Updated on 2024/02/15

写真b

 
SUETSUGU Masayuki
 
*Items subject to periodic update by Rikkyo University (The rest are reprinted from information registered on researchmap.)
Affiliation*
College of Science Department of Life Science
Graduate School of Science Doctoral Program in Life Science
Graduate School of Science Master's Program in Life Science
Title*
Professor
Degree
博士(薬学) ( 九州大学 )
Contact information
Mail Address
Research Theme*
  • 「生命とは何か?」というテーマに対し、バクテリアをはじめとした生きている細胞が自己複製する能力に着目し研究を行っている。「蛍光顕微鏡を用いて、生きている細胞内の分子のふるまいを観測する技術」、「精製蛋白質によりゲノムDNAの複製・組換え・修復・転写翻訳などのシステムを再構成する技術」により、生命が「ふえる」さらには「進化する」仕組みを人工的に再現することを目指している。また、そのアプローチの中で得られた様々な生化学的システムを人工細胞をはじめとする次世代のバイオテクノロジーに実装するための研究も進めている。

  • Research Interests
  • biochemistry

  • Molecuar biology

  • 染色体複製

  • 試験管内再構成

  • ゲノム合成

  • 合成生物学

  • Campus Career*
    • 4 2020 - Present 
      College of Science   Department of Life Science   Professor
    • 4 2020 - Present 
      Graduate School of Science   Master's Program in Life Science   Professor
    • 4 2020 - Present 
      Graduate School of Science   Doctoral Program in Life Science   Professor
    • 4 2013 - 3 2020 
      College of Science   Department of Life Science   Associate Professor
    • 4 2013 - 3 2020 
      Graduate School of Science   Master's Program in Life Science   Associate Professor
    • 4 2013 - 3 2020 
      Graduate School of Science   Doctoral Program in Life Science   Associate Professor

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    Research Areas

    • Life Science / Genome biology

    • Life Science / Molecular biology

    Research History

    • 2 2023 - Present 
      Moderna Enzymatics, inc., Director

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    • 4 2020 - Present 
      RIKKYO UNIVERSITY   Graduate School of Science Field of Study: Life Science   Professor

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    • 4 2020 - Present 
      RIKKYO UNIVERSITY   Graduate School of Science Field of Study: Life Science   Professor

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    • 4 2020 - Present 
      RIKKYO UNIVERSITY   College of Science Department of Life Science   Professor

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    • 12 2018 - 1 2023 
      OriCiro Genomics, inc., Co-ounder CSO

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    • 4 2013 - 3 2020 
      RIKKYO UNIVERSITY   Graduate School of Science Field of Study: Life Science   Associate Professor

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    • 4 2013 - 3 2020 
      RIKKYO UNIVERSITY   Graduate School of Science Field of Study: Life Science   Associate Professor

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    • 4 2013 - 3 2020 
      RIKKYO UNIVERSITY   College of Science Department of Life Science   Associate Professor

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    • 12 2011 - 3 2015 
      Japan Science and Technology Agency

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    • 10 2011 - 3 2013 
      Kyushu University   Faculty of Pharmaceutical Sciences

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    • 4 2008 - 9 2011 
      英国ニューカッスル大学   研究員

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    • 1 2003 - 4 2008 
      Kyushu University   Faculty of Pharmaceutical Sciences

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    Education

    • - 12 2002 
      Kyushu University   Graduate School, Division of Pharmaceutical Sciences

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      Country: Japan

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    Awards

    • 8 2021  
      大学発ベンチャー表彰2021 JST理事長賞  OriCiro Genomics
       
      平崎誠司、末次正幸

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    • 10 2020  
      バイオインダストリー協会  第4回「バイオインダストリー奨励賞」  染色体複製サイクル再構成による長鎖環状DNA増幅技術とその応用
       
      末次正幸

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    • 5 2010  
      EMBO Conference on Replication/repair and segregation of chromosome  EMBO poster prize 
       
      Su’etsugu, M, Errington, J

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      Award type:Award from international society, conference, symposium, etc.  Country:Germany

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    Papers

    • Enzymatic Supercoiling of Bacterial Chromosomes Facilitates Genome Manipulation

      Hironobu Fujita, Ayane Osaku, Yuto Sakane, Koki Yoshida, Kayoko Yamada, Seia Nara, Takahito Mukai, Masayuki Su’etsugu

      ACS Synthetic Biology   23 8 2022

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      Publishing type:Research paper (scientific journal)   Publisher:American Chemical Society (ACS)  

      DOI: 10.1021/acssynbio.2c00353

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    • In vitro amplification of whole large plasmids via transposon-mediated oriC insertion

      Seia Nara, Masayuki Su'etsugu

      BioTechniques (Peek behind the paper selected)71 ( 4 ) 528 - 533   10 2021

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      Publishing type:Research paper (scientific journal)   Publisher:Future Science Ltd  

      DOI: 10.2144/btn-2021-0068

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    • Amplification of over 100 kbp DNA from Single Template Molecules in Femtoliter Droplets. International journal

      Hiroshi Ueno, Hiroki Sawada, Naoki Soga, Mio Sano, Seia Nara, Kazuhito V Tabata, Masayuki Su'etsugu, Hiroyuki Noji

      ACS synthetic biology10 ( 9 ) 2179 - 2186   17 9 2021

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      Language:English   Publishing type:Research paper (scientific journal)  

      Reconstitution of the DNA amplification system in microcompartments is the primary step toward artificial cell construction through a bottom-up approach. However, amplification of >100 kbp DNA in micrometer-sized reactors has not yet been achieved. Here, implementing a fully reconstituted replisome of Escherichia coli in micrometer-sized water-in-oil droplets, we developed the in-droplet replication cycle reaction (RCR) system. For a 16 kbp template DNA, the in-droplet RCR system yielded positive RCR signals with a high success rate (82%) for the amplification from single molecule template DNA. The success rate for a 208 kbp template DNA was evidently lower (23%). This study establishes a platform for genome-sized DNA amplification from a single copy of template DNA with the potential to build more complex artificial cell systems comprising a large number of genes.

      DOI: 10.1021/acssynbio.0c00584

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    • Grand scale genome manipulation via chromosome swapping in Escherichia coli programmed by three one megabase chromosomes. International journal

      Tatsuya Yoneji, Hironobu Fujita, Takahito Mukai, Masayuki Su'etsugu

      Nucleic acids research (Breakthrough paper selected)49 ( 15 ) 8407 - 8418   28 4 2021

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      Language:English   Publishing type:Research paper (scientific journal)   Publisher:Oxford University Press ({OUP})  

      In bacterial synthetic biology, whole genome transplantation has been achieved only in mycoplasmas that contain a small genome and are competent for foreign genome uptake. In this study, we developed Escherichia coli strains programmed by three 1-megabase (Mb) chromosomes by splitting the 3-Mb chromosome of a genome-reduced strain. The first split-chromosome retains the original replication origin (oriC) and partitioning (par) system. The second one has an oriC and the par locus from the F plasmid, while the third one has the ori and par locus of the Vibrio tubiashii secondary chromosome. The tripartite-genome cells maintained the rod-shaped form and grew only twice as slowly as their parent, allowing their further genetic engineering. A proportion of these 1-Mb chromosomes were purified as covalently closed supercoiled molecules with a conventional alkaline lysis method and anion exchange columns. Furthermore, the second and third chromosomes could be individually electroporated into competent cells. In contrast, the first split-chromosome was not able to coexist with another chromosome carrying the same origin region. However, it was exchangeable via conjugation between tripartite-genome strains by using different selection markers. We believe that this E. coli-based technology has the potential to greatly accelerate synthetic biology and synthetic genomics.

      DOI: 10.1093/nar/gkab298

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    • Overcoming the Challenges of Megabase-Sized Plasmid Construction in Escherichia coli. International journal

      Takahito Mukai, Tatsuya Yoneji, Kayoko Yamada, Hironobu Fujita, Seia Nara, Masayuki Su'etsugu

      ACS synthetic biology9 ( 6 ) 1315 - 1327   19 6 2020

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      Language:English   Publishing type:Research paper (scientific journal)  

      Although Escherichia coli has been a popular tool for plasmid construction, this bacterium was believed to be "unsuitable" for constructing a large plasmid whose size exceeds 500 kilobases. We assumed that traditional plasmid vectors may lack some regulatory DNA elements required for the stable replication and segregation of such a large plasmid. In addition, the use of a few site-specific recombination systems may facilitate cloning of large DNA segments. Here we show two strategies for constructing 1-megabase (1-Mb) secondary chromosomes by using new bacterial artificial chromosome (BAC) vectors. First, the 3-Mb genome of a genome-reduced E. coli strain was split into two chromosomes (2-Mb and 1-Mb), of which the smaller one has the origin of replication and the partitioning locus of the Vibrio tubiashii secondary chromosome. This chromosome fission method (Flp-POP cloning) works via flippase-mediated excision, which coincides with the reassembly of a split chloramphenicol resistance gene, allowing chloramphenicol selection. Next, we developed a new cloning method (oriT-POP cloning) and a fully equipped BAC vector (pMegaBAC1H) for developing a 1-Mb plasmid. Two 0.5-Mb genomic regions were sequentially transferred from two donor strains to a recipient strain via conjugation and captured by pMegaBAC1H in the recipient strain to produce a 1-Mb plasmid. This 1-Mb plasmid was transmissible to another E. coli strain via conjugation. Furthermore, these 1-Mb secondary chromosomes were amplifiable in vitro by using the reconstituted E. coli chromosome replication cycle reaction (RCR). These strategies and technologies would make popular E. coli cells a productive factory for designer chromosome engineering.

      DOI: 10.1021/acssynbio.0c00008

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    • Cell-Penetrating Peptide-Mediated Transformation of Large Plasmid DNA into Escherichia coli Peer-reviewed

      Islam M.M., Odahara M., Yoshizumi T., Oikawa K., Kimura M., Su'etsugu, M. and Numata K.

      ACS Synth. Biol.8 ( 5 ) 1215 - 1218   4 2019

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      Publishing type:Research paper (scientific journal)   Publisher:American Chemical Society (ACS)  

      DOI: 10.1021/acssynbio.9b00055

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    • Efficient Arrangement of the Replication Fork Trap for In Vitro Propagation of Monomeric Circular DNA in the Chromosome-Replication Cycle Reaction

      Hasebe, T, Narita, K, Hidaka, S, Su'etsugu, M

      Life   9 2018

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    • Bacterial EndoMS/NucS acts as a clamp-mediated mismatch endonuclease to prevent asymmetric accumulation of replication errors

      Takemoto, N, Numata, I, Su'etsugu, M, Miyoshi-Akiyama, T, co, s, ondi

      Nucleic Acids Res.   6 2018

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    • Essentiality of walRK for growth in bacillus subtilis and its role during heat stress

      Hiraku Takada, Yuh Shiwa, Yuta Takino, Natsuki Osaka, Shuhei Ueda, Satoru Watanabe, Taku Chibazakura, Masayuki Su’Etsugu, Ryutaro Utsumi, Hirofumi Yoshikawa

      Microbiology (United Kingdom)164 ( 4 ) 670 - 684   1 4 2018

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      Language:English   Publishing type:Research paper (scientific journal)   Publisher:Microbiology Society  

      WalRK is an essential two-component signal transduction system that plays a central role in coordinating cell wall synthesis and cell growth in Bacillus subtilis. However, the physiological role of WalRK and its essentiality for growth have not been elucidated. We investigated the behaviour of WalRK during heat stress and its essentiality for cell proliferation. We determined that the inactivation of the walHI genes which encode the negative modulator of WalK, resulted in growth defects and eventual cell lysis at high temperatures. Screening of suppressor mutations revealed that the inactivation of LytE, an dl-endopeptidase, restored the growth of the ΔwalHI mutant at high temperatures. Suppressor mutations that reduced heat induction arising from the walRK regulon were also mapped to the walK ORF. Therefore, we hypothesized that overactivation of LytE affects the phenotype of the ΔwalHI mutant. This hypothesis was corroborated by the overexpression of the negative regulator of LytE, IseA and PdaC, which rescued the growth of the ΔwalHI mutant at high temperatures. Elucidating the cause of the temperature sensitivity of the ΔwalHI mutant could explain the essentiality of WalRK. We proved that the constitutive expression of lytE or cwlO using a synthetic promoter uncouples these expressions from WalRK, and renders WalRK nonessential in the pdaC and iseA mutant backgrounds. We propose that the essentiality of WalRK is derived from the coordination of cell wall metabolism with cell growth by regulating dl-endopeptidase activity under various growth conditions.

      DOI: 10.1099/mic.0.000625

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    • Exponential propagation of large circular DNA by reconstitution of a chromosome-replication cycle

      Su'etsugu, M, Takada, H, Katayama, T, Tsujimoto, H

      Nucleic Acids Res.   9 2017

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    • Overall Shapes of the SMC-ScpAB Complex Are Determined by Balance between Constraint and Relaxation of Its Structural Parts

      Katsuhiko Kamada, Masayuki Su'etsugu, Hiraku Takada, Makoto Miyata, Tatsuya Hirano

      STRUCTURE25 ( 4 ) 603 - +   4 2017

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      Language:English   Publishing type:Research paper (scientific journal)   Publisher:CELL PRESS  

      The SMC-ScpAB complex plays a crucial role in chromosome organization and segregation in many bacteria. It is composed of a V-shaped SMC dimer and an ScpAB subcomplex that bridges the two Structural Maintenance of Chromosomes (SMC) head domains. Despite its functional significance, the mechanistic details of SMC-ScpAB remain obscure. Here we provide evidence that ATP-dependent head-head engagement induces a lever movement of the SMC neck region, which might help to separate juxtaposed coiled-coil arms. Binding of the ScpA N-terminal domain (NTD) to the SMC neck region is negatively regulated by the ScpB C-terminal domain. Mutations in the ScpA NTD compromise this regulation and profoundly affect the overall shape of the complex. The SMC hinge domain is structurally relaxed when free from coiled-coil juxtaposition. Taken together, we propose that the structural parts of SMC-ScpAB are subjected to the balance between constraint and relaxation, cooperating to modulate dynamic conformational changes of the whole complex.

      DOI: 10.1016/j.str.2017.02.008

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    • DnaA N-terminal domain interacts with Hda to facilitate replicase clamp-mediated inactivation of DnaA Peer-reviewed

      Su'etsugu, M, Harada, Y, Keyamura, K, Matsunaga, C, Kasho, K, Abe, Y, Ueda, T, Katayama, T

      Environ. Microbiol.   7 2013

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    • The Replicase Sliding Clamp Dynamically Accumulates behind Progressing Replication Forks in Bacillus subtilis Cells Peer-reviewed

      Masayuki Su'etsugu, Jeff Errington

      MOLECULAR CELL41 ( 6 ) 720 - 732   3 2011

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      Language:English   Publishing type:Research paper (scientific journal)   Publisher:CELL PRESS  

      The sliding clamp is an essential component of the replisome required for processivity of DNA synthesis and several other aspects of chromosome metabolism. However, the in vivo dynamics of the clamp are poorly understood. We have used various biochemical and cell biological methods to study the dynamics of clamp association with the replisome in Bacillus subtilis cells. We find that clamps form large assemblies on DNA, called "clamp zones." Loading depends on DnaG primase and is probably driven by Okazaki fragment initiation on the lagging strand. Unloading, which is probably regulated, only occurs after many clamps have accumulated on the DNA. On/off cycling allows chromosomal zones of about 200 accumulated clamps to follow the replisome. Since we also show that clamp zones recruit proteins bearing a clamp-binding sequence to replication foci, the results highlight the clamp as a central organizer in the structure and function of replication foci.

      DOI: 10.1016/j.molcel.2011.02.024

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    • Hda monomerization by ADP binding promotes replicase clamp-mediated DnaA-ATP hydrolysis Peer-reviewed

      Su'etsugu, M, Nakamura, K, Keyamura, K, Kudo, Y, Katayama, T

      J. Biol. Chem.   7 2008

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    • Modes of over-initiation, dnaA gene expression and the inhibition of cell division in a novel cold-sensitive hda mutant in Escherichia coli. Peer-reviewed

      Fujimitsu, K, Su'etsugu, M, Yamaguchi, Y, Mazda, K, Fu, N, Kawakami, H, Katayama, T

      J. Bacteriol.   3 2008

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    • The interaction of DiaA and DnaA regulates the replication cycle in E. coli by directly promoting ATP-DnaA-specific initiation complexes Peer-reviewed

      Kenji Keyamura, Norie Fujikawa, Takuma Ishida, Shogo Ozaki, Masayuki Su'etsugu, Kazuyuki Fujimitsu, Wataru Kagawa, Shigeyuki Yokoyama, Hitoshi Kurumizaka, Tsutomu Katayama

      GENES & DEVELOPMENT21 ( 16 ) 2083 - 2099   8 2007

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      Language:English   Publishing type:Research paper (scientific journal)   Publisher:COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT  

      Escherichia coli DiaA is a DnaA-binding protein that is required for the timely initiation of chromosomal replication during the cell cycle. In this study, we determined the crystal structure of DiaA at 1.8 angstrom resolution. DiaA forms a homotetramer consisting of a symmetrical pair of homodimers. Mutational analysis revealed that the DnaA-binding activity and formation of homotetramers are required for the stimulation of initiation by DiaA. DiaA tetramers can bind multiple DnaA molecules simultaneously. DiaA stimulated the assembly of multiple DnaA molecules on oriC, conformational changes in ATP-DnaA-specific initiation complexes, and unwinding of oriC duplex DNA. The mutant DiaA proteins are defective in these stimulations. DiaA associated also with ADP-DnaA, and stimulated the assembly of inactive ADP-DnaA oriC complexes. Specific residues in the putative phosphosugar-binding motif of DiaA were required for the stimulation of initiation and formation of ATP-DnaA-specific-oriC complexes. Our data indicate that DiaA regulates initiation by a novel mechanism, in which DiaA tetramers most likely bind to multiple DnaA molecules and stimulate the assembly of specific ATP-DnaA-oriC complexes. These results suggest an essential role for DiaA in the promotion of replication initiation in a cell cycle coordinated manner.

      DOI: 10.1101/gad.1561207

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    • The exceptionally tight affinity of DnaA for ATP/ADP requires a unique aspartic acid residue in the AAA+ sensor 1 motif Peer-reviewed

      Kawakami, H, Ozaki, S, Suzuki, S, Nakamura, K, Senriuchi, T, Su'etsugu, M, Fujimitsu, K, Katayama, T

      Mol. Microbiol.   8 2006

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    • An isolated Hda–clamp complex is functional in the regulatory inactivation of DnaA and DNA replication Peer-reviewed

      Kawakami, H, Su'etsugu, M, Katayama, T

      J. Struct. Biol.156   220 - 229   8 2006

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    • Involvement of the Escherichia coli folate-binding protein YgfZ in RNA modification and regulation of chromosomal replication initiation (vol 59, pg 265, 2005) Peer-reviewed

      T Ote, M Hashimoto, Y Ikeuchi, M Su'etsugu, T Suzuki, T Katayama, JI Kato

      MOLECULAR MICROBIOLOGY60 ( 1 ) 252 - 252   4 2006

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      The Escherichia coli hda gene codes for a DnaA-related protein that is essential for the regulatory inactivation of DnaA (RIDA), a system that controls the initiation of chromosomal replication. We have identified the ygfZ gene, which encodes a folate-binding protein, as a suppressor of hda mutations. The ygfZ null mutation suppresses an hda null mutation. The over-initiation and abortive elongation phenotypes conferred by the hda mutations are partially suppressed in an hda ygfZ background. The accumulation of the active form of DnaA, ATP-DnaA, in the hda mutant is suppressed by the disruption of the ygfZ gene, indicating that YgfZ is involved in regulating the level of ATP-DnaA. Although ygfZ is not an essential gene, the ygfZ disruptant grows slowly, especially at low temperature, demonstrating that this gene is important for cellular proliferation. We have identified mnmE (trmE) as a suppressor of the ygfZ disruption. This gene encodes a GTPase involved in tRNA modification. Examination of RNA modification in the ygfZ mutant reveals reduced levels of 2-methylthio N-6-isopentenyladenosine, indicating that YgfZ participates in the methylthio-group formation of this modified nucleoside in some tRNAs. These results suggest that YgfZ is a key factor in regulatory networks that act via tRNA modification.

      DOI: 10.1111/j.1365-2958.2006.05067.x

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    • Involvement of the Escherichia coli folate-binding protein YgfZ in RNA modification and regulation of chromosomal replication initiation Peer-reviewed

      T Ote, M Hashimoto, Y Ikeuchi, M Su'etsugu, T Suzuki, T Katayama, J Kato

      MOLECULAR MICROBIOLOGY59 ( 1 ) 265 - 275   1 2006

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      The Escherichia coli hda gene codes for a DnaA-related protein that is essential for the regulatory inactivation of DnaA (RIDA), a system that controls the initiation of chromosomal replication. We have identified the ygfZ gene, which encodes a folate-binding protein, as a suppressor of hda mutations. The ygfZ null mutation suppresses an hda null mutation. The over-initiation and abortive elongation phenotypes conferred by the hda mutations are partially suppressed in an hda ygfZ background. The accumulation of the active form of DnaA, ATP-DnaA, in the hda mutant is suppressed by the disruption of the ygfZ gene, indicating that YgfZ is involved in regulating the level of ATP-DnaA. Although ygfZ is not an essential gene, the ygfZ disruptant grows slowly, especially at low temperature, demonstrating that this gene is important for cellular proliferation. We have identified mnmE (trmE) as a suppressor of the ygfZ disruption. This gene encodes a GTPase involved in tRNA modification. Examination of RNA modification in the ygfZ mutant reveals reduced levels of 2-methylthio-6-iodeadenosine, indicating that YgfZ participates in the methylthio-group formation of this modified nucleoside in some tRNAs. These results suggest that YgfZ is a key factor in regulatory networks that act via tRNA modification.

      DOI: 10.1111/j.1365-2958.2005.04932.x

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    • Reconstitution of in vitro inactivation and reactivation systems of DnaA protein for the control of chromosomal replication initiation in Escherichia coli

      Masayuki Su'etsugu, Kazuyuki Fujimitsu, Tsutomu Katayama

      2006 IEEE INTERNATIONAL SYMPOSIUM ON MICRO-NANOMECHATRONICS AND HUMAN SCIENCE   334 - +   2006

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      Language:English   Publishing type:Research paper (international conference proceedings)   Publisher:IEEE  

      The Escherichia coli initiator protein, DnaA binds to the replicational origin of the chromosome (oriC) and initiates the replication in a manner depending on ATP ' binding. Replication of mini-chromosome (a plasmid bearing oriC) has been reconstituted in vitro using crude extracts or purified proteins. In these in vitro systems, only ATP-bound form of DnaA is active to initiate a series of initiation reactions which leads to loading of the DNA polymerase III holoenzyme (pol III HE) onto DNA for DNA synthesis. In a manner dependent on Hda protein and DNA loading of pol III HE, ATP bound on DnaA is hydrolyzed. The resultant ADP-bound form of DnaA is inactive for the initiation. This inactivation system called RIDA (regulatory inactivation of DnaA) ensures the replication initiation only once per cell cycle. We here describe recent progress on our system to reconstitute the inactivation of DnaA by RIDA and a novel system to regenerate active DnaA. The reconstitution of the Hda-dependent DnaA-ATP hydrolysis requires the DNA-loaded form of the sliding clamp. This complex is formed by loading of the beta subunit of pol III HE onto DNA during replication process. Hda protein forms a stable complex with the sliding clamp, and most likely has a direct contact to ATP bound on DnaA. The cellular ATP-DnaA level fluctuates during the cell cycle. Regeneration from ADP-DnaA to ATP-DnaA is observed in vivo before the next round of the replication. Recently we have found a specific DNA segment DARS (DnaA-reactivating sequences) that has an activity to release ADP bound on DnaA. ATP-DnaA is regenerated by nucleotide-exchange in the presence of DARS and the elevated concentrations of ATP. The resultant ATP-DnaA was active in the mini-chromosome replication reaction in vitro. Thus, DARS can functionally reactivate ADP-DnaA. The inactivation-reactivation cycle of DnaA might be reconstituted in vitro in a manner controled by RIDA and DARS.

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    • Protein Associations in DnaA-ATP Hydrolysis Mediated by the Replicase Clamp-Hda Complex Peer-reviewed

      Su'etsugu, M, Shimuta, T, Ishida, T, Kawakami, H, Katayama, T

      J. Biol. Chem.280   6528 - 6536   8 2005

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    • Molecular mechanism of DNA replication-coupled inactivation of the initiator protein in Escherichia coli: Interaction of DnaA with the sliding clamp-loaded DNA and the sliding clamp-Hda complex Peer-reviewed

      Su'etsugu, M, Takata, M, Kubota, T, Matsuda, Y, Katayama, T

      Genes Cells9   509 - 522   8 2004

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    • Transcriptional control for initiation of chromosomal replication in Escherichia coli: fluctuation of the level of origin transcription ensures timely initiation Peer-reviewed

      Su'etsugu, M, Emoto, A, Fujimitsu, K, Keyamura, K, Katayama, T

      Genes Cells   8 2003

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    • Determination of the secondary structure in solution of the Escherichia coli DnaA DNA-binding domain Peer-reviewed

      Takayuki Obita, Takafumi Iwura, Masayuki Su'etsugu, Yoichiro Yoshida, Yoshitsugu Tanaka, Tsutomu Katayama, Tadashi Ueda, Taiji Imoto

      Biochemical and Biophysical Research Communications299 ( 1 ) 42 - 48   2002

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      DnaA protein binds specifically to a group of binding sites collectively called as DnaA boxes within the bacterial replication origin to induce local unwinding of duplex DNA. The DNA-binding domain of DnaA, domain IV, comprises the C-terminal 94 amino acid residues of the protein. We overproduced and purified a protein containing only this domain plus a methionine residue. This protein was stable as a monomer and maintained DnaA box-specific binding activity. We then analyzed its solution structure by CD spectrum and heteronuclear multi-dimensional NMR experiments. We established extensive assignments of the 1H, 13C, and 15N nuclei, and revealed by obtaining combined analyses of chemical shift index and NOE connectivities that DnaA domain IV contains six α-helices and no β-sheets, consistent with results of CD analysis. Mutations known to reduce DnaA box-binding activity were specifically located in or near two of the α-helices. These findings indicate that the DNA-binding fold of DnaA domain IV is unique among origin-binding proteins. © 2002 Elsevier Science (USA). All rights reserved.

      DOI: 10.1016/S0006-291X(02)02590-1

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    • DNA replication-coupled inactivation of DnaA protein in vitro: A role for DnaA arginine-334 of the AAA+ Box VIII motif in ATP hydrolysis Peer-reviewed

      Masayuki Su'Etsugu, Hironori Kawakami, Kenji Kurokawa, Toshio Kubota, Makoto Takata, Tsutomu Katayama

      Molecular Microbiology40 ( 2 ) 376 - 386   2001

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      The DnaA protein, which initiates chromosomal replication in Escherichia coli, is negatively regulated by both the sliding clamp of DNA polymerase III holoenzyme and the IdaB protein. We have found that, when the amount of minichromosome is limited in an in vitro replication system, minichromosomal replication-stimulated hydrolysis of DnaA-bound ATP yields the ADP-bound inactive form. The number of sliding clamps formed during replication was at least five per minichromosome, which is 2.7-fold higher than the number formed during incubation without replication. These results support the notion that coupling of DnaA-ATP hydrolysis to DNA replication is the outcome of enhanced clamp formation. We have also found that the amino acid substitution R334H in DnaA severely inhibits the hydrolysis of bound ATP in vitro. Whereas ATP bound to wild-type DnaA is hydrolysed in a DNA-dependent intrinsic manner or in a sliding clamp-dependent manner, ATP bound to DnaA R334H protein was resistant to hydrolysis under the same conditions. This arginine residue may be located in the vicinity where ATP binds, and therefore may play an essential role in ATP hydrolysis. This residue is highly conserved among DnaA homologues and also in the Box VIII motif of the AAA+ protein family.

      DOI: 10.1046/j.1365-2958.2001.02378.x

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    Misc.

    • なぜ今,長鎖DNA合成なのか Invited

      末次正幸

      実験医学(羊土社)41 ( 3 )   1 2023

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    • Synthesis of long chain DNA toward synthetic cells

      末次正幸

      実験医学40 ( 12 )   2022

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    • OriCiro Genomicsのセルフリー長鎖環状DNA合成技術

      末次正幸

      ファルマシア(日本薬学会)57 ( 3 ) 218 - 220   3 2021

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    • Purification, implantation and amplification technologies for mega-sized bacterial chromosome

      末次正幸

      日本分子生物学会年会プログラム・要旨集(Web)44th   2021

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    • ゲノム合成時代のセルフリーDNAクローニング

      末次正幸

      バイオサイエンスとインダストリー78 ( 6 ) 550 - 551   11 2020

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    • 大腸菌染色体複製サイクルの試験管内再構成と合成生物学

      末次正幸

      「生物物理」誌60 ( 5 ) 284 - 287   10 2020

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    • クローズアップ実験法(series 321) 大腸菌いらずのセルフリー長鎖DNAクローニング Invited

      奈良 聖亜, 末次 正幸

      実験医学38 ( 6 ) 1023 - 1028   4 2020

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      Authorship:Last author, Corresponding author   Language:Japanese   Publisher:(株)羊土社  

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    • 大腸菌染色体複製サイクル再構成系を基盤とした自己複製システムの構築

      奈良聖亜, 長谷部友憲, 高田啓, 末次正幸

      日本ゲノム微生物学会年会要旨集14th   2020

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    • 大腸菌ゲノム複製サイクル再構成系とその自己複製

      末次正幸, 長谷部友憲, 高田啓

      日本遺伝学会大会プログラム・予稿集91st   2019

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    • 染色体複製サイクルの自律的な繰り返しによるDNA増幅反応

      末次正幸

      化学と生物   1 2019

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      Language:Japanese   Publishing type:Article, review, commentary, editorial, etc. (scientific journal)  

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    • 細胞を使わないゲノム合成技術

      末次正幸

      月刊バイオインダストリー 8月号   8 2018

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      Language:Japanese   Publishing type:Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

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    • ゲノム合成技術、特集「生命をつくる」

      末次正幸

      現代化学1月号   1 2018

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      Language:Japanese   Publishing type:Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

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    • ゲノム複製サイクル再構成系を利用した新規DNA増幅技術

      末次正幸, 加納巧希, 倉田竜明, 篠原赳, 高田啓

      日本生化学会大会(Web)90th   2017

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    • バクテリアSMC複合体のリング開閉機構

      鎌田勝彦, 末次正幸, 高田啓, 宮田真人, 平野達也

      日本ゲノム微生物学会年会要旨集11th   2017

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    • ゲノム複製サイクル試験管内再構成系における変異誘発と分子進化

      末次正幸, 徳永翼, 高田啓, 辻本寛子

      日本進化学会大会プログラム・講演要旨集(Web)18th   2016

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    • 大腸菌染色体複製サイクルと転写翻訳反応との統合再構成

      末次正幸, 辻本寛子, 高田啓

      日本生化学会大会(Web)89th   2016

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    • 大腸菌ミニ染色体複製サイクルと転写・翻訳反応の再構成の試み

      高田啓, 辻本寛子, 末次正幸

      日本農芸化学会大会講演要旨集(Web)2016   2016

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    • 枯草菌二成分制御系Wa1RKの生育必須性の解析

      高田啓, 高田啓, 植田修平, 志波優, 志波優, 渡辺智, 千葉櫻拓, 末次正幸, 内海龍太郎, 吉川博文

      日本農芸化学会関東支部講演要旨集2016 ( Oct )   2016

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    • 大腸菌の増殖に必須な長鎖逆方向反復配列ELIXIR1の機能解析

      土田愛海, 川上広宣, 加生和寿, 末次正幸, 末次正幸, 片山勉

      日本分子生物学会年会プログラム・要旨集(Web)37th   2014

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    • Characterization of the E. coli mini-chromosome replication reactions reconstituted with the crude system and the purified system

      Masayuki Su'etsugu, Tsutomu Katayama

      GENES & GENETIC SYSTEMS87 ( 6 ) 390 - 390   12 2012

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      Language:English   Publishing type:Research paper, summary (international conference)   Publisher:GENETICS SOC JAPAN  

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    • DnaA制御サイクルの再構築による解明:新たなDnaA-ATP加水分解経路と核様体構築因子の役割

      片山勉, 加生和寿, 藤光和之, 藤光和之, 尾崎省吾, 尾崎省吾, 野口泰徳, 宮崎恵理加, 増田圭美, 末次正幸, 川上広宣

      日本生化学会大会(Web)85th   3S06 - 2   2012

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      Language:Japanese   Publisher:(公社)日本生化学会  

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    • DNA複製装置スライディングクランプの枯草菌の細胞内における動的な集合

      末次正幸

      新着論文レビュー   4 2011

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      Language:Japanese   Publishing type:Article, review, commentary, editorial, etc. (other)   Publisher:FIRST AUTHOR’S  

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    • Regulatory mechanism for replicational initiation by DiaA, a DnaA-binding factor

      Tsutomu Katayama, Kenji Keyamura, Norie Fujikawa, Takuma Ishida, Shogo Ozaki, Masayuki Suetsugu, Kazuyuki Fujimitsu, Wataru Kagawa, Shigeyuki Yokoyama, Hitoshi Kurumizaka

      GENES & GENETIC SYSTEMS82 ( 6 ) 508 - 508   12 2007

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      Language:English   Publishing type:Research paper, summary (international conference)   Publisher:GENETICS SOC JAPAN  

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    • Functional replacement of the AAA+ sensor 1 motif in DnaA, the initiator of chromosomal replication in E-coli

      Shogo Ozaki, Hironori Kawakami, Shigeo Suzuki, Kenta Nakamura, Takayuki Senriuchi, Masayuki Su'etsugu, Kazuyuki Fujimitsu, Tsutomu Katayama

      GENES & GENETIC SYSTEMS81 ( 6 ) 407 - 407   12 2006

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      Language:English   Publishing type:Research paper, summary (international conference)   Publisher:GENETICS SOC JAPAN  

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    Books and Other Publications

    • ゲノム複製サイクル再構成系とその展望、 「人工細胞の創製とその応用」(植田充美 監修)、シーエムシー出版、172-180頁

      末次 正幸( Role: Sole author)

      シーエムシー出版  31 1 2017 

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      Language:Japanese Book type:Scholarly book

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    • Chromosome replication of E.coli and B.subtilis. Escherichia coli and Bacillus subtilis; the frontiers of molecular microbiology revisited

      Katayama, T, Su'etsugu, M( Role: Joint author)

      Research Signpost  8 2012 

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      Language:English Book type:Textbook, survey, introduction

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    • 「大腸菌ゲノムDNA複製系」、『ゲノミクス・プロテオミクスの新展開−生物情報の解析と応用−』(今中忠行監修)

      片山 勉, 末次正幸, 川上広宣( Role: Joint author)

      エヌ・ティー・エス  8 2004 

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      Language:Japanese Book type:Scholarly book

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    • 「大腸菌ゲノムDNA複製系」、『ゲノミクス・プロテオミクスの新展開?生物情報の解析と応用?』(今中忠行監修)

      片山 勉, 末次正幸, 川上広宣( Role: Joint author)

      エヌ・ティー・エス  8 2004 

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      Language:Japanese Book type:Scholarly book

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    Presentations

    • Reconstituted Replication Cycle System of Escherichia coli Genome for Cell Synthesis Invited

      Masayuki Su'etsugu

      Korea-Japan Bottom-up Synthetic Biology Workshop @ELSI  6 9 2023 

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    • mRNA 医薬に貢献するセルフリー長鎖DNA合成技術 Invited

      末次正幸

      第14回日本RNAi研究会 @広島  1 9 2023 

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    • 染色体複製サイクル再構成系の技術展開

      末次正幸

      理研シンポジウム:Synthetic Biology -生物学の新たな潮流-  2 8 2023 

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    • Rapid production of plasmid DNA without relying on Escherichia coli Invited

      Masayuki Su’etsugu

      日本核酸医薬学会第8回年会 @名古屋大学  11 7 2023 

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    • OriCiro History Invited

      末次正幸

      日本ベンチャーキャピタル協会(JVCA) 産学連携ウェビナー  5 7 2023 

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    • セルフリー長鎖DNA合成技術の先端医薬への実装 Invited

      末次正幸

      東大駒場リサーチキャンパス公開シンポジウム「mRNA医薬を知る」  9 6 2023 

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    • スタートアップ起業からモデルナへ売却までのディープな4年間 Invited

      末次正幸

      東京薬科大学セミナー  8 5 2023 

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    • セルフリーDNA合成技術のディープな話 Invited

      末次正幸

      京都産業大学SPRING講演会  20 4 2023 

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    • 染色体複製サイクル再構成系と長鎖DNA合成 Invited

      末次正幸

      第4回 mRNA薬検討会  11 4 2023 

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    • ゲノム合成の技術進展 Invited

      末次 正幸

      第96回日本細菌学会総会(シンポジウム)  17 3 2023 

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    • OriCiro technology from academia research Invited

      末次正幸

      理研IMS 社会実装セミナー  24 2 2023 

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    • 染色体複製サイクル再構成系とゲノム合成 Invited

      末次正幸

      大阪公立大セミナー  12 1 2023 

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    • In vitro統合再構成で紐解く細菌ゲノムの自己複製原理 Invited

      末次正幸

      第45回日本分子生物学会年会(ワークショップ)  1 12 2022 

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    • 様々な産業に変革をもたらしていく最先端の合成生物学の潮流 Invited

      末次 正幸

      ケミカルマテリアルJapan2022  23 10 2022 

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    • In vitro 再構成で明らかになってきた複製・転写・翻訳の協調的進行 Invited

      末次正幸

      日本遺伝学会 第 94 回大会(ワークショップ原核生物の細胞増殖研究から見えてくる遺伝学の新たな課題)  14 9 2022 

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      Presentation type:Oral presentation (invited, special)  

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    • メガサイズ染色体のバクテリア細胞への出し入れおよび増幅の技術 Invited

      末次正幸

      第44回日本分子生物学会年会ワークショップ「細胞核を造る」  3 12 2021 

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      Presentation type:Oral presentation (invited, special)  

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    • なぜ今、合成生物学なのか Invited

      末次正幸

      日本科学哲学会第54回大会 特別講演  27 11 2021 

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    • 大腸菌のメガサイズゲノムの合成と移植の技術 Invited

      末次正幸

      第73回 日本生物工学会大会 シンポジウム  27 10 2021 

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    • 人工ゲノムのセルフリー合成 Invited

      末次 正幸

      バイオインダストリー協会 発酵と代謝研究会 第1回勉強会「ゲノム合成」の秘めた可能性について  21 7 2021 

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    • メガスケールのDNA合成技術とその応用 Invited

      末次正幸

      第2回 東京理科大学総合研究院合成生物学研究部門シンポジウム  19 2 2021 

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    • In vitro amplification of mega-sized circular DNA and its applications Invited

      Masayuki Su'etsugu

      I2BCParisSaclay virtual seminar  22 1 2021 

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    • DNA連結・増幅反応を用いたコンビナトリアル遺伝子ライブラリー作成 Invited

      末次正幸

      遺伝研研究会「微生物における大規模ゲノム・代謝改変技術とその利用」、遺伝研  11 2019 

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    • Cell-free assembly and amplification technology for genome-scale large DNA

      Su'etsugu, M

      The 2nd Asian Synthetic Biology Association, Chengdu, China  10 2019 

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    • Cell-free DNA technology for synthetic biology

      Su'etsugu, M

      SynbioBeta 2019, San Francisco, USA  10 2019 

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    • 大腸菌ゲノム複製サイクル再構成系とその自己複製 Invited

      末次 正幸, 長谷部 友憲, 高田 啓

      日本遺伝学会第91回大会、福島  9 2019 

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    • Cell-free assembly and amplification of genome-scale DNA

      Su'etsugu, M

      Marine Biotechnology Conference, Shimizu, Japan  9 2019 

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    • Cell-free assembly and amplification of genome-scale DNA

      Su'etsugu, M

      2019 Synthetic Biology: Engineering, Evolution & Design (SEED), New York, USA  6 2019 

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    • ゲノム合成時代のセルフリー技術 Invited

      末次 正幸, 奈良 聖亜, 倉田竜明

      日本農芸化学会 2019大会シンポジウム「ゲノム合成の世界的なうねりと現状:日本の貢献」、東京農業大学  3 2019 

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    • 染色体複製サイクルを再構成した高性能DNA増幅技術とその利用

      末次 正幸

      野地ImPACT公開シンポジウム、東京大学  3 2019 

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    • ゲノム合成の新技術

      末次 正幸

      Nano tech 2019、東京ビックサイト  1 2019 

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    • In vitro repetition of chromosome-replication cycle and its applications Invited

      Su'etsugu, M

      7th Bioscience;Biotechnology International;Symposium, Tokyo;Institute of Technology  1 2019 

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    • 長鎖DNA増幅反応RCRと共役した遺伝子編集法

      俵木彩子, 末次 正幸

      第41回日本分子生物学会年会  30 11 2018 

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    • Large plasmid synthesis using cell-free chromosome replication technology Invited International conference

      Su'etsugu, M, Kurata, T

      The 1st Asian Synthetic Biology Association  24 11 2018 

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    • Stable amplification of 200 kb circular DNA by reconstituted replication cycle of the Escherichia coli chromosome International conference

      Shinohara T, Su’etsugu M

      11th 3R+3C Symposium  12 11 2018 

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    • 無細胞長鎖DNA合成法RA-RCRによる50種以上のDNA断片の同時集積

      倉田竜明, 末次 正幸

      「細胞を創る」研究会11.0  18 10 2018 

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    • oriCトランスポゾンを利用した環状DNA の試験管内複製サイクル増幅

      奈良 聖亜, 末次 正幸

      「細胞を創る」研究会11.0  18 10 2018 

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    • 複製サイクル再構成系を利用した長鎖DNAへの塩基置換導入法

      俵木彩子, 末次 正幸

      「細胞を創る」研究会11.0  18 10 2018 

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    • 染色体複製サイクル再構成系を用いたセルフリー環状DNA合成 Invited

      末次正幸

      第56回日本生物物理学会年会 シンポジウム「ゲノム合成時代の人工細胞研究」  15 9 2018 

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    • 50種類以上に及ぶDNA断片の試験管内同時集積

      倉田竜明, 加納巧希, 末次 正幸

      第15回 21世紀大腸菌研究会  24 5 2018 

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    • oriCトランスポゾン転移に依存した長大な異種環状DNA の試験管内増幅

      奈良 聖亜, 篠原 赳, 末次 正幸

      第15回 21世紀大腸菌研究会  24 5 2018 

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    • 放線菌で見つかった新規ミスマッチ修復タンパク質EndoMSのエンドヌクレアーゼ活性

      沼田 格, 竹本 訓彦, 秋山 徹, 末次 正幸

      第15回 21世紀大腸菌研究会  24 5 2018 

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    • 複製停止配列ter を介したフォークの衝突は続く増幅反応を阻害する

      長谷部 友憲, 末次 正幸

      第15回 21世紀大腸菌研究会  24 5 2018 

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    • In vitro amplification of circular DNA mediated by oriC transposon International conference

      Nara, S, Su'etsugu, M

      International Symposium on “Artificial Cell Reactor Science and Technology”  6 4 2018 

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    • Enzymatic construction of large circular DNA from over 50 overlapping fragments International conference

      Kurata, T, Su'etsugu, M

      International Symposium on “Artificial Cell Reactor Science and Technology”  6 4 2018 

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    • Cell-free tool towards de novo genome synthesis Invited International conference

      Su'etsugu, M

      International Symposium on “Artificial Cell Reactor Science and Technology”  6 4 2018 

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    • ゲノム複製サイクル再構成による巨大環状DNA の試験管内増幅 Invited

      末次 正幸

      シンポジウム 「核酸の科学: その複製,化学修飾から損傷まで」  2 2018 

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    • PCRを凌駕!細胞を使わない次世代ゲノム合成技術 Invited

      末次 正幸

      第30回つくばライフサイエンス推進協議会ピッチ会  1 2018 

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    • 細胞を使わない次世代ゲノム合成ツール Invited

      末次正幸

      JST新技術説明会  1 2018 

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    • ゲノム複製サイクル再構成系を利用した新規DNA増幅技術 Invited

      末次正幸, 加納巧希, 倉田竜明, 篠原赳, 高田啓

      ConBio2017  12 2017 

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    • 人工ゲノム合成に向けた無細胞技術 Invited

      末次正幸

      第4回野村集団微生物制御プロジェクトERATOイノベーションセミナー  11 2017 

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    • DNA集積・増幅反応からなる無細胞ゲノム合成技術 Invited

      末次正幸, 倉田竜明

      「細胞を創る」研究会10.0「ゲノム合成時代の到来とバイオセキュリティ・セーフティ」  10 2017 

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    • 染色体複製サイクルの繰り返しによる環状DNAの試験管内自律増殖 Invited

      末次正幸, 高田 啓

      第55回日本生物物理学会年会  9 2017 

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    • Cell-free propagation of large circular DNA by reconstitution of a chromosome replication-cycle International conference

      Su'etsugu, M, Tsujimoto, H

      Gordon Research Conference "Synthetic Biology"  8 2017 

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    • 複製サイクル再構成系における転写・翻訳の共役と分子進化の試み

      高田 啓, 末次 正幸

      第14回 21世紀大腸菌研究会  6 2017 

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      Language:Japanese   Presentation type:Oral presentation (general)  

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    • ゲノム複製サイクル再構成による長鎖環状DNAの試験管内増幅技術 Invited

      末次正幸

      酵素工学研究会第77回講演会  4 2017 

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    • 10万塩基を超える長鎖環状DNAの無細胞クローニング Invited

      末次 正幸, 平田 稜, 倉田竜明, 篠原 赳, 辻本 寛子

      第11回ゲノム微生物学会年会シンポジウム「微生物での合成生物工学」  3 2017 

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    • 大腸菌染色体複製サイクルと転写翻訳反応との統合再構成 Invited

      末次 正幸, 辻本 寛子, 高田 啓

      第89回日本生化学会大会シンポジウム「生化学の基盤戦略:試験管内再構成」  9 2016 

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    • ゲノム複製サイクル試験管内再構成系における変異誘発と分子進化 Invited

      末次正幸, 徳永翼, 高田啓, 辻本寛子

      日本進化学会第18回大会ワークショップ「再構築型進化学研究-人工細胞から原始生物まで-」  8 2016 

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    • ゲノム複製の試験管内再構成系とその合成生物学的展開 Invited

      末次正幸

      第13回原子・分子・光科学(AMO)討論会  6 2016 

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    • 複製サイクル再構成によるミニ染色体の指数増殖 Invited

      末次正幸

      生命動態システム科学四拠点合同シンポジウム「生命動態の分子メカニズムと数理」  3 2016 

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    • 大腸菌染色体複製サイクルの試験管内再構成 Invited International conference

      末次正幸

      第89回日本細菌学会総会ワークショップ「バクテリア細胞増殖プロセス研究の最前線」  3 2016 

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    • 複製開始・終結・分離サイクルの統合再構成系における環状染色体のふるまい

      末次正幸, 辻本寛子

      第23回DNA複製・組換え修復ワークショップ  10 2015 

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    • 大腸菌における細胞周期依存の転写活性変動のリアルタイム観測系を用いた解析

      松本健佑, 末次正幸

      第37回分子生物学会年会  12 2014 

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    • 大腸菌遺伝子転写の細胞周期制御 Invited

      末次正幸, 松本健佑, 小林寛子

      「細胞を創る」研究会7.0  11 2014 

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    • バクテリア染色体複製のリズム Invited

      末次正幸

      生物リズム若手研究者の集い  8 2013 

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    • 試験管内再構成系における大腸菌ミニ染色体複製の開始—伸長—終結サイクル

      末次正幸, 片山勉

      第85回日本生化学会大会  12 2012 

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    • 試験管内再構成系における大腸菌ミニ染色体複製の開始?伸長?終結サイクル

      末次正幸, 片山勉

      第85回日本生化学会大会  12 2012 

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    • 大腸菌ミニ染色体複製の試験管内再構成における粗画分系と精製系の解析

      末次正幸, 片山勉

      日本遺伝学会第84回大会  9 2012 

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      Language:Japanese   Presentation type:Symposium, workshop panel (public)  

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    • 枯草菌染色体複製とその開始制御におけるDnaNクランプの役割

      末次正幸, Errington, J

      第24回微生物シンポジウム  9 2012 

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    • 枯草菌DnaNクランプの染色体複製開始制御における役割

      末次正幸, Errington, J

      2012年度グラム陽性菌ゲノム機能会議  8 2012 

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    • バクテリアゲノム複製蛋白質クランプのin vivo生化学

      末次正幸, Errington, J

      第6回日本ゲノム微生物学会年会  3 2012 

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    • Dynamic behavior of the replicase sliding clamp in Bacillus subtilis

      Su’etsugu, M, Errington, J

      第34回日本分子生物学会年会  12 2011 

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    • 枯草菌染色体の再複製開始制御におけるクランプの役割

      末次正幸, Errington, J

      第21回DNA複製・組換え・ゲノム安定性制御ワークショップ  10 2011 

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    • 枯草菌スライディングクランプの細胞内動態

      末次正幸, Errington, J

      第8回21世紀大腸菌研究会  5 2011 

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    • Subcellular and chromosomal distribution of the DnaN clamp of Bacillus subtilis during the replication cycle International conference

      Su’etsugu, M, Errington, J

      EMBO Conference on Replication/repair and segregation of chromosome  5 2010 

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    Professional Memberships

    Research Projects

    • 免疫系ヒト化動物を活用した抗感染症ヒト抗体創成基盤の確立

      AMED  AMED-CREST 

      冨塚一磨, 香月康宏, 秋田英万, 末次 正幸

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      10 2021 - 3 2027

      Authorship:Coinvestigator(s) 

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    • Roles and their molecular mechanisms of replication barrier zones programmed on chromosome for genome stability maintenance

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

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      4 2021 - 3 2024

      Grant number:21K06120

      Grant amount:\4160000 ( Direct Cost: \3200000 、 Indirect Cost:\960000 )

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    • 人工ゲノムのセルフリーOn chip合成とその起動

      文部科学省  JST CREST 「ゲノム合成」 

      末次正幸

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      10 2018 - 3 2024

      Grant type:Competitive

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    • 薬剤耐性菌を殺菌する広宿主域バイオロジクスの開発

      AMED  新興・再興感染症研究基盤創生事業(多分野融合研究領域) 

      氣駕 恒太朗, 鈴木 仁人, 新谷 政己, 末次 正幸

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      12 2020 - 3 2023

      Authorship:Coinvestigator(s) 

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    • 染色体複製サイクル再構成系の高度化で探るゲノム機能

      日本学術振興会  科学研究費助成事業 基盤研究(B) 

      末次 正幸

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      4 2020 - 3 2023

      Grant number:20H03238

      Grant amount:\17680000 ( Direct Cost: \13600000 、 Indirect Cost:\4080000 )

      大腸菌染色体複製のサイクルを再構成した複製サイクル再構成系では、ゲノムサイズの環状DNA分子の指数的な増幅が達成される。大腸菌をモデルとした研究では、複製系以外にも転写・翻訳、DNA修復、組換え、複製周期制御など、ゲノムにまつわる多くのシステムが試験管内再構成されている。本研究ではこれらシステムを複製サイクル再構成系に融合し、ゲノム動態の統合的な再現を目指す。システムだけでなくゲノム(染色体)自体を試験管内に取り出しての再現も進める。試験管内に高度に再現されたゲノム動態に対し、従来細胞を対象に用いられてきたオミックス的解析手法を適用し、新しいアプローチでゲノム機能に迫る。
      初年度となる当該年度では、染色体複製サイクル再構成系に転写再構成系を融合し、同じミニ染色体上で、複製反応、転写反応が相互に及ぼす影響について解析を進めた。従来までにT7ファージのRNAポリメラーゼを用いた転写反応を組み合わせていたが、実際の細胞内の反応を再現すべく大腸菌RNAポリメラーゼを用いた転写反応との融合を行った。この複製・転写融合系において、複製と転写が効率よく共役して進行する条件を検討し、その共役に必要ないくつかの因子を同定した。そして、これらの因子の効果についてリアルタイムPCRをもちいた定量的なデータも得た。
      さらに、染色体自体を試験管内に取り出して生化学的解析に用いる実験に対しては、染色体を大腸菌からスーパーコイルDNAとして壊さずに精製する技術を構築した。通常の大腸菌ゲノムは4.6Mbサイズで壊さずに精製することが難しいが、我々は染色体を分断する技術を独自に開発し、得られた1Mb分断染色体であればスーパーコイルDNAとして精製可能であることを見出した。

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    • 先端的順逆遺伝学手法を用いたSARS-CoV-2の伝播機構解明

      国立研究開発法人日本医療研究開発機構  新興・再興感染症研究基盤創生事業 

      末次正幸

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      9 2020 - 3 2022

      Authorship:Coinvestigator(s) 

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    • Mismatch repair system in prokaryotes lacking canonical mutS-dependent system

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

      Takemoto Norihiko

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      4 2018 - 3 2021

      Grant number:18K06190

      Grant type:Competitive

      Grant amount:\4290000 ( Direct Cost: \3300000 、 Indirect Cost:\990000 )

      MutS-dependent mismatch repair system have been known to be conserved from bacteria to human, as a replication error correction system. Disruption of MMR system results in increased mutation rate, leading to high occurrence of cancer cells or drug-resistant pathogens. However, recent advances in Next Generation Sequencing technologies shed light on the existence of exceptions, in which mutation rate is not so high regardless of the lack of canonical MMR system. In this study, we searched for conserved gene which works to decrease mutations in prokaryotes lacking canonical MMR system. We found that EndoMS works for replication error correction.

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    • In vitro reconstitution of genome self-replication system and its evolution

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

      SU'ETSUGU Masayuki

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      7 2016 - 3 2020

      Grant number:16KT0076

      Authorship:Principal investigator  Grant type:Competitive

      Grant amount:\18720000 ( Direct Cost: \14400000 、 Indirect Cost:\4320000 )

      Self-replication is a fundamental feature of living cells. Cells produce their own copy based on their genetic information. We recently developed an in vitro reconstitution system termed RCR (replication cycle reaction), in which replication cycle of the Escherichia coli chromosome repeats continuously to propagate a mini-chromosome that is a circular DNA having replication origin, oriC. RCR consists of 26 proteins .
      In this study, we combined an in vitro transcription-translation reconstitution system, PURE system with RCR (PURE-coupled RCR). All 26 RCR proteins were able to express functionally from DNA in PURE system. We constructed a mini-chromosome encoding the RCR proteins. This mini-chromosome allow us to propagate the circular DNA depending on the transcription-translation from its own DNA. We further developed a system in which the PURE-coupled RCR progress from a single mini-chromosome molecule in water-in-oil emulsion for purpose of in vitro evolution of the mini-chromosome.

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    • 人工ゲノムの試験管内合成法の開発

      その他省庁等  内閣府ImPACT 野地プログラム「人工細胞リアクタ」 

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      4 2016 - 3 2019

      Grant type:Competitive

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    • Propagation of large circular DNA by reconstitution of a chromosome-replication cycle

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

      Su'etsugu Masayuki

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      4 2014 - 3 2017

      Grant number:26640116

      Authorship:Principal investigator  Grant type:Competitive

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

      Escherichia coli has a 4.6 Mb circular chromosome with a replication origin, oriC. While the oriC replication has been reconstituted in vitro more than 30 years ago, continuous repetition of the replication cycle has not yet been achieved. Here, we reconstituted the entire replication cycle with purified enzymes that catalyze initiation at oriC, bidirectional fork progression, Okazaki-fragment maturation, and decatenation of the replicated circular products. Because decatenation provides covalently closed supercoiled monomers that are competent for the next round of replication initiation, the replication cycle repeats autonomously and continuously in an isothermal condition. This Replication-Cycle Reaction (RCR) propagates large circular DNA up to 0.2 Mb as intact covalently closed molecules. RCR provides a powerful in vitro tool to propagate large circular DNA molecules

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    • 染色体複製系の周期的駆動にむけた回路の再構成

      文部科学省  JST さきがけ 「細胞構成」 

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      12 2011 - 3 2015

      Grant type:Competitive

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    • 枯草菌細胞内への細胞周期回路の人工合成

      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) 

      末次 正幸

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      4 2012 - 3 2014

      Grant number:24119513

      Grant amount:\14040000 ( Direct Cost: \10800000 、 Indirect Cost:\3240000 )

      大腸菌染色体複製開始蛋白質DnaAは、活性型と不活性型の割合が細胞周期を通じて変動する。このDnaA活性オシレーションは複製開始のタイミングを保証するだけでなく、遺伝子転写制御にも機能している。本研究では、枯草菌という異種細胞内への合成的なアプローチによって、大腸菌DnaA活性の周期動態を再現し、その作動原理を理解することを目指して検討を行った。DnaA活性オシレーションにおいては、複製装置因子クランプのDNA上への装着・解離の動態が重要である。複製とともにDNA上には多数のクランプ分子が蓄積する。このDNA装着型クランプに依存してDnaAの不活性化反応が導かれる。複製終結後、クランプがDNAから解離すると、この不活性化は解除される。我々はまず、大腸菌クランプのDNA上への装着・解離の動態を枯草菌細胞内において再現することができるか蛍光顕微鏡を用い、検討した。枯草菌内に発現させた大腸菌クランプについて、低効率ながらもうまくその染色体DNA上への装着を導く事が可能であった。このDNA装着は枯草菌の染色体複製周期に依存しており、複製終結後にクランプがDNAから解離して、細胞全体に拡散する様子も捉えることに成功した。続いて大腸菌DnaAについても枯草菌への導入を検討した。これは細胞増殖阻害を導くものであったが、うまく発現量を抑える事で、その導入が可能であった。また、導入されたDnaAについて枯草菌核様体上への局在化が見られた。次に我々は、細胞内におけるDnaAの活性レベルを検出する系の開発を進めた。DnaAの転写制御活性を利用すれば、その活性変動を蛍光によりリアルタイム検出できると考え、独自のDnaA ChIP-seq解析の結果も踏まえ、検出系を構築した。そして、大腸菌をもちいた検討により、この系が細胞内DnaAの活性変動を検出する上で有用である事を示す事ができた。

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    • バクテリア複製蛋白質の細胞内ダイナミクス

      民間財団等  上原記念生命科学財団 海外留学リサーチフェローシップ 

      末次正幸

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      4 2010 - 3 2011

      Authorship:Principal investigator  Grant type:Competitive

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    • バクテリアにおける複製ファクトリー形成の分子機構の解明

      文部科学省  日本学術振興会 海外特別研究員 

      末次正幸

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      4 2008 - 3 2010

      Authorship:Principal investigator  Grant type:Competitive

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    • DnaA複製開始活性を複製周期と共役して抑制する因子Hdaの機能制御メカニズム

      日本学術振興会  科学研究費助成事業 

      末次正幸

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      4 2006 - 3 2008

      Authorship:Principal investigator  Grant type:Competitive

      DnaA複製開始活性を複製周期と共役して抑制する因子Hdaの機能制御メカニズムについて調べる

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    • 開始のオン・オフにより周期的に複製を行う大腸菌ミニ染色体複製サイクルの再構成

      文部科学省  科学研究費助成事業 

      末次正幸

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      4 2006 - 3 2008

      Authorship:Principal investigator  Grant type:Competitive

      開始のオン・オフにより周期的に複製を行う大腸菌ミニ染色体複製サイクルの再構成系の構築を行う

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    • 細胞周期に依存した複製開始因子不活化システムの発動を制御する蛋白質間相互作用

      日本学術振興会  科学研究費助成事業 若手研究(B) 

      末次 正幸

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      2008 - 2008

      Grant number:20770138

      Authorship:Principal investigator  Grant type:Competitive

      Grant amount:\3120000 ( Direct Cost: \2400000 、 Indirect Cost:\720000 )

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    • 開始のオン・オフにより周期的に複製を行う大腸菌ミニ染色体複製サイクルの再構成

      日本学術振興会  科学研究費助成事業 特定領域研究 

      末次 正幸, 片山 勉

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      2006 - 2007

      Grant number:18048033

      Grant amount:\3300000 ( Direct Cost: \3300000 )

      染色体複製の開始は細胞周期進行と連動して周期的に制御されている。大腸菌では、染色体複製起点を持つプラスミドDNAを鋳型とし、精製蛋白質を用いて染色体複製を再構成できる(ミニ染色体複製系)。複製開始反応は開始蛋白質DnaAのATP結合型(活性型)とADP結合型(不活性型)の変換によって制御されている。ATP-DnaAはRIDAという複製伸長反応と共役したDnaA結合性ATP加水分解機構によってADP-DnaAに変換される(不活性化システム)。またin vitroにおいては、特異的なDNA配列DARSによって、DnaA結合性ADPが解離し、ATP再結合が促されることが見いだされている(再活性化システム)。本研究では、開始のオン・オフによりコントロールされた複製サイクル系の再構成を目指すべく、不活性化システムの試験管内再構成系を確立した。この再構成系にはクランプと呼ばれるDNAポリメラーゼIII複合体のサブユニットとHdaタンパク質が必要である。不活性化システム再構成系を用いた解析から、ヌクレオチド結合によって促されるHdaの多量体構造の変換が、不活性化システムの発動に必要であることを見いだした。このHdaヌクレオチド結合は細胞内においてもDnaA活性制御に重要であった。さらに我々は、DARSが細胞内において再活性化に重要な機能を果たしていることを示し、DARS活性を調節する新規活性を見いだした。これらの結果は複製開始のオン・オフを制御する分子メカニズムを示唆するものであり、その周期性を再構成する上で重要な知見を提供するものである。

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    • DnaA複製開始活性を複製周期と共役して抑制する因子Hdaの機能制御メカニズム

      日本学術振興会  科学研究費助成事業 若手研究(B) 

      末次 正幸

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      2006 - 2007

      Grant number:18770155

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

      染色体の複製開始反応は細胞周期中ただ1回のみ起こるよう厳密に制御されている。大腸菌DnaAはATP結合型で複製開始を引き起こす。DnaAに結合したATPは、DnaA不活性化因子HdaとDNA装着型クランプ(DNAポリメラーゼIIIβサブユニット)とに依存して加水分解され、不活性なADP結合型DnaAが産生する。このDnaA制御システムはRIDAと呼ばれ、重複複製を抑制するために必須である。本研究ではまず、hda翻訳がこれまで推測されていた開始コドン(GUG)よりも15コドン下流に位置するCUGから開始していることを見いだした。大腸菌において翻訳効率が著しく低いCUGを開始コドンとして利用している遺伝子はこれが初めてであり、Hdaの細胞内量が氏いことと関連していると思われる。同定した開始コドンより翻訳したHdaは高活性体として精製された。さらにdaは、ADP特異的に結合能を示し、ATPや他のヌクレオチドとは結合しないというユニークな性質を持っていた。ADP結合型Hdaは単量体として存在し、RIDA再構成系においてDnaA結合ATPの加水分解活性を示した。一方、DP非結合型HdaはRIDAに不活性なホモ多量体を形成した。DNA装着型クランプとの結合能はADP結合型、非結合型Hdaに関わらず見られたものの、続くDnaAとの相互作用にはADP結合型Hdaが必要であった。おそらく、DP結合によるHdaの活性化には、モノマー化によるDnaA相互作用部位の露出が関与していると考えられる。さらに我々は細胞内においてもHdaがADP結合型として存在していること、またADP結合がHdaの細胞内機能に重であることを見いだした。以上の結果はHdaがADP結合1解離を通してDnaA不活性化システムの発動を制御するスイッチとして働く可能性を提唱するものである。

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    • Protein Dynamics in Complex Apparatus for the Initiation and Regulation of the Chromosomal Replication

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

      KATAYAMA Tsutomu, UEDA Tadashi, ASO Mariko, SU'ETSUGU Masayuki, KURUMIZAKA Hitoshi

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      2004 - 2006

      Grant number:16370081

      Grant amount:\13500000 ( Direct Cost: \13500000 )

      DnaA protein promotes the initiation reaction in the complex with the replication origin. In this process, the duplex DNA within the origin is unwound. Also, DnaA is a crucial target of systems to regulate replicational initiation during the cell cycle. In this research project, we had a specific aim to analyze the mechanisms in the replicational initiation and its regulatory systems from a view of dynamic and comprehensive interprotein interactions.
      1 Structure analysis of DnaA and the initiation complex
      We analyzed a hyperthermophilic eubacterium DnaA homolog and succeeded in construction of in vitro reconstituted system for the replication initiation. We promoted production of crystals of the initiation complex using this DnaA homolog. Also, we analyzed the function-structure relationship in E. coli DnaA N-terminal domains and revealed the mechanisms in inter-DnaA interaction and DnaA-DnaB helicase interaction.
      2 Interaction of DnaA, the replicative clamp, and Hda
      We found the function-structure relationship of Hda in homooligomer formation. Also, we analyzed the function-structure relationship of the clamp in interaction with Hda. Moreover, we used an advanced proteomix method to search for novel factors that interact with the clamp, resulting in identification of many candidates. We further analyzed these factors.
      3 Analysis of a novel DnaA-associating factor
      We isolated many mutant forms of DiaA, a novel DnaA-associating protein and analyzed the function-structure relationship. Also, we revealed the crystal structure of DiaA. Moreover, we for the first time revealed that DiaA enhances the process of the initiation complex formation.
      4 Design and chemical synthesis of specific inhibitors for DnaA DNA-binding domain
      We designed and synthesized oligonucleotides that were chemically modified to covalently bind to DnaA DNA-binding domain. Binding kinetics of these chemicals was analyzed.

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    • DNA複製マシンによる複製開始フィードバック制御の分子機構

      日本学術振興会  科学研究費助成事業 特別研究員奨励費 

      末次 正幸

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      2002 - 2003

      Grant number:02J09038

      Grant amount:\1000000 ( Direct Cost: \1000000 )

      当研究室では、大腸菌染色体の複製開始蛋白質DnaAに結合したATPの水解により、DnaAの不活化を導く機構、RIDA(Regulatory Inactivation of DnaA)を見出している。RIDAに8は、DNAポリメラーゼIIIβサブユニットがDNAにロードされ、βクランプを形成することが必要である。よって、複製装置にはフィードバック的に、複製開始を抑制するという概念が提唱される。最近、RIDAに必須な新奇蛋白質Hdaが同定され、RIDA構成因子が出揃った。そこで本研究では、Hisタグ融合型としてHdaを精製し、RIDA反応を生化学的に解析した。
      まず、RIDA再構成系(DNAにロードされたβクランプをゲル濾過により単離し、これを用いてHdaに依存したDnaA結合性ATPの加水分解を行う)を構築し解析した結果、HdaはADP結合により活性型となることが示唆された。実際、フィルターバインディング法により、HdaはADPに高い特異性を持って結合することがわかった(Kd値、約2μM)。
      次に、βクランプ・Hda・DnaAの3者の相互作用について検討した。まず、プルダウン法により、βサブユニットはHdaと直接結合することがわかった。さらに、ゲル濾過による解析から、DNAにロードさせたβサブユニット(βクランプ)はHdaおよびATP結合型DnaAにそれぞれ単独で結合することが見出された。よって、βクランプを足場にしてHdaとATP-DnaAが相互作用する可能性が示唆される。このときHdaがDnaA結合性ATPに作用し、その加水分解を促しているのかもしれない。DnaAおよびHdaはともにAAA+型ATPase蛋白質であるが、一部のAAA+蛋白質については、複合体を形成し、分子間の境界でATP加水分解を行うことが提唱されている。RIDAの分子機構もこれと矛盾しないと思われる。

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    Industrial property rights

    • 機能性DNAカセット及びプラスミド

      末次正幸, 向井崇人

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      Applicant:オリシロジェノミクス株式会社

      Application no:特願2022-006523  Date applied:19 1 2022

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    • 環状DNAの製造方法

      末次正幸, 奈良聖亜

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      Applicant:オリシロジェノミクス株式会社

      Application no:特願2021-148639  Date applied:13 9 2021

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    • 環状DNAの複製または増幅方法

      末次正幸

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      Publication no:WO2018/159669  Date published:7 9 2018

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    • DNA編集法

      末次正幸

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      Application no:未公開  Date applied:31 7 2018

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    • 環状DNAの増幅方法

      末次正幸, 小林寛子, 篠原赳

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      Publication no:WO2017/199991  Date published:17 11 2017

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    • DNA連結法

      末次正幸, 倉田竜明

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      Application no:未公開  Date applied:5 7 2017

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    • 環状DNAの増幅方法

      末次正幸, 小林寛子

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      Patent/Registration no:6262877  Date issued:22 12 2017

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    • JST RISTEX「ゲノム倫理」研究会ケーススタディ_末次PJ

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