Akira Motegi

Kyoto University, Kioto, Kyōto, Japan

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Publications (17)82.81 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: SUMO conjugation is a reversible posttranslational modification that regulates protein function. SENP1 is one of the six SUMO-specific proteases present in vertebrate cells and its altered expression is observed in several carcinomas. To characterize SENP1 role in genome integrity, we generated Senp1 knockout chicken DT40 cells. SENP1(-/-) cells show normal proliferation, but are sensitive to spindle poisons. This hypersensitivity correlates with increased sister chromatid separation, mitotic slippage, and apoptosis. To test whether the cohesion defect had a causal relationship with the observed mitotic events, we restored the cohesive status of sister chromatids by introducing the TOP2α(+/-) mutation, which leads to increased catenation, or by inhibiting Plk1 and Aurora B kinases that promote cohesin release from chromosomes during prolonged mitotic arrest. Although TOP2α is SUMOylated during mitosis, the TOP2α(+/-) mutation had no obvious effect. By contrast, inhibition of Plk1 or Aurora B rescued the hypersensitivity of SENP1(-/-) cells to colcemid. In conclusion, we identify SENP1 as a novel factor required for mitotic arrest and cohesion maintenance during prolonged mitotic arrest induced by spindle poisons.
    Biochemical and Biophysical Research Communications 08/2012; 426(3):310-6. · 2.41 Impact Factor
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    ABSTRACT: DPB11/TopBP1 is an essential evolutionarily conserved gene involved in initiation of DNA replication and checkpoint signaling. Here, we show that Saccharomyces cerevisiae Dpb11 forms nuclear foci that localize to sites of DNA damage in G1, S and G2 phase, a recruitment that is conserved for its homologue TopBP1 in Gallus gallus. Damage-induced Dpb11 foci are distinct from Sld3 replication initiation foci. Further, Dpb11 foci are dependent on the checkpoint proteins Mec3 (9-1-1 complex) and Rad24, and require the C-terminal domain of Dpb11. Dpb11 foci are independent of the checkpoint kinases Mec1 and Tel1, and of the checkpoint mediator Rad9. In a site-directed mutagenesis screen, we identify a separation-of-function mutant, dpb11-PF, that is sensitive to DSB-inducing agents yet remains proficient for DNA replication and the S-phase checkpoint at the permissive temperature. The dpb11-PF mutant displays altered rates of heteroallelic and direct-repeat recombination, sensitivity to DSB-inducing drugs as well as delayed kinetics of mating-type switching with a defect in the DNA synthesis step thus implicating Dpb11 in homologous recombination. We conclude that Dpb11/TopBP1 plays distinct roles in replication, checkpoint response and recombination processes, thereby contributing to chromosomal stability.
    DNA repair 02/2011; 10(2):210-24. · 4.20 Impact Factor
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    ABSTRACT: Replicative DNA polymerases are frequently stalled by DNA lesions. The resulting replication blockage is released by homologous recombination (HR) and translesion DNA synthesis (TLS). TLS employs specialized TLS polymerases to bypass DNA lesions. We provide striking in vivo evidence of the cooperation between DNA polymerase η, which is mutated in the variant form of the cancer predisposition disorder xeroderma pigmentosum (XP-V), and DNA polymerase ζ by generating POLη(-/-)/POLζ(-/-) cells from the chicken DT40 cell line. POLζ(-/-) cells are hypersensitive to a very wide range of DNA damaging agents, whereas XP-V cells exhibit moderate sensitivity to ultraviolet light (UV) only in the presence of caffeine treatment and exhibit no significant sensitivity to any other damaging agents. It is therefore widely believed that Polη plays a very specific role in cellular tolerance to UV-induced DNA damage. The evidence we present challenges this assumption. The phenotypic analysis of POLη(-/-)/POLζ(-/-) cells shows that, unexpectedly, the loss of Polη significantly rescued all mutant phenotypes of POLζ(-/-) cells and results in the restoration of the DNA damage tolerance by a backup pathway including HR. Taken together, Polη contributes to a much wide range of TLS events than had been predicted by the phenotype of XP-V cells.
    PLoS Genetics 10/2010; 6(10). · 8.52 Impact Factor
  • Akira Motegi
    Seikagaku. The Journal of Japanese Biochemical Society 08/2009; 81(7):601-5. · 0.04 Impact Factor
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    ABSTRACT: Proteasome-dependent protein degradation is involved in a variety of biological processes, including cell-cycle regulation, apoptosis, and stress-responses. Growing evidence from translational research and clinical trials proved the effectiveness of proteasome inhibitors (PIs) in treating several types of hematological malignancies. Although various key molecules in ubiquitin-dependent cellular processes have been proposed as relevant targets of therapeutic proteasome inhibition, our current understanding is far from complete. Recent rapid progress in DNA repair research has unveiled a crucial role of the ubiquitin-proteasome pathway (UPP) in regulating DNA repair. These findings thus bring up the idea that DNA repair pathways could be effective targets of PIs in mediating their cytotoxicity and enhancing the effect of radiotherapy and some DNA-damaging chemotherapeutic agents, such as cisplatin and camptothecin. In this review, we present the current perspective on the UPP-dependent regulatory mechanisms of DNA repair and discuss their therapeutic potential in the application of PIs to a broad spectrum of human cancers.
    Cancer letters 03/2009; 283(1):1-9. · 4.86 Impact Factor
  • Akira Motegi, Shunichi Takeda
    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 03/2009; 54(4 Suppl):450-8.
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    ABSTRACT: We previously proposed that mouse CD8(+)CD122(+) T cells and human CD57(+) T cells, which increase with age and exhibit potent IFN-gamma production, represent a double-edged sword as they play critical roles in host defense and the lethal IL-12/LPS-induced generalized Shwartzman reaction (GSR). However, our proposal was based solely on comparisons of young and old mice. In this study, we attempted to increase CD8(+)CD122(+) T cells in young mice with exogenous IL-15 and confirm their countervailing functions in young mice. After young mice (6 weeks) were injected with IL-15, they showed significant increases in CD8(+)CD122(+) T cells in the liver and spleen. Liver CD8(+)CD122(+) T cells from IL-15-pretreated mice had a potent capacity to produce IFN-gamma after IL-12 injection or Escherichia coli infection. IL-15-pretreated mice showed increased survival to E. coli infections and enhanced anti-tumor activities against liver metastatic EL4 cells, as well as an exacerbation of the GSR. Correspondingly, liver CD8(+)CD122(+) T cells produced more perforin than CD8(+)CD122(-) T cells in EL4-inoculated mice. Unexpectedly, comparable IL-15 treatment did not induce further increases in CD8(+)CD122(+) T cells in aged mice and did not enhance their defenses against bacterial infection or tumor growth. Interestingly, however, nontreated, aged mice (50 weeks) showed twofold higher IL-15 levels (but not TNF or IFN-gamma) in liver homogenates compared with young mice. Our results further support that CD8(+)CD122(+) T cells, which are increased physiologically or therapeutically by IL-15, are involved in antibacterial immunity, anti-tumor immunity, and the GSR.
    Journal of Leukocyte Biology 10/2008; 84(4):1047-56. · 4.57 Impact Factor
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    ABSTRACT: Chronic stalling of DNA replication forks caused by DNA damage can lead to genomic instability. Cells have evolved lesion bypass pathways such as postreplication repair (PRR) to resolve these arrested forks. In yeast, one branch of PRR involves proliferating cell nuclear antigen (PCNA) polyubiquitination mediated by the Rad5-Ubc13-Mms2 complex that allows bypass of DNA lesion by a template-switching mechanism. Previously, we identified human SHPRH as a functional homologue of yeast Rad5 and revealed the existence of RAD5-like pathway in human cells. Here we report the identification of HLTF as a second RAD5 homologue in human cells. HLTF, like SHPRH, shares a unique domain architecture with Rad5 and promotes lysine 63-linked polyubiquitination of PCNA. Similar to yeast Rad5, HLTF is able to interact with UBC13 and PCNA, as well as SHPRH; and the reduction of either SHPRH or HLTF expression enhances spontaneous mutagenesis. Moreover, Hltf-deficient mouse embryonic fibroblasts show elevated chromosome breaks and fusions after methyl methane sulfonate treatment. Our results suggest that HLTF and SHPRH are functional homologues of yeast Rad5 that cooperatively mediate PCNA polyubiquitination and maintain genomic stability.
    Proceedings of the National Academy of Sciences 09/2008; 105(34):12411-6. · 9.74 Impact Factor
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    ABSTRACT: Gross chromosomal rearrangement (GCR) is a type of genomic instability associated with many cancers. In yeast, multiple pathways cooperate to suppress GCR. In a screen for genes that promote GCR, we identified MPH1, which encodes a 3'-5' DNA helicase. Overexpression of Mph1p in yeast results in decreased efficiency of homologous recombination (HR) as well as delayed Rad51p recruitment to double-strand breaks (DSBs), which suggests that Mph1p promotes GCR by partially suppressing HR. A function for Mph1p in suppression of HR is further supported by the observation that deletion of both mph1 and srs2 synergistically sensitize cells to methyl methanesulfonate-induced DNA damage. The GCR-promoting activity of Mph1p appears to depend on its interaction with replication protein A (RPA). Consistent with this observation, excess Mph1p stabilizes RPA at DSBs. Furthermore, spontaneous RPA foci at DSBs are destabilized by the mph1Delta mutation. Therefore, Mph1p promotes GCR formation by partially suppressing HR, likely through its interaction with RPA.
    The Journal of Cell Biology 07/2008; 181(7):1083-93. · 10.82 Impact Factor
  • Akira Motegi, Kyungjae Myung
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    ABSTRACT: Gross chromosomal rearrangements (GCRs), including translocations, deletions, amplifications and aneuploidy are frequently observed in various types of human cancers. Despite their clear importance in carcinogenesis, the molecular mechanisms by which GCRs are generated and held in check are poorly understood. By using a GCR assay, which can measure the rate of accumulation of spontaneous GCRs in Saccharomyces cerevisiae, we have found that many proteins involved in DNA replication, DNA repair, DNA recombination, checkpoints, chromosome remodeling, and telomere maintenance, play crucial roles in GCR metabolism. We describe here the theoretical background and practical procedures of this GCR assay. We will explain the breakpoint structure and DNA damage that lead to GCR formation. We will also summarize the pathways that suppress and enhance GCR formation. Finally, we will briefly describe similar assays developed by others and discuss their potential in studying GCR metabolism.
    Methods 03/2007; 41(2):168-76. · 3.64 Impact Factor
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    ABSTRACT: Ionizing radiation-induced mutagenesis (IR-IM) underlies a basis for radiation associated carcinogenesis as well as resistance to radiation therapy. This process was examined in Saccharomyces cerevisiae using an array of isogenic DNA repair deficient mutants. Mutations inactivating homologous recombination (rad51, 52, 54) or nucleotide excision repair (rad1, rad10, rad4) caused elevated IR-IM whereas inactivation of TransLesion Synthesis (TLS: rad6) caused severely defective IR-IM. Of the mutations inactivating TLS polymerases, rev3 and rev1 caused equally severe defects in IR-IM whereas rad30 did not significantly affect the process. The effects of the rev3, rev1, and rad6 mutations on IR-IM were epistatic, suggesting the requirement of both polymerase zeta and Rev1p in IR-IM related TLS. Although PCNA K164 SUMOylation/ubiquitination is a proposed prerequisite for TLS, the IR-IM defect of a rev3 or a rad6 mutant was worse than and epistatic to the pol30K164R mutant, a mutant in which the PCNA had been mutated to abolish such modifications. These results suggested that IR-IM related TLS occurs in the absence of PCNA K164 modification. Further analysis of a mutant simultaneously defective in SUMOylation and mono-ubiquitination (rad18 siz1) revealed that these modifications redundantly affected TLS as well as NHEJ. A genetic model based on these observations is proposed.
    DNA Repair 01/2007; 5(12):1475-88. · 4.27 Impact Factor
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    ABSTRACT: Differential modifications of proliferating cell nuclear antigen (PCNA) determine DNA repair pathways at stalled replication forks. In yeast, PCNA monoubiquitination by the ubiquitin ligase (E3) yRad18 promotes translesion synthesis (TLS), whereas the lysine-63-linked polyubiquitination of PCNA by yRad5 (E3) promotes the error-free mode of bypass. The yRad5-dependent pathway is important to prevent genomic instability during replication, although its exact molecular mechanism is poorly understood. This mechanism has remained totally elusive in mammals because of the lack of apparent RAD5 homologues. We report that a putative tumor suppressor gene, SHPRH, is a human orthologue of yeast RAD5. SHPRH associates with PCNA, RAD18, and the ubiquitin-conjugating enzyme UBC13 (E2) and promotes methyl methanesulfonate (MMS)-induced PCNA polyubiquitination. The reduction of SHPRH by stable short hairpin RNA increases sensitivity to MMS and enhances genomic instability. Therefore, the yRad5/SHPRH-dependent pathway is a conserved and fundamental DNA repair mechanism that protects the genome from genotoxic stress.
    The Journal of Cell Biology 01/2007; 175(5):703-8. · 10.82 Impact Factor
  • American Journal of Medical Genetics Part A 10/2006; 140(18):1978-80. · 2.30 Impact Factor
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    ABSTRACT: A lethal human septic shock model, mouse generalized Shwartzman reaction (GSR), was elicited by two consecutive lippolysaccharide (LPS) injections (24 h apart) in which interferon-gamma (IFN-gamma) induced by interleukin (IL)-12 played a critical role in the priming phase, and tumor necrosis factor (TNF) was an important effector molecule in the second phase. We recently reported IL-12/LPS-induced mouse GSR age-dependently enhanced. We herein demonstrate that human peripheral blood mononuclear cells (PBMC) from healthy adults/elderly, cultured with IL-12 for 24 h and with LPS for an additional 24 h, produced a much larger amount of TNF (which increased age-dependently) than did PBMC without IL-12 priming. Whereas macrophages mainly produced TNF following LPS stimulation, macrophages and lymphocytes were necessary for a sufficient TNF production. IL-12-induced IFN-gamma up-regulated Toll-like receptor 4 (TLR-4) on macrophages of adults. Although the PBMC from children produced a substantial amount of IFN-gamma after IL-12 priming, the GSR response, with augmented TNF production and an up-regulated TLR-4 expression of macrophages, was not elicited by LPS stimulation. CD56+natural killer cells, CD56+T cells, and CD57+T cells (NK-T cells), which age-dependently increased in PBMC, produced much larger amounts of IFN-gamma after IL-12 priming than that of conventional CD56-CD57-T cells and also induced cocultured macrophages to produce TNF by subsequent LPS stimulation. The elder septic patients were consistently more susceptible to lethal shock with enhanced serum TNF levels than the adult patients. The NK cells, NK-T cells, and macrophages, which change proportionally or functionally with aging, might be involved in the enhanced GSR response/septic shock observed in elderly patients.
    Journal of Leukocyte Biology 04/2006; 79(3):463-72. · 4.57 Impact Factor
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    ABSTRACT: Gross chromosomal rearrangements (GCRs) are frequently observed in many cancers. Previously, we showed that inactivation of Rad5 or Rad18, ubiquitin ligases (E3) targeting for proliferating cell nuclear antigen (PCNA), increases the de novo telomere addition type of GCR (S. Smith, J. Y. Hwang, S. Banerjee, A. Majeed, A. Gupta, and K. Myung, Proc. Natl. Acad. Sci. USA 101:9039-9044, 2004). GCR suppression by Rad5 and Rad18 appears to be exerted by the RAD5-dependent error-free mode of bypass DNA repair. In contrast, Siz1 SUMO ligase and another ubiquitin ligase, Bre1, which target for PCNA and histone H2B, respectively, have GCR-supporting activities. Inactivation of homologous recombination (HR) proteins or the helicase Srs2 reduces GCR rates elevated by the rad5 or rad18 mutation. GCRs are therefore likely to be produced through the restrained recruitment of an HR pathway to stalled DNA replication forks. Since this HR pathway is compatible with Srs2, it is not a conventional form of recombinational pathway. Lastly, we demonstrate that selection of proper DNA repair pathways to stalled DNA replication forks is controlled by the Mec1-dependent checkpoint and is executed by cooperative functions of Siz1 and Srs2. We propose a mechanism for how defects in these proteins could lead to diverse outcomes (proper repair or GCR formation) through different regulation of DNA repair machinery.
    Molecular and Cellular Biology 03/2006; 26(4):1424-33. · 5.37 Impact Factor
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    ABSTRACT: We describe a 15-month-old girl with acute necrotizing encephalopathy (ANE) associated with HHV-6. Inflammatory cytokines were elevated in the CSF and serum and the number of CD56bright NK cells was significantly increased in the peripheral blood. CD56bright NK cells may be involved in the pathogenesis of ANE by producing inflammatory cytokines.
    Scandinavian Journal of Infectious Diseases 02/2006; 38(11-12):1122-5. · 1.71 Impact Factor
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    ABSTRACT: We examined the role of mouse CD8+ CD122+ T cells, which increase in number with age, in the generalized Shwartzman reaction. This reaction was induced by IL-12 priming and subsequent LPS challenge (after 24 h) in mice of various ages (4-50 weeks of age). Although most young mice (4 or 6 weeks of age) survived, mortality essentially increased with increasing age of the mice, and all mice of 20 weeks of age or older died within 48 h. Serum TNF-alpha levels after LPS challenge also increased age dependently. The neutralization of either IL-12-induced IFN-gamma or LPS-induced TNF-alpha improved the survival of middle-aged (25-week-old) mice. Both IFN-gamma production after IL-12 priming and TNF-alpha production from the liver mononuclear cells after LPS challenge were also prominent in the middle-aged mice. CD8+CD122+ T cells cultured with IL-12 produced a much larger amount of IFN-gamma than CD8+CD122- T cells. Although the depletion of NK/NK T cells did not decrease the IFN-gamma or TNF-alpha production in the Shwartzman reaction of the middle-aged mice, an additional depletion of CD8+CD122+ T cells did decrease such production and also improved mouse survival. Furthermore, young mice transferred with CD8+CD122+ T cells from aged B6 nude mice showed an enhanced Shwartzman reaction.
    European Journal of Immunology 03/2005; 35(2):593-602. · 4.97 Impact Factor

Publication Stats

307 Citations
82.81 Total Impact Points

Institutions

  • 2009–2011
    • Kyoto University
      • Department of Radiation Genetics
      Kioto, Kyōto, Japan
  • 2006–2008
    • National Human Genome Research Institute
      Maryland, United States
  • 2005–2008
    • National Defense Medical College
      • • Division of Microbiology
      • • Department of Pediatrics
      Tokorozawa, Saitama-ken, Japan
  • 2007
    • National Institutes of Health
      Maryland, United States