• 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.

• BMB Reports
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• v.56 no.2
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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.

• BMB Reports
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• v.40 no.3
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• pp.432-438
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• 2007

V(D)J recombination, a site-specific gene rearrangement process occurring during the lymphocyte development, begins with DNA double strand breaks by two recombination activating gene products (RAG1/2) and finishes with the repair process by several proteins including DNA-dependent protein kinase (DNA-PK). In this report, we found that RAG2 was specifically phosphorylated by DNA-PK at the $365^{th}$ serine residue, and this phosphorylated RAG2 affected the V(D)J recombination activity in cells in the GFP expression-based assay. While the V(D)J recombination activity between wild-type RAG2 and mutant S365A RAG2 in the assay using a signal joint substrate was undistinguishable in DNA-PK deficient cells (M059J), the activity with wild-type RAG2 was largely increased in DNA-PK proficient cells (M059K) in comparison with mutant RAG2, suggesting that RAG2 phosphorylation by DNA-PK plays a crucial role in the signal joint formation during V(D)J recombination.

• BMB Reports
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• v.54 no.2
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• pp.98-105
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• 2021

The clustered regularly interspaced short palindromic repeats (CRISPR) system is a family of DNA sequences originally discovered as a type of acquired immunity in prokaryotes such as bacteria and archaea. In many CRISPR systems, the functional ribonucleoproteins (RNPs) are composed of CRISPR protein and guide RNAs. They selectively bind and cleave specific target DNAs or RNAs, based on sequences complementary to the guide RNA. The specific targeted cleavage of the nucleic acids by CRISPR has been broadly utilized in genome editing methods. In the process of genome editing of eukaryotic cells, CRISPR-mediated DNA double-strand breaks (DSB) at specific genomic loci activate the endogenous DNA repair systems and induce mutations at the target sites with high efficiencies. Two of the major endogenous DNA repair machineries are non-homologous end joining (NHEJ) and homology-directed repair (HDR). In case of DSB, the two repair pathways operate in competition, resulting in several possible outcomes including deletions, insertions, and substitutions. Due to the inherent stochasticity of DSB-based genome editing methods, it was difficult to achieve defined single-base changes without unanticipated random mutation patterns. In order to overcome the heterogeneity in DSB-mediated genome editing, novel methods have been developed to incorporate precise single-base level changes without inducing DSB. The approaches utilized catalytically compromised CRISPR in conjunction with base-modifying enzymes and DNA polymerases, to accomplish highly efficient and precise genome editing of single and multiple bases. In this review, we introduce some of the advances in single-base level CRISPR genome editing methods and their applications.

• The Korean Journal of Physiology and Pharmacology
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• v.7 no.1
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• pp.9-14
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• 2003

Recent studies indicated that cancer cells become resistant to ionizing radiation (IR) and chemotherapy drugs by enhanced DNA repair of the lesions. Therefore, it is expected to increase the killing of cancer cells and reduce drug resistance by inhibiting DNA repair pathways that tumor cells rely on to escape chemotherapy. There are a number of key human DNA repair pathways which depend on multimeric polypeptide activities. For example, Ku heterodimer regulatory DNA binding subunits (Ku70/Ku80) on binding to double strand DNA breaks (DSBs) are able to interact with 470-kDa DNA-dependent protein kinase catalytic subunit (DNA-PKcs), and are essential for DNA-dependent protein kinase (DNA-PK) activity. It has been known that DNA-PK is an important factor for DNA repair and also is a sensor-transmitting damage signal to downstream targets, leading to cell cycles arrest. Our ultimate goal is to develop a treatment of breast tumors by targeting proteins involved in damage-signaling pathway and/or DNA repair. This would greatly facilitate tumor cell cytotoxic activity and programmed cell death through DNA damaging drug treatment. Therefore, we designed a domain of Ku80 mutants that binds to Ku70 but not DNA end binding activity and used the peptide in co-therapy strategy to see whether the targeted inhibition of DNA-PK activity sensitized breast cancer cells to irradiation or chemotherapy drug. We observed that the synthesized peptide (HNI-38) prevented DNA-PKcs from binding to Ku70/Ku80, thus resulting in inactivation of DNA-PK activity. Consequently, the peptide treated cells exhibited poor to no DNA repair, and became highly sensitive to IR or chemotherapy drugs, and the growth of breast cancer cells was inhibited. Additionally, the results obtained in the present study also support the physiological role of resistance of cancer cells to IR or chemotherapy.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.8
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• pp.3301-3306
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• 2015

Nucleolin (C23) is an important anti-apoptotic protein that is ubiquitously expressed in exponentially growing eukaryotic cells. In order to understand the impact of C23 in radiation therapy, we attempted to investigate the relationship of C23 expression with the radiosensitivity of human non-small cell lung cancer (NSCLC) cells. We investigated the role of C23 in activating the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), which is a critical protein for DNA double-strand breaks (DSBs) repair. As a result, we found that the expression of C23 was negatively correlated with the radiosensitivity of NSCLC cell lines. In vitro clonogenic survival assays revealed that C23 knockdown increased the radiosensitivity of a human lung adenocarcinoma cell line, potentially through the promotion of radiation-induced apoptosis and adjusting the cell cycle to a more radiosensitive stage. Immunofluorescence data revealed an increasing quantity of ${gamma}$-H2AX foci and decreasing radiation-induced DNA damage repair following knockdown of C23. To further clarify the mechanism of C23 in DNA DSBs repair, we detected the expression of DNA-PKcs and C23 proteins in NSCLC cell lines. C23 might participate in DNA DSBs repair for the reason that the expression of DNA-PKcs decreased at 30, 60, 120 and 360 minutes after irradiation in C23 knockdown cells. Especially, the activity of DNA-PKcs phosphorylation sites at the S2056 and T2609 was significantly suppressed. Therefore we concluded that C23 knockdown can inhibit DNA-PKcs phosphorylation activity at the S2056 and T2609 sites, thus reducing the radiation damage repair and increasing the radiosensitivity of NSCLC cells. Taken together, the inhibition of C23 expression was shown to increase the radiosensitivity of NSCLC cells, as implied by the relevance to the notably decreased DNA-PKcs phosphorylation activity at the S2056 and T2609 clusters. Further research on targeted C23 treatment may promote effectiveness of radiotherapy and provide new targets for NSCLC patients.

• BMB Reports
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• v.52 no.3
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• pp.208-213
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• 2019

Chemoresistance is the primary obstacle in the treatment of locally advanced and metastatic nasopharyngeal carcinoma (NPC). Recent evidence suggests that the transcription factor forkhead box M1 (FoxM1) is involved in chemoresistance. Our group previously confirmed that FoxM1 is overexpressed in NPC. In this study, we investigated the role of FoxM1 in cisplatin resistance of the cell lines 5-8F and HONE-1 and explored its possible mechanism. Our results showed that FoxM1 and NBS1 were both overexpressed in NPC tissues based on data from the GSE cohort (GSE12452). Then, we measured FoxM1 levels in NPC cells and found FoxM1 was overexpressed in NPC cell lines and could be stimulated by cisplatin. MTT and clonogenic assays, flow cytometry, ${\gamma}H2AX$ immunofluorescence, qRT-PCR, and western blotting revealed that downregulation of FoxM1 sensitized NPC cells to cisplatin and reduced the repair of cisplatin-induced DNA double-strand breaks via inhibition of the MRN (MRE11-RAD50-NBS1)-ATM axis, which might be related to the ability of FoxM1 to regulate NBS1. Subsequently, we demonstrated that enhanced sensitivity of FoxM1 knockdown cells could be reduced by overexpression of NBS1. Taken together, our data demonstrate that downregulation of FoxM1 could improve the sensitivity of NPC cells to cisplatin through inhibition of MRN-ATM-mediated DNA repair, which could be related to FoxM1-dependent regulation of NBS1.

• Journal of Life Science
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• v.13 no.3
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• pp.241-247
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• 2003

While the DNA-protein kinase (DNA-PK) complex, comprised of DNA-PKcs and Ku80, is primary involved in the repair of DNA double-strand breaks, it is also believed to participate in additional cellular processes. Here, treatment of embryo fibroblasts (MEFs) derived from either wild-type (Wt) or DNA-PKcs-null (DNA-$PKcs^{-/-}$) mice with various stress inducing agents revealed that adriamycin was markedly more cytotoxic for $Ku80^{-/-}MEFs$ and led to their long-term accumulation in the $G_2$/M phase. This differential response was not due to differences in DNA repair, since adrimycin-triggered DNA damage was repaired with comparable efficiency in both Wt and $Ku80^{-/-}MEFs$, but was associated with differences in the expression of important cell cycle regulatory genes. Our results support the notion that Ku80-mediated cytoprotection and $G_2$/M-progression are not only dependent on the cell's DNA repair but also may reflect Ku80's influence on additional cellular processes such as gene expression.

• International Journal of Stem Cells
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• v.16 no.2
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• pp.234-243
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• 2023

The recent advances in human pluripotent stem cells (hPSCs) enable to precisely edit the desired bases in hPSCs to be used for the establishment of isogenic disease models and autologous ex vivo cell therapy. The knock-in approach based on the homologous directed repair with Cas9 endonuclease, causing DNA double-strand breaks (DSBs), produces not only insertion and deletion (indel) mutations but also deleterious large deletions. On the contrary, due to the lack of Cas9 endonuclease activity, base editors (BEs) such as adenine base editor (ABE) and cytosine base editor (CBE) allow precise base substitution by conjugated deaminase activity, free from DSB formation. Despite the limitation of BEs in transition substitution, precise base editing by BEs with no massive off-targets is suggested to be a prospective alternative in hPSCs for clinical applications. Considering the unique cellular characteristics of hPSCs, a few points should be considered. Herein, we describe an updated and optimized protocol for base editing in hPSCs. We also describe an improved methodology for CBE-based C to T substitutions, which are generally lower than A to G substitutions in hPSCs.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.24
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• pp.10985-10989
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• 2015

Background: The capability for DNA double-strand breaks (DSBs) repair is crucial for inherent radiosensitivity of tumor and normal cells. We have investigated the clinicopathologic significance of DNA repair gene expression in nasopharyngeal (NP) carcinoma. Materials and Methods: A total of 65 NP cancer patients who received radiotherapy were included. The immunopositivity to Ku 70, DNA-PKcs, MRN, RAD50, XRCC4, and LIG4 were examined in all tumor tissues. Results: The patients comprised 42 males and 23 females, with a median age of 56 years (range, 18-84). The expression levels of RAD50 (0,+1,+2,+3) were 27.7%, 32.3%, 21.5%, and 18.5%. LIG4 (${\pm}$) were 43.1% and 56.9% respectively. The 5-year OS rate of patients with LIG4 (${\pm}$) were 90% and 67.9%, respectively (p=0.035). The 5-year TTP rate of patients with LIG4 (${\pm}$) were 75.9%, 55.5%, respectively (P=0.039). Conclusions: Our results suggest the possibility of predicting the radiosensitivity of NP cancer by performing immunohistochemical analysis of LIG4.