Kyushu University Academic Staff Educational and Research Activities Database
List of Presentations
Kota Mayanagi Last modified date:2019.07.30

Assistant Professor / Department of Molecular and Structural Biology / Medical Institute of Bioregulation


Presentations
1. Yoshizumi Ishino, Keisuke Oki, Mariko Nagata, Kouta Mayanagi, Tsuyoshi Shirai, Takuji Oyama, Kosuke Morikawa, Takeshi Yamagami, and Sonoko Ishino, Replisome structure and its functions in Archaea, OKAZAKI Fragment Memorial Symposium: Celebrating the 50th anniversary of the discontinuous DNA replication model, 2018.12.
2. Sonoko Ishino, Kouta Mayanagi, Tsuyoshi Shirai, Shinichi Kiyonari, Namiko Imai, Takuji Oyama, and Yoshizumi Ishino, Biochemical and Structural analyses of replication proteins bound to PCNA, 12th International Congress on Extremophiles, 2018.09.
3. Keisuke Oki, Takeshi Yamagami, Mariko Nagata, Natsuki Takashima, Kouta Mayanagi, Sonoko Ishino, and Yoshizumi Ishino, Family D DNA polymerase tethers primase and helicase in the replisome in Thermococcus kodakarensis, 12th International Congress on Extremophiles, 2018.09.
4. Kouta Mayanagi, Single particle analysis of FACT-Nucleosome complex, 新学術領域「染色体OS」第6回領域会議, 2018.02.
5. Kouta Mayanagi, Single particle analysis of FACT-Nucleosome complex, The 4th Meeting on GRANT-IN-AID FOR SCIENTIFIC RESEARCH ON INNOVATIVE AREAS “CHROMOSOME ORCHESTRATION SYSTEM”, 2017.02.
6. Kouta Mayanagi, Single particle analysis of DNA replication fork complex, 10th International 3R (DNA, Replication, Recombination, and Repair) Symposium, 2016.11,  We have been studying the mechanism of synthesis and maturation of Okazaki fragments, in which three replication factors, i.e. DNA polymerase, Flap endonuclease (FEN), and DNA ligase, are playing essential roles. As all of these three enzymes are known to interact with PCNA, a switching mechanism between these factors, bound simultaneously to a PCNA ring has been proposed, called PCNA tool belt model, which is considered to increase the efficiency of these sequential reactions. Recent biochemical studies, however, suggest sequential binding and releasing of these factors. Little is known regarding the precise switching mechanism, due to the lack of the structural knowledge of these complexes.
We investigated the structures of the core components of the replisome, such as DNA polymerase-PCNA-DNA, and DNA ligase-PCNA-DNA, by single particle analysis. Besides the known interaction through a PIP-box motif, we could find a novel contact between polymerase and PCNA, as well as between ligase and PCNA. Mutant analysis showed that these contacts are involved in the regulation of the replication factors, such as the switching between the polymerizing and editing modes of the polymerase. Our results, showing that both factors are bound to two subunits of the PCNA trimer, were inconsistent with the standard tool belt model. The third PCNA subunit, however, was still free in both complexes, thus we analyzed the complex structures with two replication factors. We could successfully visualize the 3D structure of FEN-DNA ligase-PCNA-DNA complex, and the handing over of the ds DNA from FEN to DNA ligase..
7. Mayanagi K, Ishino S Takafuji M, Mitsuoka K, Shirai T, Kiyonari S, Nishida H, Kohda D, Morikawa K, Ishino Y., Switching mechanism of the replisome as revealed by single particle analysis, 第 89 回日本生化学会大会, 2016.09, DNA replication in archaea and eukaryotes is executed by family B DNA polymerases, which exhibitfull activity when complexed with the DNA clamp, proliferating cell nuclear antigen (PCNA). PCNAhas a trimeric ring structure that encircles the DNA, and increases the processivity of the bound DNApolymerase by tethering it to the DNA. It is known now, that PCNA also interacts with multiple partnersto control DNA replication, DNA repair, and cell cycle progression, and works not only as the platform,but also as the conductor for the recruitment and release of these factors. However, the moleculararchitectures as well as the mechanism of the regulation of these replication factors are not known indetail.We have investigated the three-dimensional structure of the core components of the replisome,such as DNA polymerase-PCNA-DNA, and DNA ligase-PCNA-DNA ternary complexes, by singleparticle analysis (2- 3). Besides the authentic interaction through a PCNA-interacting protein box (PIPbox),we could find a novel contact between both polymerase-PCNA and ligase-PCNA. Mutant analysisshowed that these contacts are involved in the regulation of the replication factors, such as the switchingbetween the polymerizing and editing modes of the polymerase. Our results, showing that both factorsbound to two subunits of the PCNA trimer ring, were inconsistent with the standard tool belt model. Thethird PCNA subunit, however, was still free in both complexes, thus we analyzed the complex structureswith two replication factors bound to one PCNA ring, in order to investigate the switching mechanismbetween them in more detail..
8. Mayanagi K, Ishino S Takafuji M, Mitsuoka K, Shirai T, Kiyonari S, Nishida H, Kohda D, Morikawa K, Ishino Y., Switching mechanism of DNA replication fork complex revealed by single particle analysis, The 16th European Microscopy Congress, 2016.08.
9. Kouta Mayanagi, Molecular switching of DNA replication fork complex revealed by single particle analysis, The 1st APPA-PSSJ joint workshop, 2016.06, Okazaki fragments synthesis and maturation, during lagging strand DNA replication, is accomplished by mainly three replication factors, i.e. DNA polymerase, Flap endonuclease, and DNA ligase. All of these three enzymes are known to interact with the DNA clamp, proliferating cell nuclear antigen (PCNA), through a PCNA-interacting protein box (PIP-box) motif. PCNA forms a trimeric ring that encircles the DNA, and increases the processivity of the bound DNA polymerase by tethering it to the DNA. As up to three replication factors could be bound to a PCNA trimer, a switching mechanism between these replication factors, bound simultaneously to a PCNA ring has been proposed, called PCNA tool belt model, which is considered to increase the efficiency of the sequential reactions. However, the precise mechanism is not known in detail, due to the lack of structural data of such complexes. In order to investigate the mechanism of the regulation and switching of these replication factors bound to PCNA, we have studied the molecular architectures of the core components of the replisome, such as DNA polymerase-PCNA-DNA, and DNA ligase-PCNA-DNA complexes, by single particle analysis..
10. Kouta Mayanagi, Single particle analysis of DNA replication fork complex, The 25th Hot Spring Harbor International Symposium, “Cutting Edge of Technical Innovations in Structural and Systems Biology”, 2015.11.
11. Hiromi Ogino, Sonoko Ishino, Kouta Mayanagi, Takuji Oyama, Tsyoshi Shirai, Kosuke Morikawa, Gyri Teien Haugland, Nils-Kare Birkeland, Daisuke Kohda, Yoshizumi Ishino, Divergently evolved activation mechanism of the replicative helicase in the thermophilic archaeon, Thermoplasma acidophilum, 第36回日本分子生物学会, 2013.12.
12. Kouta Mayanagi, Electron microscopic structure analysis of protein-DNA complexes, The 3rd International conference on New Frontier of the Research on RecA-family recombinases and their accessory proteins, 2013.10.
13. Hiromi Ogino, Sonoko Ishino, Kouta Mayanagi, Takuji Oyama, Tsyoshi Shirai, Kosuke Morikawa, Gyri Teien Haugland, Nils-Kare Birkeland, Daisuke Kohda, Yoshizumi Ishino, Activation mechanism of the replicative helicase in the thermophilic archaeon, Thermoplasma acidophilum, Thermophiles 2013, 2013.09.
14. Kouta Mayanagi, Electron microscopic analysis of molecular architecture and switching mechanism of DNA replication fork complex, ICSG2013-SLS, 2013.07.
15. Kouta Mayanagi, Shinichi Kiyonari, Hirokazu Nishida, Sonoko Iahino, Mihoko Saito, Daisuke Kohda, Yoshizumi Ishino, Tsuyoshi Shirai, Kosuke Morikawa, Molecular architecture and switching mechanism of DNA replication fork complex, Gordon Research Conferences: Three Dimensional Electron Microscopy, 2013.06.
16. Shinji Aramaki, Kazuhiro Aoyama, Kouta Mayanagi, Takuo Yasunaga, 3D ultrastructure within intact cells explored by electron cryo-tomography in TEM and STEM, The 10th Asia-Pacific Microscopy Conference (APMC10), 2012.02.
17. Hiromi Ogino, Sonoko Ishino, Kouta Mayanagi, Gyri Teien Haugland, Nils-Kare Birkeland, Akihiko Yamagishi, Yoshizumi Ishino, The GINS complex from the thermoacidophilic archaeon, Thermoplasma acidophilum may function as a homotetramer in DNA replication, 第34回日本分子生物学会, 2011.12.