The Experts below are selected from a list of 2826 Experts worldwide ranked by ideXlab platform
Shunichi Takeda - One of the best experts on this subject based on the ideXlab platform.
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Impact of DNA repair pathways on the cytotoxicity of piperlongumine in chicken DT40 Cell-Lines
Genes & cancer, 2014Co-Authors: Saki Okamoto, Shunichi Takeda, Takeo Narita, Hiroyuki Sasanuma, Shin-ichiro Masunaga, Tadayoshi Bessho, Keizo TanoAbstract:Piperlongumine is a naturally-occurring small molecule with various biological activities. Recent studies demonstrate that piperlongumine selectively kills various types of transformed Cells with minimal toxicity to non-transformed Cells by inducing a high level of reactive oxygen species (ROS). ROS generates various types of DNA lesions, including base modifications and single strand breaks. In order to examine the contribution of ROS-induced DNA damage to the cytotoxicity by piperlongumine, various DNA repair-deficient chicken DT40 Cell-Lines with a single DNA repair gene deletion were tested for Cellular sensitivity to piperlongumine. The results showed that Cell Lines defective in homologous recombination (HR) display hyper-sensitivity to piperlongumine, while other Cell Lines with a deficiency in non-homologous end joining (NHEJ), base excision repair (BER), nucleotide excision repair (NER), Fanconi anemia (FA) pathway, or translesion DNA synthesis (TLS) polymerases, show no sensitivity to piperlongumine. The results strongly implicate that double strand breaks (DSBs) generated by piperlongumine are major cytotoxic DNA lesions. Furthermore, a deletion of 53BP1 or Ku70 in the BRCA1-deficient Cell Line restored Cellular resistance to piperlongumine. This strongly supports the idea that piperlongumine induces DSB- mediated Cell death. Interestingly, piperlongumine makes the wild type DT40 Cell Line hypersensitive to a PARP-inhibitor, Olaparib. The results implicate that piperlongumine inhibits HR. Further analysis with Cell-based HR assay and the kinetic study of Rad51 foci formation confirmed that piperlongumine suppresses HR activity. Altogether, we revealed novel mechanisms of piperlongumine-induced cytotoxicity.
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Chicken DT40 Cell Line lacking DJ-1, the gene responsible for familial Parkinson's disease, displays mitochondrial dysfunction.
Neuroscience research, 2013Co-Authors: Eiko N. Minakawa, Shunichi Takeda, Hodaka Yamakado, Atsushi Tanaka, Kengo Uemura, Ryosuke TakahashiAbstract:Parkinson's disease (PD) is the most common neurodegenerative movement disorder mainly due to gradual loss of dopaminergic neurons in the substantia nigra. Although the causative genes for autosomal recessive PD, Parkin, PINK1 and DJ-1, share a common pathway, at least in part, in mitochondrial quality control and protein quality control, their precise relationship remains elusive. Previous studies suggested the limitation of gene-modified mice model to solve this problem. DT40 is an avian leukosis virus-induced chicken B Cell Line with an exceptionally high ratio of targeted to random DNA integration, which enables efficient targeted disruption of multiple genes of interest. We generated DJ-1-deficient DT40 Cells and analyzed PD-related phenotypes. These Cells exhibited vulnerability to oxidative stress, mitochondrial dysfunction and fragmentation. Importantly, we showed that mitochondrial membrane potential and morphology are available for the phenotype analysis in DT40. These results suggest that genetically engineered DT40 Cells would serve as a relevant model of PD, and help understand the genetic and functional relationship among multiple causative genes. Furthermore, in Line with the recent concept of PD as a systemic disorder, elucidating the pathomechanism of PD using DT40 would lead to the development of noninvasive diagnostic tools and drug screening assays using patient-derived lymphocytes.
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SEL1L is required for endoplasmic reticulum-associated degradation of misfolded luminal proteins but not transmembrane proteins in chicken DT40 Cell Line.
Cell structure and function, 2011Co-Authors: Satoshi Ninagawa, Shunichi Takeda, Tetsuya Okada, Kazutoshi MoriAbstract:Proteins misfolded in the endoplasmic reticulum (ER) are degraded in the cytosol by a ubiquitin-dependent proteasome system, a process collectively termed ER-associated degradation (ERAD). Unraveling the molecular mechanisms of mammalian ERAD progresses more slowly than that of yeast ERAD due to the laborious procedures required for gene targeting and the redundancy of components. Here, we utilized the chicken B lymphocyte-derived DT40 Cell Line, which exhibits an extremely high homologous recombination frequency, to analyze ERAD mechanisms in higher eukaryotes. We disrupted the SEL1L gene, which encodes the sole homologue of yeast Hrd3p in both chickens and mammals; Hrd3p is a binding partner of yeast Hrd1p, an E3 ubiquitin ligase. SEL1L-knockout Cells grew only slightly more slowly than the wild-type Cells. Pulse chase experiments revealed that chicken SEL1L was required for ERAD of misfolded luminal proteins such as glycosylated NHK and unglycosylated NHK-QQQ but dispensable for that of misfolded transmembrane proteins such as NHKBACE and CD3-δ, as in mammals. The defect of SEL1L-knockout Cells in NHK degradation was restored by introduction of not only chicken SEL1L but also mouse and human SEL1L. Deletion analysis showed the importance of Sel1-like tetratricopeptide repeats but not the fibronectin II domain in the function of SEL1L. Thus, our reverse genetic approach using the chicken DT40 Cell Line will provide highly useful information regarding ERAD mechanisms in higher eukaryotes which express ERAD components redundantly.
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RAP80 Acts Independently of BRCA1 in Repair of Topoisomerase II Poison-Induced DNA Damage
Cancer research, 2010Co-Authors: Junko Iijima, Shunichi Takeda, Zhihong Zeng, Yoshihito TaniguchiAbstract:The tumor suppressor BRCA1 functions in DNA homologous recombination, and mutations in BRCA1 increase the risk of breast and ovarian cancers. RAP80 is a component of BRCA1-containing complexes that is required for recruitment of BRCA1 to sites of DNA damage. To evaluate the role of RAP80 in DNA damage repair, we genetically disrupted both RAP80 alleles in the recombinogenic avian DT40 Cell Line. The resulting RAP80(-/-) Cells were proficient at homologous recombination and nonhomologous end-joining (NHEJ), but were specifically sensitized to the topoisomerase II inhibitor etoposide. Notably, doubly mutant RAP80(-/-)BRCA1(-/-) Cells were more sensitive to etoposide than were BRCA1(-/-) Cells, revealing that RAP80 performs a BRCA1-independent repair function. Moreover, jointly impairing the function of CtIP, a distinct BRCA1 effector protein, rendered RAP80(-/-) Cells more sensitive to etoposide compared with singly mutant Cells, again illustrating a BRCA1-independent role of RAP80. Based on our findings, we propose that RAP80 exerts a specific function in repair of the topoisomerase-cleavage complex, such as the removal of covalently bound polypeptides from double-strand break ends independently of BRCA1.
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Interplay between DNA polymerases β and λ in repair of oxidation DNA damage in chicken DT40 Cells
DNA repair, 2007Co-Authors: Keizo Tano, Eiichiro Sonoda, Shunichi Takeda, Hiroshi Arakawa, Jean-marie Buerstedde, Jun Nakamura, Kenjiro Asagoshi, Elena K. Braithwaite, Rajendra Prasad, Masami WatanabeAbstract:Abstract DNA polymerase λ (Pol λ) is a DNA polymerase β (Pol β)-like enzyme with both DNA synthetic and 5′-deoxyribose-5′-phosphate lyase domains. Recent biochemical studies implicated Pol λ as a backup enzyme to Pol β in the mammalian base excision repair (BER) pathway. To examine the interrelationship between Pol λ and Pol β in BER of DNA damage in living Cells, we disrupted the genes for both enzymes either singly or in combination in the chicken DT40 Cell Line and then characterized BER phenotypes. Disruption of the genes for both polymerases caused hypersensitivity to H 2 O 2 -induced cytotoxicity, whereas the effect of disruption of either polymerase alone was only modest. Similarly, BER capacity in Cells after H 2 O 2 exposure was lower in Pol β −/− /Pol λ −/− Cells than in Pol β −/− , wild-type, and Pol λ −/− Cells, which were equivalent. These results suggest that these polymerases can complement for one another in counteracting oxidative DNA damage. Similar results were obtained in assays for in vitro BER capacity using Cell extracts. With MMS-induced cytotoxicity, there was no significant effect on either survival or BER capacity from Pol λ gene disruption. A strong hypersensitivity and reduction in BER capacity was observed for Pol β −/− /Pol λ −/− and Pol β −/− Cells, suggesting that Pol β had a dominant role in counteracting alkylation DNA damage in this Cell system.
Hideki Koyama - One of the best experts on this subject based on the ideXlab platform.
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A new system for analyzing Line retrotransposition in the chicken DT40 Cell Line widely used for reverse genetics.
Gene, 2007Co-Authors: Hiroshi Honda, Hideki Koyama, Kenji Ichiyanagi, Jun Suzuki, Takao Ono, Masaki Kajikawa, Norihiro OkadaAbstract:Long interspersed elements (Lines) are autonomous transposable elements that proliferate via retrotransposition, which involves reverse transcription of Line RNAs. It is anticipated that Line retrotransposition requires both Line-encoded proteins and host-encoded proteins. However, identification of the host factors, their roles, and the steps at which they act on retrotransposition are poorly understood because of the lack of an appropriate genetic system to study Line retrotransposition in a series of mutant hosts. To construct such a genetic system, we applied the retrotransposition-indicative cassette method to DT40 Cells, a chicken Cell Line for which a variety of isogenic mutants have been established by gene targeting. Because DT40 Cells are non-adherent, we utilized a selective soft agarose medium to allow the formation of colonies of Cells that had undergone Line retrotransposition. Colony formation was completely dependent on the activities of the Line-encoded proteins and on the presence of the essential 3' region of the Line RNA. Moreover, the selected colonies indeed carried retrotransposed Line copies in their chromosomes, with integration features similar to those of genomic (native) Line copies. This method thus allows the authentic selection of Line-retrotransposed Cells and the approximate recapitulation of retrotransposition events that occur in nature. Therefore, the DT40 Cell system established here provides a powerful tool for the elucidation of Line retrotransposition pathways, the host factors involved, and their roles.
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Genetic evidence for involvement of two distinct nonhomologous end-joining pathways in repair of topoisomerase II-mediated DNA damage
Biochemical and biophysical research communications, 2004Co-Authors: Noritaka Adachi, Susumu Iiizumi, Hideki KoyamaAbstract:In vertebrate Cells, DNA double-strand breaks are efficiently repaired by homologous recombination or nonhomologous end-joining (NHEJ). The latter pathway relies on Ku (the Ku70/Ku86 heterodimer), DNA-PKcs, Artemis, Xrcc4, and DNA ligase IV (Lig4). Here, we show that a human pre-B Cell Line nullizygous for Lig4 exhibits hypersensitivity to topoisomerase II (Top2) inhibitors, demonstrating a crucial role for the NHEJ pathway in repair of Top2-induced DNA damage in vertebrates. We also show that in the chicken DT40 Cell Line, all NHEJ mutants (i.e., Ku70-, Lig4-, and DNA-PKcs-null Cells) are equally hypersensitive to the Top2 inhibitor ICRF-193, indicating that the drug-induced damage is repaired by NHEJ involving DNA-PKcs. Intriguingly, however, DNA-PKcs-null Cells display considerably less severe phenotype than other NHEJ mutants in terms of hypersensitivity to VP-16, a Top2 poison that stabilizes cleavable complexes. The results indicate that two distinct NHEJ pathways, involving or not involving DNA-PKcs, are important for the repair of VP-16-induced DNA damage, providing additional evidence for the biological relevance of DNA-PKcs-independent NHEJ. Our results provide significant insights into the mechanisms of repair of Top2-mediated DNA damage, with implications for chemotherapy involving Top2 inhibitors.
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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.
The Journal of biological chemistry, 2003Co-Authors: Noritaka Adachi, Susumu Iiizumi, Hiromi Suzuki, Hideki KoyamaAbstract:A number of clinically useful anticancer drugs, including etoposide (VP-16), target DNA topoisomerase (topo) II. These drugs, referred to as topo II poisons, stabilize cleavable complexes, thereby generating DNA double-strand breaks. Bis-2,6-dioxopiperazines such as ICRF-193 also inhibit topo II by inducing a distinct type of DNA damage, termed topo II clamps, which has been believed to be devoid of double-strand breaks. Despite the biological and clinical importance, the molecular mechanisms for the repair of topo II-mediated DNA damage remain largely unknown. Here, we perform genetic analyses using the chicken DT40 Cell Line to investigate how DNA lesions caused by topo II inhibitors are repaired. Notably, we show that LIG4 -/- and KU70 -/- Cells, which are defective in nonhomologous DNA end-joining (NHEJ), are extremely sensitive to both VP-16 and ICRF-193. In contrast, RAD54 -/- Cells (defective in homologous recombination) are much less hypersensitive to VP-16 than the NHEJ mutants and, more importantly, are not hypersensitive to ICRF-193. Our results provide the first evidence that NHEJ is the predominant pathway for the repair of topo II-mediated DNA damage; that is, cleavable complexes and topo II clamps. The outstandingly increased cytotoxicity of topo II inhibitors in the absence of NHEJ suggests that simultaneous inhibition of topo II and NHEJ would provide a powerful protocol in cancer chemotherapy involving topo II inhibitors.
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Vertebrate Cells lacking FEN‐1 endonuclease are viable but hypersensitive to methylating agents and H2O2
Nucleic acids research, 2002Co-Authors: Yasuo Matsuzaki, Noritaka Adachi, Hideki KoyamaAbstract:The structure-specific FEN-1 endonuclease has been implicated in various Cellular processes, including DNA replication, repair and recombination. In vertebrate Cells, however, no in vivo evidence has been provided so far. Here, we knocked out the FEN-1 gene (FEN1) in the chicken DT40 Cell Line. Surprisingly, homozygous mutant (FEN1–/–) Cells were viable, indicating that FEN-1 is not essential for Cell proliferation and thus for Okazaki fragment processing during DNA replication. However, compared with wild-type Cells, FEN1–/– Cells exhibited a slow growth phenotype, probably due to a high rate of Cell death. The mutant Cells were hypersensitive to methylmethane sulfonate, N-methyl-N′-nitro-N-nitrosoguanidine and H2O2, but not to UV light, X-rays and etoposide, suggesting that FEN-1 functions in base excision repair in vertebrate Cells.
Tatsuo Nakayama - One of the best experts on this subject based on the ideXlab platform.
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GCN5 Regulates the Superoxide-Generating System in Leukocytes Via Controlling gp91-phox Gene Expression
Journal of Immunology, 2011Co-Authors: Hidehiko Kikuchi, Yasunari Takami, Futoshi Kuribayashi, Naomi Kiwaki, Tatsuo NakayamaAbstract:The superoxide anion (O 2 − )-generating system is an important mechanism of innate immune response against microbial infection in phagocytes and is involved in signal transduction mediated by various physiological and pathological signals in phagocytes and other Cells, including B lymphocytes. The O 2 − -generating system is composed of five specific proteins: p22-phox, gp91-phox, p40-phox, p47-phox, p67-phox, and a small G protein, Rac. Little is known regarding epigenetic regulation of the genes constituting the O 2 − -generating system. In this study, by analyzing the GCN5 (one of most important histone acetyltransferases)-deficient DT40 Cell Line, we show that GCN5 deficiency causes loss of the O 2 − -generating activity. Interestingly, transcription of the gp91-phox gene was drastically downregulated (to ∼4%) in GCN5-deficient Cells. To further study the involvement of GCN5 in transcriptional regulation of gp91-phox, we used in vitro differentiation system of U937 Cells. When human monoblastic U937 Cells were cultured in the presence of IFN-γ, transcription of gp91-phox was remarkably upregulated, and the Cells were differentiated to macrophage-like Cells that can produce O 2 − . Chromatin immunoprecipitation assay using the U937 Cells during cultivation with IFN-γ revealed not only that association of GCN5 with the gp91-phox gene promoter was significantly accelerated, but also that GCN5 preferentially elevated acetylation levels of H2BK16 and H3K9 surrounding the promoter. These results suggested that GCN5 regulates the O 2 − -generating system in leukocytes via controlling the gp91-phox gene expression as a supervisor. Our findings obtained in this study should be useful in understanding the molecular mechanisms involved in epigenetic regulation of the O 2 − -generating system in leukocytes.
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Lacking of Aiolos accelerates pre-mature B Cell apoptosis mediated by BCR signaling through elevation in cytochrome c release.
Biochimica et biophysica acta, 2009Co-Authors: Hidehiko Kikuchi, Kenji Toyonaga, Koki Yamashita, Masami Nakayama, Isao Tsuneyoshi, Mayumi Takasaki, Tatsuo NakayamaAbstract:Abstract Antigen binding to B Cell receptor (BCR) of pre-mature B lymphocytes leads to their apoptosis, while binding to BCR of mature B lymphocytes induces their activation and proliferation. The former binding is believed to be a mechanism so as to exclude B Cell clones leading to protection from auto-immune diseases. Cross-linking of BCR of pre-mature B Cells, including chicken DT40 Cells, with anti-immunoglobulin antibody induces their apoptosis. The PMA/ionomycin treatments, which mimic BCR stimulation, are used to study intraCellular signal transduction of B lymphocytes. Here, by analyzing the Aiolos-deficient DT40 Cell Line, Aiolos −/− , we reveal that the lack of Aiolos accelerates apoptosis of DT40 Cells mediated by BCR signaling. Moreover, the Aiolos-deficiency and BCR signaling cooperatively control this apoptosis through dramatically elevated cytochrome c release from mitochondria to cytosol and elevated caspase (caspase-3, 8 and 9) activities, resulting in drastically diminished amounts of ICAD followed by increased DNA fragmentation. Re-expression study reveals that the shorter isoform of Aiolos (Aio-2) controls PMA/ionomycin-mediated apoptosis via up-regulation and down-regulation of the PKCδ and bak genes, respectively. These findings could be a powerful trigger to resolve molecular mechanisms of negative selection of B lymphocytes and also auto-immune diseases.
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E2A participates in a fine control of pre‐mature B‐Cell apoptosis mediated by B‐Cell receptor signaling via transcriptional regulation of survivin, IAP2 and caspase‐8 genes
The FEBS journal, 2009Co-Authors: Kenji Toyonaga, Hidehiko Kikuchi, Koki Yamashita, Masami Nakayama, Kazuo Chijiiwa, Tatsuo NakayamaAbstract:Antigen binding to the B-Cell receptor (BCR) of pre-mature B lymphocytes induces their apoptotic Cell death, but binding to the BCR of mature B lymphocytes triggers activation and proliferation. Binding to pre-mature B lymphocytes is thought not only to function as a mechanism to exclude B-Cell clones that possess the ability to react with self-antigen, but also to act as a defense mechanism in auto-immune diseases. Cross-linking of BCR of pre-mature B-Cell Lines, including the chicken DT40 Cell Line, with anti-immunoglobulin IgG induces apoptotic Cell death. Treatment with phorbol 12-myristate 13-acetate/ionomycin, which mimics BCR stimulation, is used to study intraCellular signal transduction of B lymphocytes. Here, by analyzing the E2A-deficient DT40 Cell Line, E2A(-/-), we show that E2A deficiency prevents certain levels of apoptotic Cell death mediated by BCR signaling. In addition, E2A deficiency-linked BCR signaling controls the mimicked pre-mature B-Cell apoptosis by PMA/ionomycin through elevated survivin plus inhibitor of apoptosis 2 levels, and reduced caspase-3 and caspase-8 activities, resulting in increased amounts of ICAD (inhibitor of caspase-activated DNase), compared with those in the presence of E2A, followed by reduction of DNA fragmentation. These findings will contribute to the resolution of molecular mechanisms of negative selection of B Cells and also auto-immune diseases.
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GCN5 and BCR signalling collaborate to induce pre-mature B Cell apoptosis through depletion of ICAD and IAP2 and activation of caspase activities.
Gene, 2008Co-Authors: Hidehiko Kikuchi, Tatsuo NakayamaAbstract:The antigen binding to the B Cell receptor (BCR) of pre-mature B lymphocytes induces their apoptotic Cell death, although the binding to BCR of mature B lymphocytes does their activation and proliferation. The former is thought not only to function as a mechanism to exclude B Cell clones possessing the ability to react with self-antigen, but also to participate as a defense mechanism from auto-immune diseases. Cross-linking of BCR of pre-mature B Cell Lines, including the chicken DT40 Cell Line, with anti-immunoglobulin antibody induces their apoptotic Cell death. The PMA/ionomycin treatment, which mimics the BCR stimulation, is used to study intraCellular signal transduction of B lymphocytes. Here, by analyzing the GCN5-deficient DT40 Cell Line, we show that GCN5 and BCR signalling are essential for apoptotic Cell death. In addition, GCN5 and BCR signalling control cooperatively pre-mature B Cell apoptosis via both depletions of ICAD and IAP2 (inhibitors for apoptosis) and elevations of caspase-8 and caspase-3 activities, resulting in increased activity of CAD (effector for apoptosis) followed by the DNA fragmentation. These findings should be useful in understanding the molecular mechanisms involved in negative selection of B Cells as also in auto-immune diseases.
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Participation of histones, histone modifying enzymes and histone chaperones in vertebrate Cell functions.
Sub-cellular biochemistry, 2006Co-Authors: Hidehiko Kikuchi, Yasunari Takami, Masami Nakayama, Hirak Kumar Barman, Tatsuo NakayamaAbstract:Alterations in the chromatin structure are essential for easy accesses to chromosomal DNA. Such architectural alterations can be achieved by four means: (i) variants of histone subtypes, (ii) chromatin remodeling, (iii) post-translational modification, and (iv) chromatin assembly. This chapter discusses mainly on the first, third and fourth mechanisms, and especially on the acetylation of core histones, one of the third mechanisms. Taking the advantage of the gene targeting technique, we systematically established numerous mutant DT40 Cell Lines, each lacking particular gene, of interest such that encoding histones, histone deacetylases (HDACs), acetyltransferases (HATs) and chaperones, etc. Every subtype member of the histone gene family is capable of compensating the loss of others to maintain the mRNA level of each histone subtype, and most of histone variants are involved positively or negatively in the transcription regulation of particular genes. Regarding HDACs, HDAC-2 controls the amount of the IgM H-chain at the steps of both transcription and alternative pre-mRNA processing, and HDAC-3 is indispensable for Cell viability. Concerning HATs, GCN5 has tremendous impact on growth kinetics by preferentially acting as a supervisor in the normal Cell cycle progression. The distinct participatory roles of the N-terminal and C-terminal halves of HIRA, one of histone chaperones, in both Cell growth and transcription regulations of Cell cycle-related genes, have also been highlighted. Therefore, the gene targeting technique in the DT40 Cell Line can be used as a powerful tool for the functional analysis of histones, histone modifying enzymes and histone chaperones relevant to chromatin biology.
Hiroshi Arakawa - One of the best experts on this subject based on the ideXlab platform.
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A double-strand break can trigger immunoglobulin gene conversion
Nucleic acids research, 2016Co-Authors: Giulia Bastianello, Hiroshi ArakawaAbstract:All three B Cell-specific activities of the immunoglobulin (Ig) gene re-modeling system-gene conversion, somatic hypermutation and class switch recombination-require activation-induced deaminase (AID). AID-induced DNA lesions must be further processed and dissected into different DNA recombination pathways. In order to characterize potential intermediates for Ig gene conversion, we inserted an I-SceI recognition site into the complementarity determining region 1 (CDR1) of the Ig light chain locus of the AID knockout DT40 Cell Line, and conditionally expressed I-SceI endonuclease. Here, we show that a double-strand break (DSB) in CDR1 is sufficient to trigger Ig gene conversion in the absence of AID. The pattern and pseudogene usage of DSB-induced gene conversion were comparable to those of AID-induced gene conversion; surprisingly, sometimes a single DSB induced multiple gene conversion events. These constitute direct evidence that a DSB in the V region can be an intermediate for gene conversion. The fate of the DNA lesion downstream of a DSB had more flexibility than that of AID, suggesting two alternative models: (i) DSBs during the physiological gene conversion are in the minority compared to single-strand breaks (SSBs), which are frequently generated following DNA deamination, or (ii) the physiological gene conversion is mediated by a tightly regulated DSB that is locally protected from non-homologous end joining (NHEJ) or other non-homologous DNA recombination machineries.
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FancJ/Brip1 helicase protects against genomic losses and gains in vertebrate Cells
Genes to cells : devoted to molecular & cellular mechanisms, 2011Co-Authors: Hiroyuki Kitao, Hiroshi Arakawa, Indrajit Nanda, Ryuichi P. Sugino, Aiko Kinomura, Mitsuyoshi Yamazoe, Michael Schmid, Hideki Innan, Kevin Hiom, Minoru TakataAbstract:Defects in the FANCJ/BRIP1 helicase gene are associated with genome instability disorders such as familial breast cancer or Fanconi anemia (FA). Although FANCJ has an in vitro activity to resolve G-quadruplex (G4) structures, and FANCJ ortholog in C. elegans prevents G4-associated deletions during replication, how FANCJ loss affects genome integrity in higher organisms remains unclear. Here, we report that FANCJ, but not other FA genes FANCD2 or FANCC, protected against large-scale genomic deletion that occurred frequently at the rearranged immunoglobulin heavy chain (IgH) locus in chicken DT40 Cell Line, suggesting that FancJ protects the genome independently of the FA ubiquitination pathway. In a more unbiased approach using array-comparative genomic hybridization, we identified de novo deletions as well as amplifications in fancj Cells kept in culture for 2 months. A cluster of G4 sequence motifs was found near the breakpoint of one amplified region, but G4 sequence motifs were not detected at the breakpoints of two deleted regions. These results collectively suggest that, unlike in C. elegans, actions of vertebrate FANCJ to promote genome stability may not be limited to protection against the G4-mediated gene deletions.
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Activation-induced cytidine deaminase-mediated hypermutation in the DT40 Cell Line.
Philosophical transactions of the Royal Society of London. Series B Biological sciences, 2008Co-Authors: Hiroshi Arakawa, Jean-marie BuersteddeAbstract:Depending on the species and the developmental stage of B Cells, activation-induced cytidine deaminase (AID) triggers immunoglobulin (Ig) gene diversification by gene conversion, hypermutation or s...
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Interplay between DNA polymerases β and λ in repair of oxidation DNA damage in chicken DT40 Cells
DNA repair, 2007Co-Authors: Keizo Tano, Eiichiro Sonoda, Shunichi Takeda, Hiroshi Arakawa, Jean-marie Buerstedde, Jun Nakamura, Kenjiro Asagoshi, Elena K. Braithwaite, Rajendra Prasad, Masami WatanabeAbstract:Abstract DNA polymerase λ (Pol λ) is a DNA polymerase β (Pol β)-like enzyme with both DNA synthetic and 5′-deoxyribose-5′-phosphate lyase domains. Recent biochemical studies implicated Pol λ as a backup enzyme to Pol β in the mammalian base excision repair (BER) pathway. To examine the interrelationship between Pol λ and Pol β in BER of DNA damage in living Cells, we disrupted the genes for both enzymes either singly or in combination in the chicken DT40 Cell Line and then characterized BER phenotypes. Disruption of the genes for both polymerases caused hypersensitivity to H 2 O 2 -induced cytotoxicity, whereas the effect of disruption of either polymerase alone was only modest. Similarly, BER capacity in Cells after H 2 O 2 exposure was lower in Pol β −/− /Pol λ −/− Cells than in Pol β −/− , wild-type, and Pol λ −/− Cells, which were equivalent. These results suggest that these polymerases can complement for one another in counteracting oxidative DNA damage. Similar results were obtained in assays for in vitro BER capacity using Cell extracts. With MMS-induced cytotoxicity, there was no significant effect on either survival or BER capacity from Pol λ gene disruption. A strong hypersensitivity and reduction in BER capacity was observed for Pol β −/− /Pol λ −/− and Pol β −/− Cells, suggesting that Pol β had a dominant role in counteracting alkylation DNA damage in this Cell system.
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Basic Cell culture conditions.
Sub-cellular biochemistry, 2006Co-Authors: Huseyin Saribasak, Hiroshi ArakawaAbstract:The DT40 Cell Line is derived from ALV (avian leukosis virus)-transformed bursal B Cell of chicken. This Cell Line is suspension Cell, and can be cultured in Cell culture bottles and in culture plates (6-, 24- and 96-well etc.). Optimal culture temperature for the DT40 is (39 degrees C-) 41 degrees C, because chicken has higher body temperature than human and mouse. Here we summarize protocols for culture medium and condition of the DT40 Cell Line.
Tatsuo Fukagawa - One of the best experts on this subject based on the ideXlab platform.
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chromatin binding of rcc1 during mitosis is important for its nuclear localization in interphase
Molecular Biology of the Cell, 2016Co-Authors: Maiko Furuta, Tatsuo Fukagawa, Tetsuya HoriAbstract:RCC1, a guanine nucleotide exchange factor of the small GTPase Ran, plays various roles throughout the Cell cycle. However, the functions of RCC1 in biological processes in vivo are still unclear. In particular, although RCC1 has multifunctional domains, the biological significance of each domain is unclear. To examine each domain of RCC1, we established an RCC1 conditional knockout chicken DT40 Cell Line and introduced various RCC1 mutants into the knockout Cells. We found that nuclear reformation did not occur properly in RCC1-deficient Cells and examined whether specific RCC1 mutants could rescue this phenotype. Surprisingly, we found that neither the nuclear localization signal nor the chromatin-binding domain of RCC1 is essential for its function. However, codisruption of these domains resulted in defective nuclear reformation, which was rescued by artificial nuclear localization of RCC1. Our data indicate that chromatin association of RCC1 during mitosis is crucial for its proper nuclear localization in the next interphase. Moreover, proper nuclear localization of RCC1 in interphase is essential for its function through its nucleotide exchange activity.
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Structure-Specific Endonucleases Xpf and Mus81 Play Overlapping but Essential Roles in DNA Repair by Homologous Recombination
Cancer research, 2013Co-Authors: Koji Kikuchi, Tetsuya Hori, Mohiuddin, Islam Shamima Keka, Takeo Narita, Junko Iijima, Kouji Hirota, Van T. Pham, Katsuya Okawa, Tatsuo FukagawaAbstract:DNA double-strand breaks (DSBs) occur frequently during replication in sister chromatids, and are dramatically increased when Cells are exposed to chemotherapeutic agents including camptothecin. Such DSBs are efficiently repaired specifically by homologous recombination (HR) with the intact sister chromatid. HR hence plays pivotal roles in Cellular proliferation and Cellular tolerance to camptothecin. Mammalian Cells carry several structure-specific endonucleases, such as Xpf-Ercc1 and Mus81-Eme1, in which Xpf and Mus81 are the essential subunits for enzymatic activity. Here we show the functional overlap between Xpf and Mus81 by conditionally inactivating Xpf in the chicken DT40 Cell Line, which has no Mus81 ortholog. Although mammalian Cells deficient in either Xpf or Mus81 are viable, Xpf inactivation in DT40 Cells was lethal, resulting in a marked increase in the number of spontaneous chromosome breaks. Similarly, inactivation of both Xpf and Mus81 in human HeLa Cells and murine embryonic stem Cells caused numerous spontaneous chromosome breaks. Furthermore, the phenotype of Xpf-deficient DT40 Cells was reversed by ectopic expression of human Mus81-Eme1 or human Xpf-Ercc1 heterodimers. These observations indicate the functional overlap of Xpf-Ercc1 and Mus81-Eme1 in the maintenance of genomic DNA. Both Mus81-Eme1 and Xpf-Ercc1 contribute to the completion of HR as evidenced by the following data that the expression of Mus81-Eme1 or Xpf-Ercc1 diminished the number of camptothecin-induced chromosome breaks in Xpf-deficient DT40 Cells, and preventing early steps in HR by deleting XRCC3 suppressed the inviability of Xpf-deficient DT40 Cells. In summary, Xpf and Mus81 have a substantially overlapping function in completion of HR.
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The constitutive centromere component CENP-50 is required for recovery from spindle damage.
Molecular and cellular biology, 2005Co-Authors: Yukinori Minoshima, Tetsuya Hori, Tokuko Haraguchi, Yasushi Hiraoka, Masahiro Okada, Hiroshi Kimura, Ying-chun Bao, Toshiyuki Kawashima, Toshio Kitamura, Tatsuo FukagawaAbstract:We identified CENP-50 as a novel kinetochore component. We found that CENP-50 is a constitutive component of the centromere that colocalizes with CENP-A and CENP-H throughout the Cell cycle in vertebrate Cells. To determine the precise role of CENP-50, we examined its role in centromere function by generating a loss-of-function mutant in the chicken DT40 Cell Line. The CENP-50 knockout was not lethal; however, the growth rate of Cells with this mutation was slower than that of wild-type Cells. We observed that the time for CENP-50-deficient Cells to complete mitosis was longer than that for wild-type Cells. Centromeric localization of CENP-50 was abolished in both CENP-H- and CENP-I-deficient Cells. Coimmunoprecipitation experiments revealed that CENP-50 interacted with the CENP-H/CENP-I complex in chicken DT40 Cells. We also observed severe mitotic defects in CENP-50-deficient Cells with apparent premature sister chromatid separation when the mitotic checkpoint was activated, indicating that CENP-50 is required for recovery from spindle damage.
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CENP-A Is Required for Accurate Chromosome Segregation and Sustained Kinetochore Association of BubR1
Molecular and cellular biology, 2005Co-Authors: Vinciane Regnier, Tatsuo Fukagawa, William C. Earnshaw, Paola Vagnarelli, Tatiana Zerjal, Elizabeth Burns, Didier Trouche, William BrownAbstract:CENP-A is an evolutionarily conserved, centromere-specific variant of histone H3 that is thought to play a central role in directing kinetochore assembly and in centromere function. Here, we have analyzed the consequences of disrupting the CENP-A gene in the chicken DT40 Cell Line. In CENP-A-depleted Cells, kinetochore protein assembly is impaired, as indicated by mislocalization of the inner kinetochore proteins CENP-I, CENP-H, and CENP-C as well as the outer components Nuf2/Hec1, Mad2, and CENP-E. However, BubR1 and the inner centromere protein INCENP are efficiently recruited to kinetochores. Following CENP-A depletion, chromosomes are deficient in proper congression on the mitotic spindle and there is a transient delay in prometaphase. CENP-A-depleted Cells further proceed through anaphase and cytokinesis with unequal chromosome segregation, suggesting that some kinetochore function remains following substantial depletion of CENP-A. We furthermore demonstrate that CENP-A-depleted Cells exhibit a specific defect in maintaining kinetochore localization of the checkpoint protein BubR1 under conditions of checkpoint activation. Our data thus point to a specific role for CENP-A in assembly of kinetochores competent in the maintenance of mitotic checkpoint signaling.
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dicer is essential for formation of the heterochromatin structure in vertebrate Cells
Nature Cell Biology, 2004Co-Authors: Tatsuo Fukagawa, Yasunari Takami, Tatsuo Nakayama, Masahiro Nogami, Mitsuko Yoshikawa, Masashi Ikeno, Tuneko Okazaki, Mitsuo OshimuraAbstract:RNA interference is an evolutionarily conserved gene-silencing pathway in which the nuclease Dicer cleaves double-stranded RNA into small interfering RNAs1. The biological function of the RNAi-related pathway in vertebrate Cells is not fully understood. Here, we report the generation of a conditional loss-of-function Dicer mutant in a chicken–human hybrid DT40 Cell Line that contains human chromosome 21. We show that loss of Dicer results in Cell death with the accumulation of abnormal mitotic Cells that show premature sister chromatid separation. Aberrant accumulation of transcripts from α-satellite sequences, which consist of human centromeric repeat DNAs, was detected in Dicer-deficient Cells. Immunocytochemical analysis revealed abnormalities in the localization of two heterochromatin proteins, Rad21 cohesin protein and BubR1 checkpoint protein, but the localization of core kinetochore proteins such as centromere protein (CENP)-A and -C was normal. We conclude that Dicer-related RNA interference machinery is involved in the formation of the heterochromatin structure in higher vertebrate Cells.
