA double-strand break repair system, was highly accumulated in reprogramming intermediates upon co-expression of Rad51. This study identified a novel role of Rad51 in enhancing the reprogramming efficiency, possibly by facilitating mesenchymal-to-epithelial transition and by regulating a DNA damage repair pathway during the early phase of the reprogramming process.

• 생명과학회지
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• 제15권4호
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• pp.542-547
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• 2005

시스플래틴이나 마이토마이신 C (MMC)와 같은 DNA 사슬간 교차결합 (interstrand cross-link ; ICL) 물질에 대해 Brca1 결손세포들이 보이는 높은 감수성은 Brca1 단백질이 세포의 ICL복구반응에 중요한 역할을 담당하고 있음을 암시하고 있다. Brca1 단백질은 재조합 의존성 또는 재조합 비의존성 경로를 통한 DNA 이중사슬 절단(double-strand break ; DSB) 복구에 필수적인 역할을 담당한다. 최근 본인이 속한 연구그룹에서 재조합 의존성 경로를 통한 세포의 ICL복구반응에 Brca1이 관여한다는 것을 밝혀 보고한바 있다. 본 연구에서는 Brca1 단백질의 재조합 비의존성 복구반응에 대한 관여여부를 $p53^{-/-}$와 $p53^{-/-}\;Brcal^{-/-}$ 세포주를 사용하여 연구하였다. 교차결합 복구 실험에서 Brca1 결손 세포주는 Brca1 정상 세포주보다 현저히 낮은 활성을 보였다. 또한, Brca1 결손세포 주의 MMC 에 대한 감수성과 ICL복구능이 Brca1 단백질 발현을 통해 회복되는 것을 확인하였다. 흥미롭게도, Brca1의 11번 엑손 결손세포주 $(Brca1^{\Delta11})$는 높은 MMC저항성과 ICL 복구능을 보였다. 이러한 결과들을 종합하여 볼 때, Brca1 단백질은 ICL복구에 재조합 의존성 경로뿐만 아니라 재조합 비의존성 경로를 통해서도 관여하며, 이러한 활성에는 엑손 11 부분이 아닌 N 말단의 RING 핑거 도메인이나 C 말단의 BRCT도메인이 중요하다는 것을 알 수 있다.

• Journal of Microbiology and Biotechnology
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• 제25권5호
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• pp.598-605
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• 2015

The cohesin complex holds sister chromatids together and prevents premature chromosome segregation until the onset of anaphase. Mcd1 (also known as Scc1), the α-kleisin subunit of cohesin, is a key regulatory subunit of the mitotic cohesin complex and is required for maintaining sister chromatid cohesion, chromosome organization, and DNA repair. We investigated the function of Mcd1 in meiosis by ectopically expressing Mcd1 during early meiotic prophase I in Saccharomyces cerevisiae. Mcd1 partially regulated the progression of meiotic recombination, sister chromatid separation, and nuclear division. DNA physical analysis during meiotic recombination showed that Mcd1 induced double-strand breaks (DSBs) but negatively regulated homologous recombination during DSB repair; Mcd1 expression delayed post-DSB stages, leading to inefficiencies in the DSB-to-joint molecule (JM) transition and subsequent crossover formation. These findings indicate that meiotic cells undergo Mcd1-mediated DSB formation during prophase I, and that residual Mcd1 could regulate the progression of JM formation during meiotic recombination.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제34권5호
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• pp.509-517
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• 2008

DNA 손상 치유 유전자 연구를 기초로 한 임상적 접근이 새로운 치료방법으로 떠오르고 있다. 많은 연구들이 중요한 DNA 수복유전자의 다형성을 찾아내어 각각의 단백질의 활동성에 대한 영향을 알아내고 특정한 치료법을 찾아내고 임상적 적용을 시도하고 결과를 평가하였다. 그 결과 암 치료에서 정상 세포와 암세포에서 DNA 수복 유전자의 발현 분석은 화학요법이나 방사선 치료에서 개인맞춤형 치료법을 가능하게 하고 있다. 예를 들어, NER이 결핍된 종양은 cisplatin 치료에 민감성을 나타내고, MMR 결핍세포는 알킬화 화학요법 약제에 높은 내성을 나타낸다. 선천성 비폴립성 결장암과 같은 MMR 결손종양 또한 알킬화 화학요법 약제에 의한 치료에 내성을 가진다. 신경교종(glioma)에서 MGMT 유전자 프로모터가 흔히 메틸화되는데 이것은 유전자 발현이 억제되고 알킬화 화학요법제에 대한 반응성을 증가시킨다. 향후 구강악안면외과 영역에서도 구강암의 발생의 위험성을 증가시킬 수 있는 더 많은 DNA 수복 유전자의 다형성을 발굴하고 임상적으로 개인맞춤형 치료법을 개발하고 적용할 수 있는 많은 연구가 필요할 것으로 사료된다.

• Molecules and Cells
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• 제39권7호
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• pp.550-556
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• 2016

During meiosis, exchange of DNA segments occurs between paired homologous chromosomes in order to produce recombinant chromosomes, helping to increase genetic diversity within a species. This genetic exchange process is tightly controlled by the eukaryotic RecA homologs Rad51 and Dmc1, which are involved in strand exchange of meiotic recombination, with Rad51 participating specifically in mitotic recombination. Meiotic recombination requires an interaction between homologous chromosomes to repair programmed double-strand breaks (DSBs). In this study, we investigated the budding yeast meiosis-specific proteins Hop2 and Sae3, which function in the Dmc1-dependent pathway. This pathway mediates the homology searching and strand invasion processes. Mek1 kinase participates in switching meiotic recombination from sister bias to homolog bias after DSB formation. In the absence of Hop2 and Sae3, DSBs were produced normally, but showed defects in the DSB-to-single-end invasion transition mediated by Dmc1 and auxiliary factors, and mutant strains failed to complete proper chromosome segregation. However, in the absence of Mek1 kinase activity, Rad51-dependent recombination progressed via sister bias in the $hop2{\Delta}$ or $sae3{\Delta}$ mutants, even in the presence of Dmc1. Thus, Hop2 and Sae3 actively modulate Dmc1-dependent recombination, effectively progressing homolog bias, a process requiring Mek1 kinase activation.

• BMB Reports
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• 제56권5호
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• pp.265-274
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• 2023

Defects in DNA double-strand break (DSB) repair signaling permit cancer cells to accumulate genomic alterations that confer their aggressive phenotype. Nevertheless, tumors depend on residual DNA repair abilities to survive the DNA damage induced by genotoxic stress. This is why only isolated DNA repair signaling is inactivated in cancer cells. DNA DSB repair signaling contributes to general mechanism for various types of lesions in diverse cell cycle phases. DNA DSB repair genes are frequently mutated and amplified in cancer; however, limited data exist regarding the overall genomic prospect and functional result of these modifications. We list the DNA repair genes and related E3 ligases. Mutation and expression frequencies of these genes were analyzed in COSMIC and TCGA. The 11 genes with a high frequency of mutation differed between cancers, and mutations in many DNA DSB repair E3 ligase genes were related to a higher total mutation burden. DNA DSB repair E3 ligase genes are involved in tumor suppressive or oncogenic functions, such as RNF168 and FBXW7, by assisting the functionality of these genomic alterations. DNA damage response-related E3 ligases, such as RNF168, FBXW7, and HERC2, were generated with more than 10% mutation in several cancer cells. This study provides a broad list of candidate genes as potential biomarkers for genomic instability and novel therapeutic targets in cancer. As a DSB related proteins considerably appear the possibilities for targeting DNA repair defective tumors or hyperactive DNA repair tumors. Based on recent research, we describe the relationship between unstable DSB repairs and DSB-related E3 ligases.

• Journal of Microbiology and Biotechnology
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• 제27권2호
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• pp.405-411
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• 2017

Homologous recombination occurs between homologous chromosomes and is significantly involved in programmed double-strand break (DSB) repair. Activation of two recombinases, Rad51 and Dmc1, is essential for an interhomolog bias during meiosis. Rad51 participates in both mitotic and meiotic recombination, and its strand exchange activity is regulated by an inhibitory factor during meiosis. Thus, activities of Rad51 and Dmc1 are coordinated to promote homolog bias. It has been reported that Hed1, a meiosis-specific protein in budding yeast, regulates Rad51-dependent recombination activity. Here, we investigated the role of Hed1 in meiotic recombination by ectopic expression of the protein after pre-meiotic replication in Saccharomyces cerevisiae. DNA physical analysis revealed that the overexpression of Hed1 delays the DSB-to-joint molecule (JM) transition and promotes interhomolog JM formation. The study indicates a possible role of Hed1 in controlling the strand exchange activity of Rad51 and, eventually, meiotic crossover formation.

• 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.