Updated on 2025/06/24

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写真a

 
Noritaka Adachi
 
Organization
Graduate School of Nanobioscience Department of Life and Environmental System Science Professor
School of Science Department of Science
Title
Professor
External link

Degree

  • 博士(薬学) ( 東京大学 )

Research Interests

  • Human Genome

  • Aging

  • 分子生物学

  • DNA修復

  • 組換え

  • 細胞周期

  • DNA Recombination

  • Cell Cycle

  • Cancer Genetics

  • Cancer Chemotherapy

  • がん

  • 老化

  • ヒトゲノム

  • Molecular Biology

  • DNA Repair

Research Areas

  • Life Science / Pharmaceutical hygiene and biochemistry

  • Life Science / Genetics

  • Life Science / Molecular biology

  • Life Science / Cell biology

Education

  • The University of Tokyo   Graduate School of Pharmaceutical Sciences

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  • The University of Tokyo   Faculty of Pharmaceutical Sciences

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    Country: Japan

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Research History

Professional Memberships

Papers

  • Homology-arm length of donor DNA affects the impact of Msh2 loss on homologous recombination-mediated gene targeting. International journal

    Shinta Saito, Tetsuya Suzuki, Takehiko Nohmi, Noritaka Adachi

    Proc. Natl. Acad. Sci. USA   122 ( 24 )   e2508507122   2025.6

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    Efficient targeted integration (TI) of homologous donor DNA is crucial for precise genome editing. Although mismatch repair (MMR) is known to suppress TI as well as homologous recombination (HR) when sequence divergence is present, it remains controversial as to whether MMR affects TI of isogenic donor DNA. In this study, we investigated whether and how the MMR protein Msh2 affects TI of isogenic donor DNA. We found that HR-dependent TI is suppressed by Msh2 only when a homology arm of donor DNA is as short as 1.7 kb. In contrast, single-strand annealing-mediated TI, which is cell cycle-independent and becomes prominent when HR or non-homologous end joining is inactivated, is weakly but constantly affected by Msh2 irrespective of the size of homology arms. Our results reveal a previously unrecognized type of HR suppression by Msh2 and provide implications for precise genome editing using short-arm donor DNA vectors.

    DOI: 10.1073/pnas.2508507122

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  • HR eye & MMR eye: one-day assessment of DNA repair-defective tumors eligible for targeted therapy. International journal

    Shinta Saito, Shingo Kato, Usaki Arai, Atsuki En, Jun Tsunezumi, Taichi Mizushima, Kensuke Tateishi, Noritaka Adachi

    Nature Communications   16 ( 1 )   4239 - 4239   2025.5

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    Homologous recombination (HR) and mismatch repair (MMR) act as guardians of the human genome, and defects in HR or MMR are causative in at least a quarter of all malignant tumors. Although these DNA repair-deficient tumors are eligible for effective targeted therapies, fully reliable diagnostic strategies based on functional assay have yet to be established, potentially limiting safe and proper application of the molecular targeted drugs. Here we show that transient transfection of artificial DNA substrates enables ultrarapid detection of HR and MMR. This finding led us to develop a diagnostic strategy that can determine the cellular HR/MMR status within one day without the need for control cells or tissues. Notably, the accuracy of this method allowed the discovery of a pathogenic RAD51D mutation, which was missed by existing companion diagnostic tests. Our methods, termed HR eye and MMR eye, are applicable to frozen tumor tissues and roughly predict the response to therapy. Overall, the findings presented here could pave the way for accurately assessing malignant tumors with functional defects in HR or MMR, a step forward in accelerating precision medicine.

    DOI: 10.1038/s41467-025-59462-2

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  • Characterization and regulation of cell cycle-independent noncanonical gene targeting. International journal

    Shinta Saito, Noritaka Adachi

    Nature Communications   15 ( 1 )   5044 - 5044   2024.6

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    Homology-dependent targeted DNA integration, generally referred to as gene targeting, provides a powerful tool for precise genome modification; however, its fundamental mechanisms remain poorly understood in human cells. Here we reveal a noncanonical gene targeting mechanism that does not rely on the homologous recombination (HR) protein Rad51. This mechanism is suppressed by Rad52 inhibition, suggesting the involvement of single-strand annealing (SSA). The SSA-mediated gene targeting becomes prominent when DSB repair by HR or end-joining pathways is defective and does not require isogenic DNA, permitting 5% sequence divergence. Intriguingly, loss of Msh2, loss of BLM, and induction of a target-site DNA break all significantly and synergistically enhance SSA-mediated targeted integration. Most notably, SSA-mediated integration is cell cycle-independent, occurring in the G1 phase as well. Our findings provide unequivocal evidence for Rad51-independent targeted integration and unveil multiple mechanisms to regulate SSA-mediated targeted as well as random integration.

    DOI: 10.1038/s41467-024-49385-9

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  • Non-homologous DNA end joining and alternative pathways to double-strand break repair Reviewed

    Howard H. Y. Chang, Nicholas R. Pannunzio, Noritaka Adachi, Michael R. Lieber

    Nature Reviews Molecular Cell Biology   18 ( 8 )   495 - 506   2017.8

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  • Dual loss of human POLQ and LIG4 abolishes random integration Reviewed

    Shinta Saito, Ryo Maeda, Noritaka Adachi

    Nature Communications   8   16112   2017.7

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    DOI: 10.1038/ncomms16112

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  • Bidirectional gene organization: A common architectural feature of the human genome Reviewed

    N Adachi, MR Lieber

    Cell   109 ( 7 )   807 - 809   2002.6

  • DNA ligase IV-deficient cells are more resistant to ionizing radiation in the absence of Ku70: Implications for DNA double-strand break repair Reviewed

    Noritaka Adachi, Takayuki Ishino, Yasuyuki Ishii, Shunichi Takeda, Hideki Koyama

    Proc. Natl. Acad. Sci. USA   98 ( 21 )   12109 - 12113   2001.10

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    DOI: 10.1073/pnas.201271098

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  • Sulforaphane Induces Transient Reactive Oxygen Species-Mediated DNA Damage in HeLa Cells. Reviewed International journal

    Sakine Kobayashi, Seiya Nishiba, Chinatsu Sato, Kazuya Toriumi, Yuduki Someya, Noritaka Adachi, Shigeki Takeda, Aya Kurosawa

    Genes to Cells   30 ( 1 )   e13190   2025.1

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    Sulforaphane (SFN), an isothiocyanate found in plants of the Brassicaceae family, possesses antioxidant, apoptosis-inducing, and radiosensitizing effects. As one of the mechanisms of cytotoxicity by SFN, SFN has been suggested to be involved in the induction of DNA damage and inhibition of DNA repair. Recently, we reported on the potency of SFN in inducing single-ended double-strand breaks (DSBs) that are caused by the collision of replication forks with single-strand breaks (SSBs). However, the mechanism of SSB accumulation by SFN remains unclear. In this study, we examined the effect of SFN on SSB-inducing factors in HeLa cells. Although the inhibitory effect of SFN on DNA topoisomerase I was not observed, we found that the reduced form of glutathione (GSH; an antioxidant) level was decreased in an SFN concentration-dependent manner. Furthermore, the addition of ascorbic acid partially increased the viability of SFN-treated HeLa cells. We subsequently observed that poly(ADP-ribose) accumulated in SFN-treated HeLa cells, which occurs during early SSB repair. Collectively, these findings suggest that SFN may transiently induce SSBs via reactive oxygen species in HeLa cells.

    DOI: 10.1111/gtc.13190

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  • Quercetin exhibits cytotoxicity in cancer cells by inducing two-ended DNA double-strand breaks

    Yuduki Someya, Shinta Saito, Shigeki Takeda, Noritaka Adachi, Aya Kurosawa

    Biochemical and Biophysical Research Communications   739   150977 - 150977   2024.12

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    DOI: 10.1016/j.bbrc.2024.150977

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  • SMAD2/3 signaling regulates initiation of mouse Wolffian ducts and proximal differentiation in Müllerian ducts

    Tadaaki Nakajima, Akihiro Imai, Chihiro Ishii, Kota Tsuruyama, Risa Yamanaka, Yasuhiro Tomooka, Shinta Saito, Noritaka Adachi, Satomi Kohno, Tomomi Sato

    FEBS Open Bio   2023.11

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    Male and female reproductive tracts develop from anterior intermediate mesoderm with similar differentiation processes. The anterior intermediate mesoderm develops into the mesonephros, and the Wolffian duct initiates by epithelialization in the mesonephros. The Müllerian duct invaginates from the coelomic epithelium of the cranial mesonephros for ductal formation and is then regionalized into proximal to caudal female reproductive tracts. In this study, we focused on the epithelialization of the Wolffian duct, initiation of the Müllerian duct, and the regionalization step of the Müllerian ducts as a continuous process. By using intermediate mesodermal cells from mouse pluripotent stem cells, we identified that inhibition of SMAD2/3 signaling might be involved in the differentiation into mesenchymal cells, after which mesonephric cells might be then epithelialized during differentiation of the Wolffian duct. Aggregation of coelomic epithelial cells might be related to initiation of the Müllerian duct. Transcriptomic analysis predicted that consensus sequences of SMAD3/4 were enriched among highly expressed genes in the proximal Müllerian duct. SMAD2/3 signaling to regulate differentiation of the Wolffian duct was continuously activated in the proximal Müllerian duct and was involved in proximal and oviductal regionalization. Therefore, SMAD2/3 signaling may be finely tuned to regulate differentiation from initiation to regionalization steps.

    DOI: 10.1002/2211-5463.13729

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  • Arsenic affects homologous recombination and single‐strand annealing but not end‐joining pathways during DNA double‐strand break repair

    Aya Kurosawa, Shinta Saito, Mikiko Sakurai, Mizuki Shinozuka, Yuduki Someya, Noritaka Adachi

    FEBS J.   2023.8

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    Arsenic is a carcinogen that can cause skin, lung, and bladder cancer. While DNA double‐strand breaks (DSBs) have been implicated in arsenic‐induced carcinogenesis, the exact mechanism remains unclear. In this study, we performed genetic analysis to examine the impact of arsenic trioxide (As<sub>2</sub>O<sub>3</sub>) on four different DSB repair pathways using the human pre‐B cell line Nalm‐6. Random integration analysis showed that As<sub>2</sub>O<sub>3</sub> does not negatively affect non‐homologous end joining or polymerase theta‐mediated end joining. In contrast, chromosomal DSB repair analysis revealed that As<sub>2</sub>O<sub>3</sub> decreases the efficiency of homologous recombination (HR) and, less prominently, single‐strand annealing. Consistent with this finding, As<sub>2</sub>O<sub>3</sub> decreased gene‐targeting efficiency, owing to a significant reduction in the frequency of HR‐mediated targeted integration. To further verify the inhibitory effect of arsenic on HR, we examined cellular sensitivity to olaparib and camptothecin, which induce one‐ended DSBs requiring HR for precise repair. Intriguingly, we found that As<sub>2</sub>O<sub>3</sub> significantly enhances sensitivity to those anticancer agents in HR‐proficient cells. Our results suggest that arsenic‐induced genomic instability is attributed to HR suppression, providing valuable insights into arsenic‐associated carcinogenesis and therapeutic options.

    DOI: 10.1111/febs.16922

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  • A Cell System-Assisted Strategy for Evaluating the Natural Antioxidant-Induced Double-Stranded DNA Break (DSB) Style. International journal

    Yuduki Someya, Sakine Kobayashi, Kazuya Toriumi, Shigeki Takeda, Noritaka Adachi, Aya Kurosawa

    Genes   14 ( 2 )   2023.2

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    Natural antioxidants derived from plants exert various physiological effects, including antitumor effects. However, the molecular mechanisms of each natural antioxidant have not yet been fully elucidated. Identifying the targets of natural antioxidants with antitumor properties in vitro is costly and time-consuming, and the results thus obtained may not reliably reflect in vivo conditions. Therefore, to enhance understanding regarding the antitumor effects of natural antioxidants, we focused on DNA, one of the targets of anticancer drugs, and evaluated whether antioxidants, e.g., sulforaphane, resveratrol, quercetin, kaempferol, and genistein, which exert antitumor effects, induce DNA damage using gene-knockout cell lines derived from human Nalm-6 and HeLa cells pretreated with the DNA-dependent protein kinase inhibitor NU7026. Our results suggested that sulforaphane induces single-strand breaks or DNA strand crosslinks and that quercetin induces double-strand breaks. In contrast, resveratrol showed the ability to exert cytotoxic effects other than DNA damage. Our results also suggested that kaempferol and genistein induce DNA damage via unknown mechanisms. Taken together, the use of this evaluation system facilitates the analysis of the cytotoxic mechanisms of natural antioxidants.

    DOI: 10.3390/genes14020420

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  • Error-prone bypass patch by a low-fidelity variant of DNA polymerase zeta in human cells. Reviewed International journal

    Tetsuya Suzuki, Akira Sassa, Petr Grúz, Ramesh C Gupta, Francis Johnson, Noritaka Adachi, Takehiko Nohmi

    DNA Repair   100   103052 - 103052   2021.4

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    DNA polymerase ζ (Pol ζ) is a specialized Pol that is involved in translesion DNA synthesis (TLS), in particular, in the extension of primer DNA after bypassing DNA lesions. Previously, we established human cells that express a variant form of Pol ζ with an amino acid change of leucine 2618 to methionine (L2618M) in the catalytic subunit REV3L (DNA Repair, 45, 34-43, 2016). This amino acid change made the cells more sensitive to the mutagenicity of benzo[a]pyrene diol epoxide (BPDE). In this study, we embedded BPDE-N2-guanine at a defined position in the supF gene on the shuttle plasmid and introduced it to REV3 L2618M cells or the wild-type (WT) cells to examine how far Pol ζ L2618M extends the primer DNA after bypassing the lesion. The adduct induced primarily G to T and G to C at the adducted site in both cell lines, but generated additional sequence changes such as base substitutions, deletions and additions in the extension patch much more often in REV3 L2618M cells than in the WT cells. Mutations in the extension patch in REV3 L2618M cells occurred most often within 10 bps from the adducted site. Then, the number of mutations gradually decreased and no mutations were observed between 30 and 40 bps from the lesion. We concluded that human Pol ζ L2618M and perhaps WT Pol ζ extend the primer DNA up to approximately 30 bps from the lesion in vivo. The possibility of involvement of Pol ζ L2618M in the insertion step of TLS is discussed.

    DOI: 10.1016/j.dnarep.2021.103052

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  • Correction: LEDGF/p75-Independent HIV-1 Replication Demonstrates a Role for HRP-2 and Remains Sensitive to Inhibition by LEDGINs. Reviewed International journal

    Rik Schrijvers, Jan De Rijck, Jonas Demeulemeester, Noritaka Adachi, Sofie Vets, Keshet Ronen, Frauke Christ, Frederic D Bushman, Zeger Debyser, Rik Gijsbers

    PLoS Pathogens   16 ( 9 )   e1008894   2020.9

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    [This corrects the article DOI: 10.1371/journal.ppat.1002558.].

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  • Complex genetic interactions between DNA polymerase β and the NHEJ ligase. Reviewed International journal

    Aya Kurosawa, Hiroyuki Kuboshima, Noritaka Adachi

    FEBS J.   287 ( 2 )   377 - 385   2020.1

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    Mammalian cells possess multiple pathways for repairing various types of DNA damage. Although the molecular mechanisms of each DNA repair pathway have been analyzed by biochemical analysis and cell biological analysis, interplay between different pathways has not been fully elucidated. In this study, using human Nalm-6-mutant cell lines, we analyzed the relationship between the base excision repair factor DNA polymerase β (POLβ) and DNA ligase IV (LIG4), which is essential for DNA double-strand break (DSB) repair by non-homologous end-joining (NHEJ). We found that cells lacking both POLβ and LIG4 grew significantly more slowly than either single mutant, indicating cooperative functions of the two proteins in normal cell growth. To further investigate the genetic interaction between POLβ and LIG4, we examined DNA damage sensitivity of the mutant cell lines. Our results suggested that NHEJ acts as a backup pathway for repairing alkylation damage (when converted into DSBs) in the absence of POLβ. Surprisingly, despite the critical role of POLβ in alkylation damage repair, cells lacking POLβ exhibited increased resistance to camptothecin (a topoisomerase I inhibitor that induces DNA single-strand breaks), irrespective of the presence or absence of LIG4. A LIG4-independent increased resistance associated with POLβ loss was also observed with ionizing radiation; however, cells lacking both POLβ and LIG4 were more radiosensitive than either single mutant. Taken together, our findings provide novel insight into the complex interplay between different DNA repair pathways.

    DOI: 10.1111/febs.15012

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  • Reply: Radiation Dose Does Matter: Mechanistic Insights into DNA Damage and Repair Support the Linear No-Threshold Model of Low-Dose Radiation Health Risks. Reviewed

    Duncan JR, Lieber MR, Adachi N, Wahl RL

    Journal of Nuclear Medicine   60 ( 3 )   437 - 438   2019.3

  • Reply: Radiation Dose Does Matter: Mechanistic Insights into DNA Damage and Repair Support the Linear No-Threshold Model of Low-Dose Radiation Health Risks Reviewed

    Duncan JR, Lieber MR, Adachi N, Wahl RL

    Journal of Nuclear Medicine   59 ( 11 )   1780 - 1781   2018.11

  • Radiation Dose Does Matter: Mechanistic Insights into DNA Damage and Repair Support the Linear No-Threshold Model of Low-Dose Radiation Health Risks. Reviewed

    Duncan JR, Lieber MR, Adachi N, Wahl RL

    Journal of Nuclear Medicine   59 ( 7 )   1014 - 1016   2018.7

  • Dynamic behavior of DNA topoisomerase IIβ in response to DNA double-strand breaks. Reviewed

    Morotomi-Yano K, Saito S, Adachi N, Yano KI

    Scientific Reports   8 ( 1 )   10344   2018.7

  • DNA repair after exposure to ionizing radiation is not error-free Reviewed

    James R. Duncan, Michael R. Lieber, Noritaka Adachi, Richard L. Wahl

    Journal of Nuclear Medicine   59 ( 2 )   348   2018.2

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    DOI: 10.2967/jnumed.117.197673

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  • Mechanistic basis for increased human gene targeting by promoterless vectors-roles of homology arms and Rad54 paralogs Reviewed

    Shinta Saito, Aya Kurosawa, Noritaka Adachi

    FEBS J.   284 ( 17 )   2748 - 2763   2017.9

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    DOI: 10.1111/febs.14137

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  • TBP-like Protein (TLP) Disrupts the p53-MDM2 Interaction and Induces Long-lasting p53 Activation Reviewed

    Ryo Maeda, Hiroyuki Tamashiro, Kazunori Takano, Hiro Takahashi, Hidefumi Suzuki, Shinta Saito, Waka Kojima, Noritaka Adachi, Kiyoe Ura, Takeshi Endo, Taka-aki Tamura

    J. Biol. Chem.   292 ( 8 )   3201 - 3212   2017.2

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    DOI: 10.1074/jbc.M116.763318

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  • DNA polymerase kappa protects human cells against MMC-induced genotoxicity through error-free translesion DNA synthesis Reviewed

    Yuki Kanemaru, Tetsuya Suzuki, Akira Sassa, Kyomu Matsumoto, Noritaka Adachi, Masamitsu Honma, Satoshi Numazawa, Takehiko Nohmi

    Genes and Environment   39 ( 1 )   6   2017

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    DOI: 10.1186/s41021-016-0067-3

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  • Sensitivity of human cells expressing low-fidelity or weak-catalytic-activity variants of DNA polymerase zeta to genotoxic stresses Reviewed

    Tetsuya Suzuki, Petr Gruz, Masamitsu Honma, Noritaka Adachi, Takehiko Nohmi

    DNA REPAIR   45   34 - 43   2016.9

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    DOI: 10.1016/j.dnarep.2016.06.002

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  • The role of DNA polymerase zeta in translesion synthesis across bulky DNA adducts and cross-links in human cells Reviewed

    Tetsuya Suzuki, Petr Gruz, Masamitsu Honma, Noritaka Adachi, Takehiko Nohmi

    MUTATION RESEARCH-FUNDAMENTAL AND MOLECULAR MECHANISMS OF MUTAGENESIS   791   35 - 41   2016.9

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    DOI: 10.1016/j.mrfmmm.2016.08.004

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  • Mutations in XRCC4 cause primordial dwarfism without causing immunodeficiency Reviewed

    Shinta Saito, Aya Kurosawa, Noritaka Adachi

    JOURNAL OF HUMAN GENETICS   61 ( 8 )   679 - 685   2016.8

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  • Advances in the Development of Gene-Targeting Vectors to Increase the Efficiency of Genetic Modification Reviewed

    Shinta Saito, Noritaka Adachi

    BIOLOGICAL & PHARMACEUTICAL BULLETIN   39 ( 1 )   25 - 32   2016.1

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    DOI: 10.1248/bpb.b15-00701

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  • Catalytic and non-catalytic roles of DNA polymerase kappa in the protection of human cells against genotoxic stresses Reviewed

    Yuki Kanemaru, Tetsuya Suzuki, Naoko Niimi, Petr Gruz, Kyomu Matsumoto, Noritaka Adachi, Masamitsu Honma, Takehiko Nohmi

    ENVIRONMENTAL AND MOLECULAR MUTAGENESIS   56 ( 8 )   650 - 662   2015.10

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    DOI: 10.1002/em.21961

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  • XRCC4遺伝子変異を原因とする遺伝性疾患:免疫不全を伴わない原発性小人症 Reviewed

    SAITO SHINTA, KUROSAWA AYA, ADACHI NORITAKA

    放射線生物研究   50 ( 3 )   269 - 280   2015.9

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  • Role for Artemis nuclease in the repair of radiation-induced DNA double strand breaks by alternative end joining Reviewed

    Mario Moscariello, Radi Wieloch, Aya Kurosawa, Fanghua Li, Noritaka Adachi, Emil Mladenov, George Iliakis

    DNA REPAIR   31   29 - 40   2015.7

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    DOI: 10.1016/j.dnarep.2015.04.004

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  • Construction and applications of exon-trapping gene-targeting vectors with a novel strategy for negative selection Biotechnology Reviewed

    Shinta Saito, Kiyoe Ura, Miho Kodama, Noritaka Adachi

    BMC Research Notes   8 ( 1 )   278   2015.6

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    DOI: 10.1186/s13104-015-1241-6

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  • In vivo protective roles of DNA polymerase kappa against genotoxic insults Reviewed

    Kanemaru Yuki, Takeiri Akira, Suzuki Tetsuya, Wada Naoko A, Niimi Naoko, Gruz Petr, Motoyama Shigeki, Matsuzaki Kaori, Tateishi Hiromi, Matsumoto Kaoru, Sassa Akira, Masumura Kenichi, Yamada Masami, Mishima Masayuki, Jishage Kou-ichi, Matsumoto Kyomu, Adachi Noritaka, Honma Masamitsu, Nohmi Takehiko

    DNA REPAIR   28   143   2015.4

  • Role of Auf1 in elimination of oxidatively damaged messenger RNA in human cells Reviewed

    Takashi Ishii, Hiroshi Hayakawa, Takeshi Sekiguchi, Noritaka Adachi, Mutsuo Sekiguchi

    FREE RADICAL BIOLOGY AND MEDICINE   79   109 - 116   2015.2

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    DOI: 10.1016/j.freeradbiomed.2014.11.018

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  • Analysis of the Role of Homology Arms in Gene-Targeting Vectors in Human Cells Reviewed

    Ayako Ishii, Aya Kurosawa, Shinta Saito, Noritaka Adachi

    PLOS ONE   9 ( 9 )   e108236   2014.9

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    DOI: 10.1371/journal.pone.0108236

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  • ヒト細胞におけるDNA二本鎖切断の修復 Invited

    KUROSAWA AYA, ADACHI NORITAKA

    Isotope News   ( 721 )   8 - 14   2014.5

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  • In vivo evidence that phenylalanine 171 acts as a molecular brake for translesion DNA synthesis across benzo[a]pyrene DNA adducts by human DNA polymerase kappa Reviewed

    Akira Sassa, Tetsuya Suzuki, Yuki Kanemaru, Naoko Niimi, Hirofumi Fujimoto, Atsushi Katafuchi, Petr Gruz, Manabu Yasui, Ramesh C. Gupta, Francis Johnson, Toshihiro Ohta, Masamitsu Honma, Noritaka Adachi, Takehiko Nohmi

    DNA REPAIR   15   21 - 28   2014.3

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    DOI: 10.1016/j.dnarep.2013.12.008

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  • C-terminal region of DNA ligase IV drives XRCC4/DNA ligase IV complex to chromatin Reviewed

    Sicheng Liu, Xunyue Liu, Radhika Pankaj Kamdar, Rujira Wanotayan, Mukesh Kumar Sharma, Noritaka Adachi, Yoshihisa Matsumoto

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   439 ( 2 )   173 - 178   2013.9

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    DOI: 10.1016/j.bbrc.2013.08.068

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  • DNA Ligase IV and Artemis Act Cooperatively to Suppress Homologous Recombination in Human Cells: Implications for DNA Double-Strand Break Repair Reviewed

    Aya Kurosawa, Shinta Saito, Sairei So, Mitsumasa Hashimoto, Kuniyoshi Iwabuchi, Haruka Watabe, Noritaka Adachi

    PLoS ONE   8 ( 8 )   e72253   2013.8

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    DOI: 10.1371/journal.pone.0072253

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  • Interference in DNA Replication Can Cause Mitotic Chromosomal Breakage Unassociated with Double-Strand Breaks Reviewed

    Mari Fujita, Hiroyuki Sasanuma, Kimiyo N. Yamamoto, Hiroshi Harada, Aya Kurosawa, Noritaka Adachi, Masato Omura, Masahiro Hiraoka, Shunichi Takeda, Kouji Hirota

    PLOS ONE   8 ( 4 )   e60043   2013.4

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    DOI: 10.1371/journal.pone.0060043

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  • Restoration of Mismatch Repair Functions in Human Cell Line Nalm-6, Which Has High Efficiency for Gene Targeting Reviewed

    Tetsuya Suzuki, Akiko Ukai, Masamitsu Honma, Noritaka Adachi, Takehiko Nohmi

    PLOS ONE   8 ( 4 )   e61189   2013.4

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    DOI: 10.1371/journal.pone.0061189

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  • Both CpG Methylation and Activation-Induced Deaminase Are Required for the Fragility of the Human bcl-2 Major Breakpoint Region: Implications for the Timing of the Breaks in the t(14;18) Translocation Reviewed

    Xiaoping Cui, Zhengfei Lu, Aya Kurosawa, Lars Klemm, Andrew T. Bagshaw, Albert G. Tsai, Neil Gemmell, Markus Mueschen, Noritaka Adachi, Chih-Lin Hsieh, Michael R. Lieber

    MOLECULAR AND CELLULAR BIOLOGY   33 ( 5 )   947 - 957   2013.3

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  • Generation and use of genetically modified human cell lines: a promising approach for in vitro toxicology studies. Reviewed

    Adachi N

    J Clinic Toxicol.   3 ( 5 )   45   2013

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  • Repair of accidental DNA double-strand breaks in the human genome and its relevance to vector DNA integration. Reviewed

    Adachi N, Saito S, Kurosawa A

    Gene Technology.   3   e107   2013

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  • Endogenous factors causative of spontaneous DNA damage that leads to random integration in human cells. Reviewed

    Kamekawa H, Kurosawa A, Umehara M, Toyoda E, Adachi N

    Gene Technology.   2   105   2013

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  • DNA repair and translesion DNA synthesis as possible mechanisms underlying genotoxic thresholds Reviewed

    Takehiko Nohmi, Kenichi Masumura, Petr Gruz, Naomi Toyoda-Hokaiwado, Makiko Takamune, Naoko Niimi, Tetsuya Suzuki, Yuki Kanemaru, Manabu Yasui, Masami Yamada, Masamitsu Honma, Noritaka Adachi

    MUTAGENESIS   27 ( 6 )   793 - 793   2012.11

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  • The helicase domain and C-terminus of human RecQL4 facilitate replication elongation on DNA templates damaged by ionizing radiation Reviewed

    Masaoki Kohzaki, Maria Chiourea, Gwennaelle Versini, Noritaka Adachi, Shunichi Takeda, Sarantis Gagos, Thanos D. Halazonetis

    CARCINOGENESIS   33 ( 6 )   1203 - 1210   2012.11

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  • Model for MLL translocations in therapy-related leukemia involving topoisomerase II beta-mediated DNA strand breaks and gene proximity Reviewed

    Ian G. Cowell, Zbyslaw Sondka, Kayleigh Smith, Ka Cheong Lee, Catriona M. Manville, Malgorzata Sidorczuk-Lesthuruge, Holly Ashlene Rance, Kay Padget, Graham Hunter Jackson, Noritaka Adachi, Caroline A. Austin

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   109 ( 23 )   8989 - 8994   2012.6

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  • Artemis C-terminal region facilitates V(D)J recombination through its interactions with DNA Ligase IV and DNA-PKcs Reviewed

    Shruti Malu, Pablo De Ioannes, Mikhail Kozlov, Marsha Greene, Dailia Francis, Mary Hanna, Jesse Pena, Carlos R. Escalante, Aya Kurosawa, Hediye Erdjument-Bromage, Paul Tempst, Noritaka Adachi, Paolo Vezzoni, Anna Villa, Aneel K. Aggarwal, Patricia Cortes

    JOURNAL OF EXPERIMENTAL MEDICINE   209 ( 5 )   955 - 963   2012.5

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  • LEDGF/p75-Independent HIV-1 Replication Demonstrates a Role for HRP-2 and Remains Sensitive to Inhibition by LEDGINs Reviewed

    Rik Schrijvers, Jan De Rijck, Jonas Demeulemeester, Noritaka Adachi, Sofie Vets, Keshet Ronen, Frauke Christ, Frederic D. Bushman, Zeger Debyser, Rik Gijsbers

    PLOS PATHOGENS   8 ( 3 )   e1002558   2012.3

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  • Impaired cell adhesion, apoptosis, and signaling in WASP gene-disrupted Nalm-6 pre-B cells and recovery of cell adhesion using a transducible form of WASp Reviewed

    Rikiya Sato, Susumu Iiizumi, Eun-Sung Kim, Fumiko Honda, Sang-Kyou Lee, Noritaka Adachi, Hideki Koyama, Shuki Mizutani, Tomohiro Morio

    INTERNATIONAL JOURNAL OF HEMATOLOGY   95 ( 3 )   299 - 310   2012.3

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    Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency disease affecting cell morphology and signal transduction in hematopoietic cells. The function of Wiskott-Aldrich syndrome protein (WASp) and its partners in protein interaction have been studied intensively in mice; however, detailed biochemical characterization of its signal transduction and assessment of its functional consequence in human WASp-deficient lymphocytes remain difficult. In this study, we generated Nalm-6 cells in which the WAS protein gene (WASP) was disrupted by homologous recombination-based gene targeting and a cell-permeable form of recombinant WASp for functional study. The WASP(-/-) cells showed impaired adhesive capacity and polarization to plate-bound anti-CD47 mAb, anti-CD9 mAb, or to fibronectin. The defective morphological changes were accompanied by impaired intracellular signaling. In addition, the WASp-deficient cells displayed augmented apoptosis induced by CD24 cross-linking. A recombinant fusion protein composed of Hph-1 cell-permeable peptide and WASp prepared in Escherichia coli. Hph-1-WASp was efficiently transduced and expressed in WASP(-/-) Nalm-6 cells in a dose-dependent manner. The wild-type WASp, but not the mutant restored adhesion capacity, spreading morphology, and cytoskeletal reorganization. Additionally, the recombinant protein was successfully transduced into normal lymphocytes. These findings suggest that gene-disrupted model cell lines and cell-permeable recombinant proteins may serve as important tools for the detailed analysis of intracellular molecules involved in PID.

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  • Impaired cell adhesion, apoptosis, and signaling in WASP gene-disrupted Nalm-6 pre-B cells and recovery of cell adhesion using a transducible form of WASp. Reviewed

    Rikiya Sato, Susumu Iiizumi, Eun-Sung Kim, Fumiko Honda, Sang-Kyou Lee, Noritaka Adachi, Hideki Koyama, Shuki Mizutani, Tomohiro Morio

    Int. J. Hematol.   95 ( 3 )   299 - 310   2012.3

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  • Nucleofection-based gene targeting in human pre-B cells Reviewed

    Aya Kurosawa, Shinta Saito, Mikako Mori, Noritaka Adachi

    GENE   492 ( 1 )   305 - 308   2012.1

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    DOI: 10.1016/j.gene.2011.11.003

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  • Functions and Regulation of Artemis: A Goddess in the Maintenance of Genome Integrity Reviewed

    Aya Kurosawa, Noritaka Adachi

    JOURNAL OF RADIATION RESEARCH   51 ( 5 )   503 - 509   2010.9

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  • OGFOD1, a member of the 2-oxoglutarate and iron dependent dioxygenase family, functions in ischemic signaling Reviewed

    Ken Saito, Noritaka Adachi, Hideki Koyama, Masayuki Matsushita

    FEBS LETTERS   584 ( 15 )   3340 - 3347   2010.8

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  • Chromosomal Manipulation by Site-Specific Recombinases and Fluorescent Protein-Based Vectors Reviewed

    Munehiro Uemura, Youko Niwa, Naoki Kakazu, Noritaka Adachi, Kazuo Kinoshita

    PLOS ONE   5 ( 3 )   e9846   2010.3

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  • FEN1 functions in long patch base excision repair under conditions of oxidative stress in vertebrate cells. Reviewed International journal

    Kenjiro Asagoshi, Keizo Tano, Paul D Chastain 2nd, Noritaka Adachi, Eiichiro Sonoda, Koji Kikuchi, Hideki Koyama, Kenji Nagata, David G Kaufman, Shunichi Takeda, Samuel H Wilson, Masami Watanabe, James A Swenberg, Jun Nakamura

    Molecular cancer research : MCR   8 ( 2 )   204 - 15   2010.2

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    From in vitro studies, flap endonuclease 1 (FEN1) has been proposed to play a role in the long patch (LP) base excision repair (BER) subpathway. Yet the role of FEN1 in BER in the context of the living vertebrate cell has not been thoroughly explored. In the present study, we cloned a DT40 chicken cell line with a deletion in the FEN1 gene and found that these FEN1-deficient cells exhibited hypersensitivity to H(2)O(2). This oxidant produces genotoxic lesions that are repaired by BER, suggesting that the cells have a deficiency in BER affecting survival. In experiments with extracts from the isogenic FEN1 null and wild-type cell lines, the LP-BER activity of FEN1 null cells was deficient, whereas repair by the single-nucleotide BER subpathway was normal. Other consequences of the FEN1 deficiency were also evaluated. These results illustrate that FEN1 plays a role in LP-BER in higher eukaryotes, presumably by processing the flap-containing intermediates of BER.

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  • Loss of Bloom syndrome protein destabilizes human gene cluster architecture Reviewed

    Michael W. Killen, Dawn M. Stults, Noritaka Adachi, Les Hanakahi, Andrew J. Pierce

    HUMAN MOLECULAR GENETICS   18 ( 18 )   3417 - 3428   2009.9

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  • Topoisomerase II alpha inhibition following DNA transfection greatly enhances random integration in a human pre-B lymphocyte cell line Reviewed

    Eriko Toyoda, Aya Kurosawa, Haruna Kamekawa, Noritaka Adachi

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   382 ( 3 )   492 - 496   2009.5

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  • Heterozygous Disruption of the DNA Topoisomerase I Gene Confers Cellular Resistance to Camptothecin in Human Cells Reviewed

    Eriko Toyoda, Aya Kurosawa, Michihiko Fujii, Noritaka Adachi

    BIOLOGICAL & PHARMACEUTICAL BULLETIN   32 ( 4 )   724 - 727   2009.4

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  • [FeFe]-hydrogenase-like gene is involved in the regulation of sensitivity to oxygen in yeast and nematode Reviewed

    Michihiko Fujii, Noritaka Adachi, Kazuki Shikatani, Dai Ayusawa

    GENES TO CELLS   14 ( 4 )   457 - 468   2009.4

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    Oxygen is essential for the life of aerobic organisms, but reactive oxygen species (ROS) derived from oxygen can be a threat for it. Many genes are involved in generation of ROS, but not much attention has been focused on the reactions from which ROS are generated. We therefore screened for mutants that showed an increased sensitivity to oxidative stress in the nematode Caenorhabditis elegans, and isolated a novel mutant, oxy-4(qa5001). This mutant showed an increased sensitivity to a high concentration of oxygen, and decreased longevity at 20 degrees C but not at 26 degrees C. The genetic analysis has revealed that oxy-4 had a causative mutation in an [FeFe]-hydrogenase-like gene (Y54H5A.4). In the yeast Saccharomyces cerevisiae, a deletion of NAR1, a possible homologue of oxy-4, also caused a similar increased sensitivity to oxygen. [FeFe]-hydrogenases are enzymes that catalyze both the formation and the splitting of molecular hydrogen, and function in anaerobic respiration in anaerobes. In contrast, [FeFe]-hydrogenase-like genes identified in aerobic eukaryotes do not generate hydrogen, and its functional roles are less understood. Our results suggested that [FeFe]-hydrogenase-like genes were involved in the regulation of sensitivity to oxygen in S. cerevisiae and C. elegans.

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  • [FeFe]-hydrogenase-like gene is involved in the regulation of sensitivity to oxygen in yeast and nematode

    Michihiko Fujii, Noritaka Adachi, Kazuki Shikatani, Dai Ayusawa

    GENES TO CELLS   14 ( 4 )   457 - 468   2009.4

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    Oxygen is essential for the life of aerobic organisms, but reactive oxygen species (ROS) derived from oxygen can be a threat for it. Many genes are involved in generation of ROS, but not much attention has been focused on the reactions from which ROS are generated. We therefore screened for mutants that showed an increased sensitivity to oxidative stress in the nematode Caenorhabditis elegans, and isolated a novel mutant, oxy-4(qa5001). This mutant showed an increased sensitivity to a high concentration of oxygen, and decreased longevity at 20 degrees C but not at 26 degrees C. The genetic analysis has revealed that oxy-4 had a causative mutation in an [FeFe]-hydrogenase-like gene (Y54H5A.4). In the yeast Saccharomyces cerevisiae, a deletion of NAR1, a possible homologue of oxy-4, also caused a similar increased sensitivity to oxygen. [FeFe]-hydrogenases are enzymes that catalyze both the formation and the splitting of molecular hydrogen, and function in anaerobic respiration in anaerobes. In contrast, [FeFe]-hydrogenase-like genes identified in aerobic eukaryotes do not generate hydrogen, and its functional roles are less understood. Our results suggested that [FeFe]-hydrogenase-like genes were involved in the regulation of sensitivity to oxygen in S. cerevisiae and C. elegans.

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  • Genetic Evidence That the Non-Homologous End-Joining Repair Pathway Is Involved in LINE Retrotransposition Reviewed

    Jun Suzuki, Katsumi Yamaguchi, Masaki Kajikawa, Kenji Ichiyanagi, Noritaka Adachi, Hideki Koyama, Shunichi Takeda, Norihiro Okada

    PLOS GENETICS   5 ( 4 )   e1000461   2009.4

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  • Generation of tetracycline-inducible conditional gene knockout cells in a human Nalm-6 cell line Reviewed

    Tatsuya Ono, Hitoshi Nishijima, Noritaka Adachi, Susumu Iizumi, Akane Morohoshi, Hideki Koyama, Kei-ichi Shibahara

    JOURNAL OF BIOTECHNOLOGY   141 ( 1-2 )   1 - 7   2009.4

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  • Molecular Mechanism of Protein Assembly on DNA Double-strand Breaks in the Non-homologous End-joining Pathway Reviewed

    Ken-ichi Yano, Keiko Morotomi-Yano, Noritaka Adachi, Hidenori Akiyama

    JOURNAL OF RADIATION RESEARCH   50 ( 2 )   97 - 108   2009.3

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  • Mechanisms of chromosomal DNA strand break repair in human cells. Reviewed

    Noritaka ADACHI

    Tanpakushitsu Kakusan Koso.   54 ( 4 Suppl )   472 - 478   2009.3

  • The Iron Chelator Dp44mT Causes DNA Damage and Selective Inhibition of Topoisomerase II alpha in Breast Cancer Cells Reviewed

    V. Ashutosh Rao, Sarah R. Klein, Keli K. Agama, Eriko Toyoda, Noritaka Adachi, Yves Pommier, Emily B. Shacter

    CANCER RESEARCH   69 ( 3 )   948 - 957   2009.2

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    DOI: 10.1158/0008-5472.CAN-08-1437

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  • Improved applications of the tetracycline-regulated gene depletion system Reviewed

    Hitoshi Nishijima, Takami Yasunari, Tatsuo Nakayama, Noritaka Adachi, Kei-ichi Shibahara

    BIOSCIENCE TRENDS   3 ( 5 )   161 - 167   2009

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  • Impact of non-homologous end-joining deficiency on random and targeted DNA integration: implications for gene targeting Reviewed

    Susumu Iiizumi, Aya Kurosawa, Sairei So, Yasuyuki Ishii, Yuichi Chikaraishi, Ayako Ishii, Hideki Koyama, Noritaka Adachi

    NUCLEIC ACIDS RESEARCH   36 ( 19 )   6333 - 6342   2008.11

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  • KU70/80, DNA-PKcs, and Artemis are essential for the rapid induction of apoptosis after massive DSB formation Reviewed

    Takuya Abe, Masamichi Ishiai, Yoshifumi Hosono, Akari Yoshimura, Shusuke Tada, Noritaka Adachi, Hideki Koyama, Minoru Takata, Shunichi Takeda, Takemi Enomoto, Masayuki Seki

    CELLULAR SIGNALLING   20 ( 11 )   1978 - 1985   2008.11

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  • Loss of DNA ligase IV prevents recognition of DNA by double-strand break repair proteins XRCC4 and XLF Reviewed

    Sumithra Jayaram, Gary Ketner, Noritaka Adachi, Les A. Hanakahi

    NUCLEIC ACIDS RESEARCH   36 ( 18 )   5773 - 5786   2008.10

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    DOI: 10.1093/nar/gkn552

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  • NK314, a topoisomerase II inhibitor that specifically targets the alpha isoform Reviewed

    Eriko Toyoda, Shigehide Kagaya, Ian G. Cowell, Aya Kurosawa, Keiichi Kamoshita, Kiyohiro Nishikawa, Susumu Iiizumi, Hideki Koyama, Caroline A. Austin, Noritaka Adachi

    JOURNAL OF BIOLOGICAL CHEMISTRY   283 ( 35 )   23711 - 23720   2008.8

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    DOI: 10.1074/jbc.M803936200

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  • Cell sorting analysis of cell cycle-dependent X-ray sensitivity in end joining-deficient human cells Reviewed

    Kuniyoshi Iwabuchi, Mitsumasa Hashimoto, Tadashi Matsui, Aya Kurosawa, Noritaka Adachi, Takayasu Date

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   372 ( 4 )   662 - 667   2008.8

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  • The requirement of artemis in double-strand break repair depends on the type of DNA damage Reviewed

    Aya Kurosawa, Hideki Koyama, Shinichi Takayama, Kensuke Miki, Dai Ayusawa, Michihiko Fujii, Susumu Iiizumi, Noritaka Adachi

    DNA AND CELL BIOLOGY   27 ( 1 )   55 - 61   2008.1

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    DOI: 10.1089/dna.2007.0649

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  • Gene targeting in cultured human cells Reviewed

    Susumu Iiizumi, Noritaka Adachi

    Seikagaku   80 ( 7 )   651 - 657   2008

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  • Highly proficient gene targeting by homologous recombination in the human Pre-B cell line Nalm-6 Reviewed

    Noritaka Adachi, Aya Kurosawa, Hideki Koyama

    Methods in Molecular Biology   435   17 - 29   2008

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    DOI: 10.1007/978-1-59745-232-8_2

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  • Gene targeting using the human Nalm-6 pre-B cell line Reviewed

    Noritaka Adachi, Hitoshi Nishijima, Kei-Ichi Shibahara

    BIOSCIENCE TRENDS   2 ( 5 )   169 - 180   2008

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  • Overexpression of HAM1 gene detoxifies 5-bromodeoxyuridine in the yeast Saccharomyces cerevisiae

    Shinichi Takayama, Michihiko Fujii, Aya Kurosawa, Noritaka Adachi, Dai Ayusawa

    CURRENT GENETICS   52 ( 5-6 )   203 - 211   2007.11

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    5- Bromodeoxyuridine (BrdU) is known to modulate expression of particular genes, and eventually arrest cell division in mammalian and yeast cells. To study a molecular basis for these phenomena, we adopted a genetic approach with a yeast cell system. We screened multicopy suppressor genes that confer resistance to BrdU with a thymidine-auxotrophic strain of the yeast Saccharomyces cerevisiae. One of such genes was found to encode Ham1 protein, which was originally identified as a possible triphosphatase for N-6- hydroxylaminopurine triphosphate. Consistent with this, overexpression of the HAM1 gene reversed growth arrest caused by BrdU, and blocked incorporation of BrdU into genomic DNA. On the contrary, disruption of the gene sensitized cells to BrdU. A crude extract from Ham1- overproducing cells showed a high activity to hydrolyze BrdUTP to BrdUMP and pyrophosphate in addition to abnormal purine nucleotides. Purified recombinant Ham1 protein showed the same activity. These results demonstrate that Ham1 protein detoxifies abnormal pyrimidine as well as purine nucleotides.

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  • Overexpression of HAM1 gene detoxifies 5-bromodeoxyuridine in the yeast Saccharomyces cerevisiae Reviewed

    Shinichi Takayama, Michihiko Fujii, Aya Kurosawa, Noritaka Adachi, Dai Ayusawa

    CURRENT GENETICS   52 ( 5-6 )   203 - 211   2007.11

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    5- Bromodeoxyuridine (BrdU) is known to modulate expression of particular genes, and eventually arrest cell division in mammalian and yeast cells. To study a molecular basis for these phenomena, we adopted a genetic approach with a yeast cell system. We screened multicopy suppressor genes that confer resistance to BrdU with a thymidine-auxotrophic strain of the yeast Saccharomyces cerevisiae. One of such genes was found to encode Ham1 protein, which was originally identified as a possible triphosphatase for N-6- hydroxylaminopurine triphosphate. Consistent with this, overexpression of the HAM1 gene reversed growth arrest caused by BrdU, and blocked incorporation of BrdU into genomic DNA. On the contrary, disruption of the gene sensitized cells to BrdU. A crude extract from Ham1- overproducing cells showed a high activity to hydrolyze BrdUTP to BrdUMP and pyrophosphate in addition to abnormal purine nucleotides. Purified recombinant Ham1 protein showed the same activity. These results demonstrate that Ham1 protein detoxifies abnormal pyrimidine as well as purine nucleotides.

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  • MK314は安定したDNA切断複合体を形成するトポイソメラーゼIIα特異的阻害剤である(NK314 is a topoisomerase IIα specific inhibitorforming stable DNA cleavage complex)

    豊田 恵利子, 加々谷 重英, 岡本 一也, 横山 浩史, 長幡 武光, 西川 清広, 黒沢 綾, 小山 秀機, 足立 典隆

    日本癌学会総会記事   66回   204 - 205   2007.8

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  • 遺伝性癌の分子遺伝学と疫学 ヒト遺伝子ノックアウト細胞株を用いたトポイソメラーゼ介在性DNA損傷の修復に関する遺伝的研究(Molecular Genetics and Epidemiology of Hereditary Cancer Genetic studies on the repair of topoisomerase-mediated DNA damage using human gene knockout cell lines)

    黒沢 綾, 豊田 恵利子, 小山 秀機, 足立 典隆

    日本癌学会総会記事   66回   258 - 258   2007.8

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  • Absence of p53 enhances growth defects and etoposide sensitivity of human cells lacking the bloom syndrome helicase BLM Reviewed

    Sairei So, Noritaka Adachi, Hideki Koyama

    DNA AND CELL BIOLOGY   26 ( 7 )   517 - 525   2007.7

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  • ヒト重症複合型免疫不全症候群原因遺伝子ARTEMISの機能と制御

    足立 典隆

    放射線生物研究   印刷中 ( 4 )   386 - 401   2007

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  • Simple one-week method to construct gene-targeting vectors: application to production of human knockout cell lines Reviewed

    Susumu Iiizumi, Yuji Nomura, Sairei So, Koichi Uegaki, Kayoko Aoki, Kei-ichi Shibahara, Noritaka Adachi, Hideki Koyama

    BIOTECHNIQUES   41 ( 3 )   311 - +   2006.9

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  • 53BP1 contributes to survival of cells irradiated with X-ray during G1 without Ku70 or Artemis Reviewed

    Kuniyoshi Iwabuchi, Mitsumasa Hashimoto, Tadashi Matsui, Takayuki Kurihara, Hiroko Shimizu, Noritaka Adachi, Masamichi Ishiai, Ken-ichi Yamamoto, Hiroshi Tauchi, Minoru Takata, Hideki Koyama, Takayasu Date

    GENES TO CELLS   11 ( 8 )   935 - 948   2006.8

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  • Enhanced gene targeting efficiency by siRNA that silences the expression of the Bloom syndrome gene in human cells Reviewed

    S So, Y Nomura, N Adachi, Y Kobayashi, T Hori, Y Kurihara, H Koyama

    GENES TO CELLS   11 ( 4 )   363 - 371   2006.4

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  • Heterozygous inactivation ofhuman Ku70/Ku86 heterodimer does not affect cell growth, double-strand break repair, or genome integrity Reviewed

    K Uegaki, N Adachi, S So, S Iiizumi, H Koyama

    DNA REPAIR   5 ( 3 )   303 - 311   2006.3

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    DOI: 10.1016/j.dnarep.2005.10.008

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  • Parp-1 protects homologous recombination from interference by Ku and ligase IV in vertebrate cells Reviewed

    H Hochegger, D Dejsuphong, T Fukushima, C Morrison, E Sonoda, Schreiber, V, GY Zhao, A Saberi, M Masutani, N Adachi, H Koyama, G de Murcia, S Takeda

    EMBO JOURNAL   25 ( 6 )   1305 - 1314   2006.3

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    DOI: 10.1038/sj.emboj.7601015

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  • The human pre-B cell line Nalm-6 is highly proficient in gene targeting by homologous recombination Reviewed

    N Adachi, S So, S Iiizumi, Y Nomura, K Murai, C Yamakawa, K Miyagawa, H Koyama

    DNA AND CELL BIOLOGY   25 ( 1 )   19 - 24   2006.1

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    DOI: 10.1089/dna.2006.25.19

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  • DNA二重鎖切断修復の新守護神

    足立 典隆

    実験医学   24   2006

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  • Fen-1 facilitates homologous recombination by removing divergent sequences at DNA break ends Reviewed

    K Kikuchi, Y Taniguchi, A Hatanaka, E Sonoda, H Hochegger, N Adachi, Y Matsuzaki, H Koyama, DC van Gent, M Jasin, S Takeda

    MOLECULAR AND CELLULAR BIOLOGY   25 ( 16 )   6948 - 6955   2005.8

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    DOI: 10.1128/MCB.25.16.6948-6955.2005

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  • Evidence for a role of vertebrate Rad52 in the repair of topoisomerase II-mediated DNA damage Reviewed

    N Adachi, S Iiizumi, H Koyama

    DNA AND CELL BIOLOGY   24 ( 6 )   388 - 393   2005.6

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    DOI: 10.1089/dna.2005.24.388

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  • Chromosomal instability in the cattle clones derived by somatic cell nuclear-transfer Reviewed

    H Hanada, K Takeda, T Tagami, K Nirasawa, S Akagi, N Adachi, S Takahashi, Y Izaike, M Iwamoto, DI Fuchimoto, N Miyashita, M Kubo, A Onishi, WA King

    MOLECULAR REPRODUCTION AND DEVELOPMENT   71 ( 1 )   36 - 44   2005.5

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    DOI: 10.1002/mrd.20283

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  • Generation and characterization of endonuclease G null mice Reviewed

    RA Irvine, N Adachi, DK Shibata, GD Cassell, KF Yu, ZE Karanjawala, CL Hsieh, MR Lieber

    MOLECULAR AND CELLULAR BIOLOGY   25 ( 1 )   294 - 302   2005.1

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    DOI: 10.1128/MCB.25.1.294-302.2005

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  • Genetic interactions between BLM and DNA ligase IV in human cells Reviewed

    S So, N Adachi, MR Lieber, H Koyama

    JOURNAL OF BIOLOGICAL CHEMISTRY   279 ( 53 )   55433 - 55442   2004.12

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    DOI: 10.1074/jbc.M409827200

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  • Loss of nonhomologous end joining confers camptothecin resistance in DT40 cells - Implications for the repair of topoisomerase I-mediated DNA damage Reviewed

    N Adachi, So, SR, H Koyama

    JOURNAL OF BIOLOGICAL CHEMISTRY   279 ( 36 )   37343 - 37348   2004.9

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    DOI: 10.1074/jbc.M313910200

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  • Genetic evidence for involvement of two distinct nonhomologous end-joining pathways in repair of topoisomerase II-mediated DNA damage Reviewed

    N Adachi, S Iiizumi, S So, H Koyama

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   318 ( 4 )   856 - 861   2004.6

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    DOI: 10.1016/j.bbrc.2004.04.099

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  • Hypersensitivity of nonhomologous DNA end-joining mutants to VP-16 and ICRF-193 - Implications for the repair of topoisomerase II-mediated DNA damage Reviewed

    N Adachi, H Suzuki, S Iiizumi, H Koyama

    JOURNAL OF BIOLOGICAL CHEMISTRY   278 ( 38 )   35897 - 35902   2003.9

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    DOI: 10.1074/jbc.M306500200

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  • Enforced cytokinesis without complete nuclear division in embryonic cells depleting the activity of DNA topoisomerase II alpha Reviewed

    N Akimitsu, N Adachi, H Hirai, MS Hossain, H Hamamoto, M Kobayashi, Y Aratani, H Koyama, K Sekimizu

    GENES TO CELLS   8 ( 4 )   393 - 402   2003.4

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  • The embryonic lethality in DNA ligase IV-deficient mice is rescued by deletion of Ku: implications for unifying the heterogeneous phenotypes of NHEJ mutants Reviewed

    ZE Karanjawala, N Adachi, RA Irvine, EK Oh, D Shibata, K Schwarz, CL Hsieh, MR Lieber

    DNA REPAIR   1 ( 12 )   1017 - 1026   2002.12

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  • Vertebrate cells lacking FEN-1 endonuclease are viable but hypersensitive to methylating agents and H2O2 Reviewed

    Y Matsuzaki, N Adachi, H Koyama

    NUCLEIC ACIDS RESEARCH   30 ( 14 )   3273 - 3277   2002.7

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    DOI: 10.1093/nar/gkf440

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  • 5-bromo-2 '-deoxyuridine efficiently suppresses division potential of the yeast Saccharomyces cerevisiae Reviewed

    M Fujii, H Ito, T Hasegawa, T Suzuki, N Adachi, D Ayusawa

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   66 ( 4 )   906 - 909   2002.4

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    DOI: 10.1271/bbb.66.906

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  • Two overlapping divergent transcription units in the human genome: The FEN1/C11orf10 locus Reviewed

    Noritaka Adachi, Zarir E. Karanjawala, Yasuo Matsuzaki, Hideki Koyama, Michael R. Lieber

    OMICS A Journal of Integrative Biology   6 ( 3 )   273 - 279   2002

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    DOI: 10.1089/15362310260256927

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  • Decreased topoisomerase IIα expression confers increased resistance to ICRF-193 as well as VP-16 in mouse embryonic stem cells Reviewed

    Masao Kobayashi, Noritaka Adachi, Yasuaki Aratani, Akihiko Kikuchi, Hideki Koyama

    Cancer Letters   166 ( 1 )   71 - 77   2001.5

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    DOI: 10.1016/S0304-3835(01)00447-5

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  • Bleomycin enhances random integration of transfected DNA into a human genome Reviewed

    Chikako Nakayama, Noritaka Adachi, Hideki Koyama

    Mutation Research - DNA Repair   409 ( 1 )   1 - 10   1998.10

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    DOI: 10.1016/S0921-8777(98)00036-6

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  • Characterization of the 3' untranslated region of mouse DNA topoisomerase IIα mRNA Reviewed

    Masao Kobayashi, Noritaka Adachi, Hideki Koyama

    Gene   215 ( 2 )   329 - 337   1998.7

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    DOI: 10.1016/S0378-1119(98)00283-2

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  • Cell cycle-dependent regulation of the mouse DNA topoisomerase IIα gene promoter Reviewed

    Noritaka Adachi, Masao Kobayashi, Hideki Koyama

    Biochemical and Biophysical Research Communications   230 ( 1 )   105 - 109   1997.1

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    DOI: 10.1006/bbrc.1996.5893

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▼display all

Books

  • Complete suppression of random integration in human cells

    Noritaka ADACHI( Role: ContributorRecent advances in genome editing)

    2019.8 

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  • 進化するゲノム編集技術

    斎藤慎太, 黒沢綾, 足立典隆( Role: Contributor遺伝子改変技術の効率化を指向したターゲティングベクターの開発)

    NTS  2015.10 

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  • 【分子標的薬を極める-基礎から臨床まで-】分子標的薬の基礎 相同組換え修復

    斎藤 慎太, 足立 典隆

    産科と婦人科   87 ( 10 )   1138 - 1144   2020.10

  • 研究者の最新動向 ランダム挿入反応の分子機構と完全阻害

    足立 典隆

    Precision Medicine   3 ( 6 )   590 - 593   2020.6

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  • 非相同末端連結とその阻害剤 Reviewed

    黒沢 綾, 足立 典隆

    放射線生物研究   52 ( 2 )   162 - 172   2017.6

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  • 高LET線ならびに放射線類似薬により誘発されるDNA二本鎖切断の修復における修復経路の重要度の解析(Requirement of DNA double-strand break repair pathways for repairing high LET radiation- and radiomimetic agents-induced DNA damage)

    黒沢 綾, 高橋 昭久, 武田 茂樹, 柴田 淳史, 足立 典隆

    日本放射線影響学会大会講演要旨集   63回   75 - 75   2020.10

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  • DNA polymerase θ is essential for alternative end-joining

    斎藤慎太, 足立典隆

    日本分子生物学会年会プログラム・要旨集(Web)   43rd   2020

  • Functional analysis of human WHSC1 in DNA double-strand break repair

    坂本裕太, 斎藤慎太, 足立典隆

    日本環境変異原学会大会プログラム・要旨集   49th (CD-ROM)   2020

  • ヒト二本鎖DNA切断修復機構とゲノム安定性維持

    足立 典隆

    上原記念生命科学財団研究報告集   33   1 - 5   2019.12

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  • ベクターDNAの非特異的な組込みを100%抑制する

    足立 典隆

    Medical Science Digest   45 ( 9 )   560 - 562   2019.8

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  • ヒト細胞におけるDNAポリメラーゼθの機能解析

    吉川真子, 斎藤慎太, 足立典隆

    日本分子生物学会年会プログラム・要旨集(Web)   42nd   2019

  • DNA二本鎖切断修復におけるAPOBEC3Gの役割

    門口美咲, 斎藤慎太, 斎藤慎太, 村山美織, 足立典隆, 足立典隆

    日本分子生物学会年会プログラム・要旨集(Web)   42nd   2019

  • 二重鎖切断修復および外来DNA組み込みにおけるヒトDNA polymerase θの役割(Role of human DNA polymerase θ in double-strand break repair and foreign DNA integration)

    斎藤 慎太, 足立 典隆

    日本癌学会総会記事   77回   1289 - 1289   2018.9

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  • ヒストンメチル化酵素NSD2とDNA損傷修復

    原田佳歩, 佐々彰, 竹内心, 足立典隆, 浦聖恵

    日本環境変異原学会大会プログラム・要旨集   47th   2018

  • ヒストンメチル化酵素NSD2のDNA損傷修復への関与の解明

    原田佳歩, 佐々彰, 鈴木慈, 前田亮, 足立典隆, 浦聖恵

    日本分子生物学会年会プログラム・要旨集(Web)   41st   2018

  • エトポシドにより促進されるランダムインテグレーションのメカニズムの解析

    森本 精太, 斎藤 慎太, 足立 典隆

    生命科学系学会合同年次大会   2017年度   [1P - 0723]   2017.12

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  • DNA二本鎖切断修復におけるDNAポリメラーゼθの機能解析

    吉川 真子, 斎藤 慎太, 足立 典隆

    生命科学系学会合同年次大会   2017年度   [1P - 0720]   2017.12

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  • ヒストンH3K36メチル化酵素NSD2を介したゲノム維持機構の解析

    鈴木 慈, 原田 佳歩, 佐々 彰, 福戸 敦彦, 田代 聡, 足立 典隆, 浦 聖恵

    生命科学系学会合同年次大会   2017年度   [2P - 0588]   2017.12

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  • DNAポリメラーゼθは異なる末端の接続に必須である(DNA polymerise θis essential for alternative end-joining)

    斎藤 慎太, 足立 典隆

    生命科学系学会合同年次大会   2017年度   [4LT16 - 0595)]   2017.12

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  • 二重鎖DNA切断修復と外部DNA統合におけるヒトDNAポリメラーゼθの役割(Role of human DNA polymerise θ in double-strand break repair and foreign DNA integration)

    斎藤 慎太, 足立 典隆

    日本癌学会総会記事   76回   P - 2013   2017.9

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  • ヒトiPS細胞を用いた遺伝子ターゲティングの効率化

    阿部 雄太, 斎藤 慎太, 足立 典隆

    日本薬学会年会要旨集   136年会 ( 3 )   122 - 122   2016.3

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  • DNA二本鎖切断修復におけるヒトWHSC1遺伝子の機能解析

    小川敬史, 斎藤慎太, 足立典隆, 足立典隆

    日本分子生物学会年会プログラム・要旨集(Web)   39th   2016

  • ヒトDNA二本鎖切断修復におけるXRCC4とLIG4の機能解析

    山下顕裕, 斎藤慎太, 内田夏海, 足立典隆, 足立典隆

    日本分子生物学会年会プログラム・要旨集(Web)   39th   2016

  • DNA二重鎖切断修復におけるヒトのDNA-PKcsの調節的な役割(Regulatory role of human DNA-PKcs in DNA double-strand break repair)

    渡部 瑶, 斎藤 慎太, 黒沢 綾, 足立 典隆

    日本生化学会大会・日本分子生物学会年会合同大会講演要旨集   88回・38回   [3LBA029] - [3LBA029]   2015.12

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  • ヒト体細胞における高効率の遺伝子標的化システムの開発 ベクターの染色体への統合のメカニズム解析(Development of a high-efficiency gene-targeting system in human somatic cells: mechanistic analysis of vector integration)

    斎藤 慎太, 足立 典隆

    日本生化学会大会・日本分子生物学会年会合同大会講演要旨集   88回・38回   [3LBA030] - [3LBA030]   2015.12

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  • Protective Roles of DNA Polymerase Zeta in Human Cells Against Environmental Genotoxins.

    T. Suzuki, P. Gruz, M. Honma, N. Adachi, T. Nohmi

    ENVIRONMENTAL AND MOLECULAR MUTAGENESIS   56   S59 - S59   2015.8

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  • 放射線によって誘導されるDNAの複製異常に対してはBLMヘリカーゼではなくRecQL4ヘリカーゼが重要な働きをする(Human RecQL4, but not BLM, helicase facilitate replication elongation on DNA templates damaged by ionizing radiation)

    香崎 正宙, 足立 典隆, 武田 俊一

    日本癌学会総会記事   73回   P - 3002   2014.9

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  • ヒト遺伝子ノックアウト細胞を用いたDNA ligase IV/XRCC4複合体の機能解析

    内田夏海, 斎藤慎太, 足立典隆, 足立典隆

    日本薬学会年会要旨集(CD-ROM)   134th   2014

  • DNA polymerase kappaのヒトゲノム恒常性維持における構造的および触媒的役割の検討

    兼丸祐紀, 鈴木哲矢, 新見直子, GRUZ Petr, 松元郷六, 足立典隆, 本間正充, 能美健彦

    日本分子生物学会年会プログラム・要旨集(Web)   37th   1P-0183 (WEB ONLY)   2014

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    J-GLOBAL

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  • Phenylalanine 171 of Human DNA Polymerase Kappa Is a Molecular Brake for Translesion DNA Synthesis across Benzo[a]pyrene Adducts.

    A. Sassa, T. Suzuki, Y. Kanemaru, N. Niimi, H. Fujimoto, A. Katafuchi, P. Gruz, M. Yasui, R. Gupta, F. Johnson, T. Ohta, M. Honma, N. Adachi, T. Nohmi

    ENVIRONMENTAL AND MOLECULAR MUTAGENESIS   54   S31 - S31   2013.9

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  • 酸化RNAに結合するタンパク質AUF1の働き:遺伝子ノックアウト細胞の作製と解析

    石井健士, 早川浩, 関口猛, 足立典隆, 関口睦夫

    日本分子生物学会年会プログラム・要旨集(Web)   36th   2013

  • 生化学的分画法によるXRCC4-DNAリガーゼIV複合体のクロマチン結合の解析(Chromatin binding of XRCC4-DNA ligase IV complex revealed by biochemical fractionation analysis)

    Liu Sicheng, Kamdar Radhika Pankaj, 足立 典隆, 松本 義久

    日本放射線影響学会大会講演要旨集   55回   117 - 117   2012.9

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  • 低線量率放射線照射下におけるDNA2重鎖切断修復因子の役割

    冨田雅典, 小林純也, 岩淵邦芳, 松本義久, 足立典隆, 高田穣, 内海博司

    日本放射線影響学会大会講演要旨集   55th   2012

  • 損傷を誘発する化学物質の細胞毒性に対するヒトDNAポリメラーゼζの役割

    鈴木哲矢, 足立典隆, 能美健彦, 能美健彦

    日本分子生物学会年会プログラム・要旨集(Web)   35th   2012

  • RecQL4のヘリカーゼドメインを含むC末部位はヒトにおいて放射線によるDNAダメージ損傷後のDNA複製伸長を促進する

    香崎正宙, CHIOUREA Maria, VERSINI Gwennaelle, 足立典隆, 武田俊一, GAGOS Sarantis, HALAZONETIS Thanos D.

    日本分子生物学会年会プログラム・要旨集(Web)   35th   2012

  • P-089 Evaluation of genotoxic and cytotoxic sensitivity of DNA polymerase κ knockout and knockin human cells to DNA damaging agents(Poster Presentation)

    Kanemaru Yuki, Suzuki Tetsuya, Niimi Naoko, Gruz Petr, Matsumoto Kyomu, Adachi Noritaka, Honma Masamitsu, Nohmi Takehiko

    日本環境変異原学会大会プログラム・要旨集   ( 40 )   137 - 137   2011

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  • ヒト細胞における遺伝子標的効率の改善(Improvement of gene-targeting efficiency in human cells)

    黒沢 綾, 足立 典隆

    日本癌学会総会記事   69回   117 - 117   2010.8

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  • High-efficiency gene targeting in a human pre-B cell line: towards the human gene knockout project

    Noritaka Adachi

    GENOME BIOLOGY   11   2010

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    DOI: 10.1186/gb-2010-11-S1-P1

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  • Cre組換え酵素による染色体間組換え頻度測定により明らかとなったテロメア近傍遺伝子のアクセシビリティー

    植村宗弘, 丹羽陽子, 嘉数直樹, 足立典隆, 木下和生

    生化学   2010

  • P-007 Characterization of human DNA polymerase κ(Poster Session)

    Niimi Naoko, Iiizumi Susumu, Adachi Noritaka, Koyama Hideki, Nohmi Takehiko

    日本環境変異原学会大会プログラム・要旨集   ( 37 )   110 - 110   2008

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  • Genetic analysis of an interaction between human Mus81 and cross-link repair factors

    NOMURA YUJI, ADACHI NORITAKA, SO SAIREI, AOKI KAYOKO, KOYAMA HIDEKI

    25 ( 1 )   73 - 73   2006.3

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  • Gene knockout studies on DNA strand break repair in human cells

    ADACHI NORITAKA, IIIZUMI SUSUMU, NOMURA YUJI, SO SAIREI, KOYAMA HIDEAKI

    25 ( 1 )   72 - 72   2006.3

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  • High-efficiency gene targeting with BLM siRNA in human cell lines

    SO SAIREI, NOMURA YUJI, ADACHI NORITAKA, IIIZUMI SUSUMU, KOBAYASHI YUKI, HORI TAMAKI, KURIHARA HARUYUKI, KOYAMA HIDEKI

    25 ( 1 )   74 - 74   2006.3

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  • Impact of DNA ligase IV deficiency on random and targeted integration

    IIIZUMI SUSUMU, ADACHI NORITAKA, ISHII YASUYUKI, CHIKARAISHI YUICHI, SO SAIREI, KOYAMA HIDEKI

    25 ( 1 )   71 - 71   2006.3

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  • ゲノムDNA鎖切断の修復における二本鎖切断修復経路の役割

    足立 典隆, 小山 秀機

    放射線生物研究   39 ( 3 )   231 - 243   2004.9

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    Language:Japanese   Publisher:放射線生物研究会  

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    Other Link: http://search.jamas.or.jp/link/ui/2004319827

  • Analysis of Genomic Instability and DNA Double-strand Break Repair in Human BLM Knockout Cells

    SO Sairei, ADACHI Noritaka, LIEBER Michael R, KOYAMA Hideki

    23 ( 1 )   44 - 44   2004.3

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  • O6-2 Repair efficiency of DNA single strand breaks in base excision repair deficient cells as determined by a real-time strand break assay.

    Buersutatte A-M.

    ( 33 )   118 - 118   2004

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  • DNA複製関連酵素の変異体作製による,in vivo及びin vitroでの酵素機能解析 哺乳動物細胞のS期開始におけるDNAトポイソメラーゼIIαの役割

    関水 和久, 秋光 信佳, シャヒッドホサイン・ムクタディール, 濱本 洋, 足立 典隆, 小林 正男, 荒谷 康昭, 小山 秀機, 高木 亨, 平井 洋

    生化学   73 ( 8 )   650 - 650   2001.8

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  • Analysis on the mechanism of DNA topoisomerase IIα gene expression

    ADACHI Noritaka, NOMOTO Minoru, KOYAMA Hideki

    21   407 - 407   1998.12

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  • トポイソメラ-ゼの発現制御機構と変異株を利用した機能解析 (特集 DNAトポロジ---細胞生物学へ踏み込むトポイソメラ-ゼ)

    足立 典隆, 小林 正男, 小山 秀機

    細胞工学   16 ( 11 )   1597 - 1605   1997.11

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  • Enhancement by Bleomycin of Nonhomologous Recombination in Human Cells

    NAKAYAMA C., ADACHI N., KOYAMA H.

    16 ( 1 )   80 - 80   1997.3

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  • マウスDNAトポイソメラーゼIIα遺伝子の構造と発現

    足立 典隆, 小林 正男, 小山 秀機

    日本分子生物学会年会プログラム・講演要旨集   19   434 - 434   1996.8

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  • 細胞増殖におけるDNAトポイソメラーゼIIαの必須性の検討

    小林 正男, 足立 典隆, 小山 秀機

    日本分子生物学会年会プログラム・講演要旨集   19   294 - 294   1996.8

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  • The Int'l Symp. on DNA Topoisomerases in Chemotherapy

    ADACHI NORITAKA

    Farumashia   28 ( 3 )   280 - 280   1992.3

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▼display all

Industrial property rights

  • 非相同末端連結欠損細胞及びその利用

    足立 典隆

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    Applicant:公立大学法人横浜市立大学

    Application no:特願2017-091736  Date applied:2017.5

    Announcement no:特開2017-201978  Date announced:2017.11

    Patent/Registration no:特許第6925003号  Date registered:2021.8 

    J-GLOBAL

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  • 遺伝子ターゲティングベクター及びその利用方法

    足立 典隆

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    Applicant:公立大学法人横浜市立大学

    Application no:JP2012082160  Date applied:2012.12

    Announcement no:WO2013-089123  Date announced:2013.6

    Publication no:WO2013-089123  Date published:2013.6

    Patent/Registration no:特許第6037290号  Date registered:2016.11 

    J-GLOBAL

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  • 遺伝子ターゲティングベクター、その作製方法及び利用方法

    足立 典隆

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    Applicant:公立大学法人横浜市立大学

    Application no:JP2012063248  Date applied:2012.5

    Announcement no:WO2012-165270  Date announced:2012.12

    Publication no:WO2012-165270  Date published:2012.12

    Patent/Registration no:特許第5999602号  Date registered:2016.9 

    J-GLOBAL

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Research Projects

  • 条件的遺伝子発現系の理論的構築による新規DNA修復欠損がん診断・治療戦略の開発

    Grant number:22K19382  2022.6 - 2024.3

    日本学術振興会  科学研究費助成事業  挑戦的研究(萌芽)

    足立 典隆

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    Grant amount:\6500000 ( Direct Cost: \5000000 、 Indirect Cost:\1500000 )

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  • 生体環境からみたゲノム組換え修復に関する包括的理解

    Grant number:19H01151  2019.4 - 2023.3

    日本学術振興会  科学研究費助成事業  基盤研究(A)

    足立 典隆

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    Grant amount:\37830000 ( Direct Cost: \29100000 、 Indirect Cost:\8730000 )

    二本鎖DNA切断は、細胞や個体にとって最も脅威となるゲノム損傷であり、速やかに修復される必要がある。本研究の目的は、ゲノム編集ヒト細胞を駆使した遺伝学的解析により、二本鎖DNA切断修復システムの全体像、特にalt-EJとSSAの分子機構を明らかにし、組換え修復機構についての包括的理解を目指すことにある。今年度は、ヒトゲノム編集細胞を用いてPolQやMsh2、Blm、末端の削り込み(end resection)に関わる新規因子に関する機能解析を行った。特にPolQについては構造と機能の相関に関する詳細な解析を進めた。特定の重金属が組換え修復に及ぼす影響についても明らかにした。

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  • Efficient suppression of random DNA integration

    Grant number:18K19407  2018.6 - 2020.3

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Challenging Research (Exploratory)

    Adachi Noritaka

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    Grant amount:\6240000 ( Direct Cost: \4800000 、 Indirect Cost:\1440000 )

    In this research project, we have conducted a functional analysis of DNA polymerase theta, an essential protein for alternative end-joining-mediated DNA double-strand break repair. Additionally, using a cell-based screening method, we have screened compounds that inhibit human DNA polymerase theta. Our study will help develop a unique system to suppress DNA polymerase theta-mediated random integration, which will lead to human cell gene targeting with high efficiency.

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  • Development of high-efficiency gene targeting technologies

    Grant number:15H04323  2015.4 - 2018.3

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (B)

    Noritaka Adachi

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    Grant amount:\17550000 ( Direct Cost: \13500000 、 Indirect Cost:\4050000 )

    In this research project, we have successfully identified a human protein that is essential for alternative end-joining, a minor pathway of DNA double-strand break repair. This discovery has led us to develop a unique system to suppress random integration of transfected DNA, making it possible to perform human cell gene targeting with 100% efficiency.

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  • Genes that enable high-efficiency gene disruption in human cells

    Grant number:26670031  2014.4 - 2016.3

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Challenging Exploratory Research

    Adachi Noritaka

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    Grant amount:\3770000 ( Direct Cost: \2900000 、 Indirect Cost:\870000 )

    In this research project, we have identified several human genes that increase the efficiency of gene targeting by homologous recombination. We have shown for the first time that an enzyme with cytidine deaminase activity can enhance gene targeting in NHEJ-null cells.

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  • Role of DNA-PKcs in neurogenesis

    Grant number:26740019  2014.4 - 2016.3

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Young Scientists (B)

    Kurosawa Aya, ADACHI Noritaka

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    Grant amount:\3900000 ( Direct Cost: \3000000 、 Indirect Cost:\900000 )

    The aim of this study was to investigate the role of the PRKDC, one of the causative genes for microcephaly, in neurogenesis. As human gene-knockout iPS cells are useful in understanding disease mechanisms, we established the protocol for generation of human gene-knockout iPS cells using a plasmid-based targeting vector.

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  • Development and applications of high-efficiency gene targeting in human cells

    Grant number:24310149  2012.4 - 2015.3

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (B)

    ADACHI NORITAKA

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    Grant amount:\19630000 ( Direct Cost: \15100000 、 Indirect Cost:\4530000 )

    We have been developing systems that enable rapid production of human cell lines with the use of plasmid-based DNA vectors. In this research project, we have conducted basic studies aimed at developing ultrahigh-efficiency gene-targeting systems, particularly in terms of controlling random integration, a mechanism that acts to reduce the efficiency of gene targeting.

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  • Analysis of human genes that enable high-efficiency gene targeting

    Grant number:24659035  2012.4 - 2014.3

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Challenging Exploratory Research

    ADACHI Noritaka

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    Grant amount:\3900000 ( Direct Cost: \3000000 、 Indirect Cost:\900000 )

    We have recently constructed an efficient system that enables rapid production of genetically modified human cell lines by gene targeting. Human cell mutants thus made will provide invaluable tools for human medicine and future drug development. In this research project, we have identified several human genes, which, when down-regulated, increase the efficiency of gene targeting by homologous recombination.

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  • ゲノム切断修復におけるクロマチン構造の役割

    Grant number:23131511  2011.4 - 2013.3

    日本学術振興会  科学研究費助成事業  新学術領域研究(研究領域提案型)

    足立 典隆

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    Grant amount:\13260000 ( Direct Cost: \10200000 、 Indirect Cost:\3060000 )

    本研究では、普遍的な細胞内クロマチン制御機構の解明を目指した「次世代DNA修復研究」の一環として、クロマチン構造変換・維持やDNA二本鎖切断修復に重要な役割を果たすヒト遺伝子のノックアウト細胞の作製と解析を行うことで、クロマチン構造(特にヘテロクロマチン形成や転写抑制)の変化が及ぼすゲノム安定性(特にDNA損傷/変異誘発率や反復配列の安定性)や二本鎖切断修復機構への影響を明らかすることを目標とした。まず、これまで開発してきたヒト遺伝子ノックアウト系を利用して、クロマチン構造維持に関わる因子(NAD+依存性脱アセチル化酵素、転写コリプレッサーKAP1、HP1等)の遺伝子破壊株の作製を進めた。さらに、これまでに作製したDNA二本鎖切断修復の変異株の表現型解析をさらに詳細に行った。その結果、各種NHEJ欠損株がそれぞれ異なる表現型を示すことや、53BP1とArtemisが共にNHEJ経路から他修復経路へのスイッチングに作用している可能性を示すことができた。また、作製した変異株における自発的DNA損傷の変化やHDAC阻害剤感受性についても解析を行った。さらに、トポイソメラーゼ阻害剤などの抗癌剤への感受性の変化を調べることで、変異株におけるDNA修復能の解析を行った。一方、ゲノム上のさまざまな部位にI-SceI認識部位を含む組換え基質を導入(ノックイン)した細胞株を作製していくための準備を進めた。これは、各細胞株のさまざまな部位に生じた二本鎖切断の修復効率やその特性を解析していく上で重要なツールとなる。また、こうした研究の過程においてヒトNalm-6細胞において遺伝子ターゲティング効率を最大100%にまで上昇させることに成功した。

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  • Epigenetic marking by histone variant H3.3 during myeloid cell differentiation

    Grant number:23659492  2011 - 2012

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Challenging Exploratory Research

    TAMURA Tomohiko, ADACHI Noritaka, NISHIYAMA Akira

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    Grant amount:\3640000 ( Direct Cost: \2800000 、 Indirect Cost:\840000 )

    Cell differentiation in multicellular organisms is achieved by dynamic changes in gene expression pattern. Dysregulation of such processes can cause various diseases including cancers. Histone variant H3.3 plays an important role in epigenetic memory of active genes. In this study we mapped genome-wide distribution of H3.3 during macrophage differentiation. The results indicated that H3.3 may regulate several steps of transcription including elongation. We also identified H3.3K36 as an amino acid residue required for H3.3 incorporation into chromatin.

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  • GeneticanalysisofDNAstrandbreakrepairinhumancelIs

    Grant number:21590066  2009 - 2011

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (C)

    ADACHI Noritaka

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    Grant amount:\4680000 ( Direct Cost: \3600000 、 Indirect Cost:\1080000 )

    In this study, we have generated a series of human cell mutants that lack one or two genes implicated in DNA strand break repair. These gene-knockout mutants allowed us to perform comprehensive geneticanalysis of human DNA repair genes, especially those involved in DNA double-strand break repair and genetic recombination.

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  • ヒト遺伝子ノックアウトシステムの効率化と疾患遺伝子機能解析への応用

    Grant number:20018022  2008 - 2009

    日本学術振興会  科学研究費助成事業  特定領域研究

    足立 典隆

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    Grant amount:\7300000 ( Direct Cost: \7300000 )

    DNA鎖切断修復経路の異常や欠損はさまざまな遺伝病やがんと密接に関連している.我々は最近,ヒトpre-B由来リンパ球細胞株Nalm-6を用いて,遺伝子ノックアウト細胞を効率良く取得できるシステムの開発に成功した.そこで本研究では,DNA鎖切断修復に関わるヒト遺伝子について系統的に遺伝子ノックアウトを行い,得られた変異細胞株の表現型解析を行った.特に本研究では,遺伝子間あるいは経路間の相互作用の解析を目的として,二重変異株の作製と解析を進め,主に以下の成果を得た.
    ・NHEJ欠損細胞は,エトポシドなどのトポイソメラーゼII阻害剤に高感受性を示す一方,カンプトテシン(トポイソメラーゼI阻害剤)やParp阻害剤に対しては耐性を示す.
    ・ヒト細胞でNHEJを欠損させるとジーンターゲティング効率の上昇がみられる.
    ・TDP1やAPTX(ともに脊髄小脳失調症の原因遺伝子)を欠損した細胞は,ブレオマイシンに対して高感受性を示す.
    ・DNA ligaseIVとDNAポリメラーゼβは,DNA修復において部分的にオーバーラップした機能をもつ.
    一方,ヒト細胞には複数のランダムインテグレーション機構が存在しており,ターゲティングベクターと非ターゲティングベクターとでインテグレーションの機構が異なっている可能性が高いことがわかったため,ベクターの構造,特に相同領域中の反復配列がインテグレーション反応に及ぼす影響を詳細に解析した.この成果は,今後の遺伝子ノックアウト研究に重要な示唆を与えると期待される.

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  • ヒト染色体DNA切断とその修復機構

    Grant number:20055017  2008 - 2009

    日本学術振興会  科学研究費助成事業  特定領域研究

    足立 典隆

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    Grant amount:\4500000 ( Direct Cost: \4500000 )

    染色体DNA鎖切断の修復には多種多様な経路が関わっているため,さまざまなタイプのDNA鎖切断に対しての修復における各経路の役割や制御機構についてはよくわかっていない.本研究では,我々が最近開発したヒトNalm-6細胞を使った逆遺伝学的解析システムを利用して,染色体DNA鎖切断修復に関わる遺伝子のノックアウト細胞を系統的に作製し,得られた変異細胞の表現型を解析することにより,DNA鎖切断の生成機構やこれに対する応答・修復機構をヒト細胞で解析することを目的として研究を行った.本年度は,昨年度に引き続き,DNA鎖切断修復に関わる遺伝子を系統的にノックアウトしホモ変異細胞の取得を行った.得られたホモ変異細胞の解析から,相同組換え経路と非相同末端連結(NHEJ)経路との相対的役割に関するいくつかの知見を得ることができた.また,新規抗がん剤である化合物NK314や鉄キレート剤Dp44mTの細胞内ターゲットがトポイソメラーゼIIαであることを示すとともに,こうした薬剤や放射線類似作用物質が細胞内での非相同組換えに及ぼす影響を詳細に解析した.一方,NHEJ経路で働くと考えられているエンドヌクレアーゼArtemisがNHEJ非依存性の機能をもつ可能性が示唆されたため,他遺伝子との二重変異株の作製を通じた遺伝学的解析にも着手した.

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  • ヒトDNA鎖切断修復の逆遺伝学的解析と医療への応用

    Grant number:18018034  2006 - 2007

    日本学術振興会  科学研究費助成事業  特定領域研究

    足立 典隆, 小山 秀機

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    Grant amount:\7700000 ( Direct Cost: \7700000 )

    DNA鎖切断修復経路の異常や欠損はさまざまな遺伝病やがんと密接に関連する.我々は最近,ヒトpre-B由来リンパ球細胞株Nalm-6を用いて,遺伝子ノックアウトを郊率良く行えるシステムの開発に成功した.そこで本研究では,DNA鎖切断修復に関わる遺伝子について系統的に遺伝子ノックアウトを行い,得られた変異細胞株の表現型解析を行った.特に,二重変異株の作製と解析により,遺伝子間あるいは経路間の相互作用の解析を進めた.主な成果の概要は以下の通りである.
    ・二本鎖切断修復に関わる遺伝子の機能解析を行い,特にBLM(ブルーム症候群原因遺伝子)やp53,DNA ligase IV,Mus81エンドヌクレアーゼ,FANCB(ファンコニー貧血原因遺伝子の一つ)の変異株の詳細な表現型解析から,ヒト細胞におけるこれら遺伝子群の相互作用を明らかにした.
    ・DNA二本鎖切断の修復には,相同組換えとエンドジョイニングの双方が重要な働きをしているが,傷の種類や量によって各経路の働きが大きく異なることを明らかにした.
    ・TDP1(脊髄小脳失調症SCAN1原因遺伝子)欠損細胞は,カンプトテシンやブレオマイシンなどの抗がん剤に対して高感受性を示すが,APTX(脊髄小脳失調症AOA1/EAOH原因遺伝子)欠損細胞ではこうした表現型が観察されないことを明らかにした.
    ・DNA ligase IVとArtemis(ともに重症複合免疫不全症の原因遺伝子)はともにエンドジョイニング経路において機能しているが,Artemisはこれとは別の機能,すなわち修復経路選択のステップにも関わっていることを明らかにした。

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  • Analysis of DNA strand break repair using human knockout cells

    Grant number:18590063  2006 - 2007

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (C)

    ADACHI Noritaka

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    Grant amount:\3890000 ( Direct Cost: \3500000 、 Indirect Cost:\390000 )

    We recently developed a system using the human cell line Nalm-6 that enables rapid production of human gene knockout cells. In this study, we used this system to genetically analyze the function of human genes that have been implicated in DNA strand break repair. Specifically, we disrupted a series of genes involving those for Rad54, FancB, Mus81, Tdp1, Ku70/80, and Lig3, which all have some roles in DNA single and/or double-strand break repair in human cells. In addition, we tried to produce as many double-knockout mutant cells as possible, particularly those with a Lig4 mutation. Using these mutants, we have successfully analyzed the cellular sensitivity to anticancer agents involving cisplatin and topoisomerase inhibitors as well as the genetic interaction between p53 and BLM and between Mus81 and FancB, We also performed gene knockdown experiments with siRNAs for DNA ligases that are potentially involved in random integration and/or gene targeting. The results suggested that suppressing nonhomologous end-joining reactions could enhance the efficiency of gene targeting. Our findings also indicated that there must be another mechanism for random integration that does not rely on the nonhomologous end-joining pathway.

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  • Genetic studies on the interaction between base excision repair and recombinational repair using human gene knockout cells

    Grant number:18570163  2006 - 2007

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (C)

    KOYAMA Hideki, ADACHI Noritaka

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    Grant amount:\3960000 ( Direct Cost: \3600000 、 Indirect Cost:\360000 )

    In this study, we have performed genetic analysis on the interaction between base excision repair and recombinational repair in human cells. Specifically, we disrupted, by means of gene targeting using Nalm-6 cells, the PolB, Rad54, and Lig4 genes involved in base excision repair homologous recombination, or nonhomologous end-joining, respectively. Additionally, we tried to make double-knockout mutant cell lines for those genes. We investigated the growth rate, cell cycle distributions, and recombination capacity of these mutants. Furthermore, using these mutants, we have successfully analyzed cellular sensitivity to a variety of genotoxic agents such as X-rays, UV light, alkylating agents, hydrogen peroxide, topoisomerase inhibitors, and replication stress. These studies led us to conclude that homologous recombination is important for repairing DNA double-strand breaks that arose from single-strand breaks. Most importantly, this work has highlighted the genetic interaction between DNApolymerase beta and DNAligase W in human cells.

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  • ヒト染色体DNA切断とその修復機構

    Grant number:18058019  2006 - 2007

    日本学術振興会  科学研究費助成事業  特定領域研究

    足立 典隆, 小山 秀機

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    Grant amount:\5000000 ( Direct Cost: \5000000 )

    染色体DNA鎖切断修復には多種多様な経路が存在しているため,さまざまな種類のDNA鎖切断に対する各経路の役割分担やその制御機構については明らかにされていない.そこで本研究では,我々が最近開発したヒトNalm-6細胞を使った逆遺伝学的解析システムを利用して,染色体DNA鎖切断修復に関わる遺伝子のノックアウト細胞を系統的に作製し,得られた変異細胞の表現型を解析することにより,鎖切断に対する応答・修復機構をヒト細胞で明らかにすることを目的として研究を行った.本年度は,DNA鎖切断修復に関わる遺伝子を系統的にノックアウトし,計画していたほとんどの遺伝子についてホモ変異細胞の作製を完了することができた.次に得られた変異細胞を詳細に解析し,相同組換え経路と非相同末端連結経路との相対的役割に関するいくつかの新たな知見を得た.特に,カンプトテシンやエトポシドなどのトポイソメラーゼ阻害剤が誘発する一本鎖・二本鎖切断に対する各経路の役割を解析し,トポイソメラーゼI阻害剤によるダメージに対しては相同組換え経路が,トポイソメラーゼII阻害剤によるダメージに対しては非相同末端連結経路が重要であることを明らかにした.また,非相同末端連結に関わるエンドヌクレアーゼであるArtemisがDNA ligase IVに依存した非相同末端連結経路における役割とは別の機能を担っていることを突き止めた.さらに,こうしたヒト細胞変異株における組換え頻度の解析等から,二本鎖切断修復に関わる第三の経路の存在を明らにした.

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  • 動物細胞のDNA鎖切断修復と非相同組換えに関する逆遺伝学的解析

    Grant number:16790058  2004 - 2005

    日本学術振興会  科学研究費助成事業  若手研究(B)

    足立 典隆

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    Grant amount:\3500000 ( Direct Cost: \3500000 )

    ヒトNalm-6細胞およびニワトリDT40細胞を用いて,一本鎖DNA切断修復/塩基除去修復機構の解析を行った。具体的には,DT40細胞のFEN1変異株,DNAポリメラーゼβ変異株,FEN1/DNAポリメラーゼβ二重変異株の表現型解析と,Nalm-6細胞のDNAポリメラーゼβ変異株の作製と表現型解析を行った。その結果,アルキル化剤によるDNA損傷の修復においてショートパッチ経路,ロングパッチ経路がともに重要であるのに対し,酸化的損傷に対してはロングパッチ経路のみが重要であることを明らかにした。この結果は生化学的手法によっても確認できた。また,二本鎖DNA切断修復についても詳細な解析を行った。エンドジョイニング変異株の解析から,エンドジョイニング阻害によりランダムインテグレーションを抑制できることを明らかにした。さらに,本研究では,Nalm-6細胞を用いて,Rad54や,Xrcc3,Ku70,Ku80,DNA ligase III, DNA ligase IなどDNA一本鎖/二本鎖切断修復への関与が示唆される遺伝子およびp53遺伝子について,順次ジーンターゲティングを行った。また,これら遺伝子の二重変異株の作製を進めた。これら一連の変異株を利用して,トポイソメラーゼ阻害剤などの抗がん剤の細胞傷害作用を増強させる方策を考察した。また,DNAligase IV破壊株を詳しく解析した結果,ヒトとニワトリとで表現型が異なることを明らかにした。

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  • トポイソメラーゼ阻害剤によるゲノム損傷の修復におけるDNA鎖切断修復機構の役割

    Grant number:16023257  2004

    日本学術振興会  科学研究費助成事業  特定領域研究

    足立 典隆

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    Grant amount:\4000000 ( Direct Cost: \4000000 )

    トポイソメラーゼ阻害剤によるDNA損傷修復の機構解明を目的として,ヒトNalm-6細胞およびニワトリDT40,細胞由来の培養細胞変異株を用いて遺伝学的な解析を行った。その結果,トポイソメラーゼII阻害剤によるDNA損傷に対してニンドジョイニングが必須の役割を果たしていることが明らかとなった。これに対し相同組換えは,トポイソメラーゼII阻害剤による不可逆的な損傷を増やす方向に作用していた。以上の結果は酵母と全く逆であり,動物細胞を使った研究がいかに重要であるかを再認識させられた。また以上の結果から,エンドジョイニングを抑制すればトポイソメラーゼII阻害剤の作用を著しく増強できることが強く示唆された。一方,トポイソメラーゼI損傷修復には相同組換えが重要であり,エンドジョイニングは細胞の生存にとって不利に作用することがわかった。ただし,トポイソメラーゼI阻害剤によるDNA損傷は一本鎖切断が主であるため,今後はDNA一本鎖切断修復経路の関与について詳細な解析を進める必要がある。また,エンドジョイニングを欠損したDT40細胞がカンプトテシン耐性を示したことから,エンドジョイニング活性の低下ががん細胞の耐性獲得の原因となっている可能性が示唆された。次に,p53ステータスとトポイソメラーゼ阻害剤感受性との相関を明らかにするため,ヒトNalm-6細胞においてp53遺伝子のノックアウトを行った。さらに,Rad52や,Xrcc3,Ku70,DNA ligaseなどDNA鎖切断修復への関与が示唆される遺伝子について,順次ジーンターゲティングを行った。今後、ヒト細胞を用いた系統的な逆遺伝学的解析によって,より効果的な化学療法が確立されることが期待される。

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  • ヒト遺伝子の系統的な機能解析に向けた高効率ジーンターゲティング系の開発

    Grant number:16012252  2004

    日本学術振興会  科学研究費助成事業  特定領域研究

    小山 秀機, 足立 典隆

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    Grant amount:\5000000 ( Direct Cost: \5000000 )

    ヒト細胞からジーンターゲティングによりノックアウト株の作製は、ターゲット効率低く困難である。最近、我々はヒトプレB細胞腫由来のNalm-6株が高いターゲット効率を持つことを見出した。そこで本研究はヒト遺伝子の系統的な機能解析に応用するため、Nalm-6株を用いた高効率ターゲティング系を開発することを目的とする。今年度、常染色体遺伝子変異・有糸分裂組換え系を作製と、増殖必須遺伝子の条件致死変異株の作製を行った。その結果、(1)10種類の遺伝子座のターゲティングを行い、2〜10%の高効率でターゲットクローンを得た。したがって、Nalm-6細胞が高効率のターゲティングが可能な株であることを確認した。(2)16番染色体に座乗するAPRT遺伝子のターゲティングを行い、ヘテロ破壊(APRT+/-)株を得た。この株を用いて、8-アザアデニン耐性でホモ破壊(APRT-/-)細胞の自然突然変異を検出するFluctuation test系をほぼ作製した。(3)変異遺伝子の構造解析のため、APRT遺伝子領域を増幅できるPCR解析系を作製した。また、APRT座にリンクし相同染色体を識別できるミニサテライトマーカーを見出した。(4)一方、DNAの2重鎖切断修復やテロメア維持に働くKU70遺伝子の欠損株を取得するためターゲティングを行い、ヘテロ変異株を得た。ついで、第2のターゲティングを行ったがホモ変異株が得られず、KU70は増殖・生存に必須と思われた。(5)そこで、Tet制御系を用いて条件致死株として分離するため、まずヘテロ変異株にTetベクターを導入した。ついで、Tet応答性ヒトKU70 cDNAベクターを導入し、DOXで発現制御できる株を分離した。この株にKU70ターゲティングを行ったがターゲットクローンを得られず、さらに分離法を検討している。

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  • ヒトプレB細胞よりBLM破壊株の作製とその非相同組換え能の解析

    Grant number:16021247  2004

    日本学術振興会  科学研究費助成事業  特定領域研究

    小山 秀機, 足立 典隆

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    Grant amount:\5100000 ( Direct Cost: \5100000 )

    高発がん性遺伝病ブルーム症候群は、高頻度の姉妹染色体交換とともに欠失、転座などの染色体異常を示す。この異常の原因となり発がんにつながる可能性のあるエラープローンの非相同的末端連結(NHEJ)の解析は、わずかである。我々はヒトプレB細胞腫由来Nalm-6株およびNHEJに必須のDNAリガーゼIV(Lig4)欠損株から、ジーンターゲティングによりBLM破壊株と2重破壊株を取得し、性状解析を行った。また、4株のHPRT座へSceI部位を導入した株を作製し、ついでSceI発現ベクターを導入・誘発した2重鎖切断(DSB)のNHEJによる修復能を解析した。その結果、BLM破壊株は野生株に比べて増殖遅延や1本鎖切断を生じるMMSやUVに対する高感受性、突然変異率の上昇などの表現型を示したが、興味深いことに、これら表現型はLig4欠損との2重破壊株では抑制された。これはBLM破壊株において、DNA複製の際に1本鎖切断からone-ended DSBを多数生じ、NHEJ修復による異常な組換えにより細胞死を起こすためと考える。一方、染色体SceI部位に誘導したtwo-ended DSBのNHEJ修復において、BLM破壊株は野生株とほぼ同じ修復パターンを示した。この結果から、ブルーム細胞においてone-ended DSBのNHEJ修復が、染色体異常を引き起こす原因であることが示唆される。一方、NHEJの主要因子KU70の破壊株作製のため、野生株をターゲティングしてヘテロ破壊株を得たがホモ破壊株が得られず、KU70は増殖・生存に必須と考える。そこでTet制御系を用いた条件致死株の作製を試み、ヘテロ破壊株にTet制御ベクターを導入した。ついでTet応答性ヒトKU70 cDNAベクターを導入し、DOXで制御できる株を分離した。この株を用いてKU70遺伝子のターゲティングを行った。

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  • Studies of base excision repair in cell mutants deficient. in either DNA polymerase, or flap endonuclease-1 or both, generated from chicken DT40 cells.

    Grant number:15570146  2003 - 2004

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (C)

    KOYAMA Hideki, ADACHI Noritaka

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    Grant amount:\3700000 ( Direct Cost: \3700000 )

    Base excision repair (BER) is the major pathway in repair of DNA base lesions such as apurinic/apyrimidinic (AP) sites, or deaminated, alkylated or oxidative bases. The BER pathway is divided into DNA polymerase β(Polβ)-dependent short-patch BER and PCNA/flap endonuclease-1 (FEN-1)-dependent long-patch BER. We generated knockout cell lines deficient in either FEN-1 or Polβ, or both from the chicken DT40 cell line and studied differential roles of the proteins in the two subpathways. Surprisingly, double mutant cells could survive nevertheless of deficiency in the subpathways, implying the redundancy of both proteins or the existence of alternative pathways to backup the defects. Three mutant cell lines were all hypersensitive to methyl methanesulfonate, but FEN1-null and double mutants were hypersensitive to hydrogen peroxide compared with wild-type cells. In vitro BER assay, using cell-free extracts and a double-stranded DNA substrate containing one uracil, revealed that while wild-type and Polβ-null cells had an activity to repair the defect, FEN1-null and double mutant cells showed almost no activity. Importantly, this result indicates that FEN1, but not Polβ, is essential for repairing one base defect. Therefore, we are further studying the reason why cells lacking FEN1 but not Polβ are unable to repair the defect.

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  • ヒトプレB細胞よりBLM破壊株の作製とそ非相同的末端連結能の解析

    Grant number:15023249  2003

    日本学術振興会  科学研究費助成事業  特定領域研究

    小山 秀機, 足立 典隆

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    Grant amount:\3400000 ( Direct Cost: \3400000 )

    高発がん性の遺伝病ブルーム症候群(BS)は、高頻度の姉妹染色体交換とともに欠失、転座などの染色体異常を示す。従来、この染色体異常を引き起こし発がんの原因となる可能性の高いエラープローンの非相同的末端連結(NHEJ)の解析はわずかしか報告がない。最近、我々はヒトプレB細胞Nalm-6およびNalm-6由来でNHEJに必須のDNAリガーゼIV(LIG4)欠損株から、ジーンターゲティングによりBLM破壊株と2重破壊株を取得した。本研究は、これら破壊株を用いて染色体上におけるNHEJ能を解析し、BSの高発がん性の原因とメカニズムを明らかにすることを目的として行い、以下の結果を得た。(1)各細胞株のNHEJ能を解析するため、hprt遺伝子座にSceI部位を組み込んだ株を作製した。この各株にpSceIベクターを導入し生き残ったクローン中でSceI部位の消失クローンを選択、PCRで連結部位を増幅し塩基配列を決定した。この方法により各株のNHEJ修復頻度と連結部位の構造を調べ、BLM破壊株は野生株とほぼ同じ頻度とサイズの欠失を示し、BLM破壊株でNHEJがほぼ正常に機能していることを見出した。一方、LIG4破壊株は高い頻度とより大きな欠失を示し、2重破壊株はそれがより顕著であった。一方、各破壊株の表現型を解析すると、(2)BLM破壊株の増殖能の大幅な低下、(3)姉妹染色体交換頻度の増大、(4)hprt座における突然変異頻度の増大、(5)DNA傷害剤MMS、HU、UVに高感受性を示した。しかし、(6)BLMとLIG4の2重破壊株において、増殖能、突然変異頻度、DNA傷害剤感受性がLIG4欠損により抑制されるという興味深い結果を得た。(7)Ku70遺伝子のヘテロ破壊株を作製できたが完全破壊株が得られず、Ku70が生存に必須の可能性がある。そこで、条件致死破壊株として作製を試みている。

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  • DT40細胞を用いた非相同組換え機構の遺伝的解析

    Grant number:14771289  2002 - 2003

    日本学術振興会  科学研究費助成事業  若手研究(B)

    足立 典隆

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    Grant amount:\3200000 ( Direct Cost: \3200000 )

    ニワトリBリンパ細胞株DT40を主材料に用いて,非相同組換えへの関与が示唆される遺伝子の破壊株(ホモ変異株)をジーンターゲティング法により作製し,解析を行った。
    1.DNA ligase IV:ホモ変異株においてランダムインテグレーション頻度が著しく低下していること,またジーンターゲティング効率が上昇していることがわかった。これらの表現型はKu変異株や二重変異株においても全く同様であった。またターゲティング効率の低い遺伝子座において特に上昇率が高かった。したがって,非相同組換え(エンドジョイニング)がランダムインテグレーションの主要機構であり,この反応を抑制することでジーンターゲティングを効率良く行える可能性が示された。一方以上の結果をもとに,ヒト細胞への応用を試みた。
    2.DNA ligase III:得られたヘテロ変異株を用いてホモ変異株の取得を試みた,必須遺伝子である可能性が高いため,テトラサイクリン制御系を利用した条件致死変異株の作製に着手した。
    3.FEN-1:ホモ変異株においてランダムインテグレーション頻度が上昇していることがわかった。これはFEN-1欠損によりゲノムが不安定になるためと考えられる。またDNAポリメラーゼβとの二重変異株の解析から,脱塩基部位の修復に関わる新規経路が存在する可能性が示唆された。
    4.DNAポリメラーゼβ:ホモ変異株においてランダムインテグレーション頻度が約半分に低下している可能性が示された。さらに詳細な解析を行う必要があるが,動物細胞のランダムインテグレーションに関わるDNAポリメラーゼはいまだ同定されていないため,非常に興味深い。
    5.DNAトポイソメラーゼII:ヘテロ変異株において抗癌剤エトポシドに対する感受性が約半分に低下していることを明らかにした。一方,DNA ligase IV変異株やKu変異株において,エトポシドに対する感受性が著しく増大していることを見い出した。DNA ligase IV変異株においてトポイソメラーゼIIをヘテロにしてもやはりエトポシド感受性は約半分に低下した。これらの結果はトポイソメラーゼII損傷修復においてエンドジョイニングが必須の役割を果たすことを示している。

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  • ニワトリDT40細胞を用いた非相同組換え機構の遺伝的解析

    Grant number:12771409  2000 - 2001

    日本学術振興会  科学研究費助成事業  奨励研究(A)

    足立 典隆

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    Grant amount:\2000000 ( Direct Cost: \2000000 )

    ニワトリBリンパ細胞株DT40を用いて,非相同組換えへの関与が示唆される遺伝子の破壊株(ホモ変異株)をジーンターゲティング法により作製し,解析を行った.
    1.DNA ligase IV : DNA ligase IV遺伝子破壊株およびKu70遺伝子との二重破壊株の解析を引き続き行った.DNA ligase IVが,Kuに依存した非相同組換え(エンドジョイニング)の過程に必須の役割を果たしていることを明らかにした.また,これらの破壊株におけるランダムインテグレーションおよびジーンターゲティングの頻度を詳しく調べた.
    2.DNA ligase III:ヘテロ変異株を取得し、解析を行った.また,ホモ変異株の取得を試みるとともに,DNA ligaseIIIが細胞増殖に必須である可能性を考慮し,条件致死変異株作製を試みた.
    3.FEN-1:ホモ変異株を取得し,解析を行った.FEN-1はDNA複製に必須ではないが,塩基除去修復に必須であることを明らかにした.また,組換え,特にランダムインテグレーションにおけるFEN-1の役割についても調べた.さらに,DNAポリメラーゼβとの二重破壊株を作製するため,DNAポリメラーゼβ遺伝子をクローニング,導入ベクターを作製して破壊株の分離を試みた.
    4.DNAトポイソメラーゼ(トポ)IIα:ヘテロ変異株の解析を引き続き行った.マウスES細胞での結果と同様,ヘテロ変異株の増殖速度は野生株と変わらなかったが,VP-16やICRF-193等のトポII阻害剤に対する感受性が低下していた.また,組換え,特にランダムインテグレーションにおけるトポIIαの役割についても調べた.

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  • DNAトポイソメラーゼIIαの生理的機能と発現制御機構

    Grant number:10771295  1998 - 1999

    日本学術振興会  科学研究費助成事業  奨励研究(A)

    足立 典隆

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    Grant amount:\1700000 ( Direct Cost: \1700000 )

    1.トポIIαの生理的機能の解明 ジーンターゲティング法により取得したヘテロ変異ES細胞を解析し,トポIIαの発現量低下により細胞の増殖速度が遅れることを明らかにした。また,トポIIα発現量とトポII阻害剤(VP-16,ICRF-193)感受性との間に正の相関関係があることを示した。さらに,個体レベルの解析を目指してノックアウトマウスの作製を試みたところ,ホモ変異マウスは致死となることがわかった。ヘテロ変異マウスでは全く異常はみられなかった。
    2.トポIIαの発現制御機構の解明 トポIIαプロモーターの活性が細胞周期に依存して変化しておりG2/M期に最大に達することをマウスNIH3T3細胞を用いて示した。この転写調節に関わるシス配列を検索した結果,複数の逆向きCCAAT配列(ICB)がG2/M期での転写活性化に必要であることがわかった。ゲルシフト法により,ICBに結合する転写因子としてNF-Yを同定した。しかし,細胞周期を通じてNF-Yの発現量およびICB結合能に大きな変化はみられなかった。また,プロモーター上における転写因子の結合様式は細胞周期を通じてほとんど変化しなかった。そこで,ヒストンアセチル化の関与を調べたところ,脱アセチル化酵素阻害剤であるトリコスタチンAによりプロモーター活性が著しく上昇することがわかった。この活性化にも複数のICBが必要であった。このことから,G2/M期での転写活性化にNF-Yを介したアセチル化が重要であることが強く示唆された。

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