| 1. |
＃Keisuke Oki, Takeshi Yamagami, Mariko Nagata, Kouta Mayanagi, ＠Tsuyoshi Shirai, ＠Naruhiko Adachi, Tomoyuki Numata, Sonoko Ishino, Yoshizumi Ishino, DNA polymerase D temporarily connects primase to the CMG-like helicase before interacting with proliferating cell nuclear antigen, Nucleic Acids Research, 10.1093/nar/gkab243., 2021.04, The eukaryotic replisome is comprised of three family-B DNA polymerases (Polα, δ and ϵ). Polα forms a stable complex with primase to synthesize short RNA-DNA primers, which are subsequently elongated by Polδ and Polϵ in concert with proliferating cell nuclear antigen (PCNA). In some species of archaea, family-D DNA polymerase (PolD) is the only DNA polymerase essential for cell viability, raising the question of how it alone conducts the bulk of DNA synthesis. We used a hyperthermophilic archaeon, Thermococcus kodakarensis, to demonstrate that PolD connects primase to the archaeal replisome before interacting with PCNA. Whereas PolD stably connects primase to GINS, a component of CMG helicase, cryo-EM analysis indicated a highly flexible PolD-primase complex. A conserved hydrophobic motif at the C-terminus of the DP2 subunit of PolD, a PIP (PCNA-Interacting Peptide) motif, was critical for the interaction with primase. The dissociation of primase was induced by DNA-dependent binding of PCNA to PolD. Point mutations in the alternative PIP-motif of DP2 abrogated the molecular switching that converts the archaeal replicase from de novo to processive synthesis mode.. |
| 2. |
Kouta Mayanagi, Keisuke Oki, Naoyuki Miyazaki, Sonoko Ishino, Takeshi Yamagami, Kosuke Morikawa, Kenji Iwasaki, Daisuke Kohda, Tsuyoshi Shirai, Yoshizumi Ishino, Two conformations of DNA polymerase D-PCNA-DNA, an archaeal replisome complex, revealed by cryo-electron microscopy, BMC Biology, in press, 2020.09. |
| 3. |
Kouta Mayanagi,Kazumi Saikusa, Naoyuki Miyazaki,Satoko Akashi, Kenji Iwasaki, Yoshifumi Nishimura, Kosuke Morikawa, Yasuo Tsunaka, Structural visualization of key steps in nucleosome reorganization by human FACT, Scientific Reports, 10.1038/s41598-019-46617-7., 9, 1, 10183, 2019.07, Facilitates chromatin transcription (FACT) is a histone chaperone, which accomplishes both nucleosome assembly and disassembly. Our combined cryo-electron microscopy (EM) and native mass spectrometry (MS) studies revealed novel key steps of nucleosome reorganization conducted by a Mid domain and its adjacent acidic AID segment of human FACT. We determined three cryo-EM structures of respective octasomes complexed with the Mid-AID and AID regions, and a hexasome alone. We discovered extensive contacts between a FACT region and histones H2A, H2B, and H3, suggesting that FACT is competent to direct functional replacement of a nucleosomal DNA end by its phosphorylated AID segment (pAID). Mutational assays revealed that the aromatic and phosphorylated residues within pAID are essential for octasome binding. The EM structure of the hexasome, generated by the addition of Mid-pAID or pAID, indicated that the dissociation of H2A-H2B dimer causes significant alteration from the canonical path of the nucleosomal DNA.. |
| 4. |
Takashima N, Ishino S, Oki K, Takafuji M, Yamagami T, Matsuo R, Mayanagi K, Ishino Y., Elucidating functions of DP1 and DP2 subunits from the Thermococcus kodakarensis family D DNA polymerase, Extremophiles, 10.1007/s00792-018-1070-3, 23, 1, 161-172, 2019.01. |
| 5. |
Kouta Mayanagi, Sonoko Ishino, Tsuyoshi Shirai, Takuji Oyama, Shinichi Kiyonari, Daisuke Kohda, Kosuke Morikawa Yoshizumi Ishino, Direct visualization of DNA baton pass between replication factors bound to PCNA, Scientific Reports, 10.1038/s41598-018-34176-2, 8, 1, 16209, 2018.11, In Eukarya and Archaea, the lagging strand synthesis is accomplished mainly by three key factors, DNA polymerase (Pol), flap endonuclease (FEN), and DNA ligase (Lig), in the DNA replication process. These three factors form important complexes with proliferating cell nuclear antigen (PCNA), thereby constructing a platform that enable each protein factor to act successively and smoothly on DNA. The structures of the Pol-PCNA-DNA and Lig-PCNA-DNA complexes alone have been visualized by single particle analysis. However, the FEN-PCNA-DNA complex structure remains unknown. In this report, we for the first time present this tertiary structure determined by single particle analysis. We also successfully visualized the structure of the FEN-Lig-PCNA-DNA complex, corresponding to a putative intermediate state between the removal of the DNA flap by FEN and the sealing of the nicked DNA by Lig. This structural study presents the direct visualization of the handing-over action, which proceeds between different replication factors on a single PCNA clamp bound to DNA. We detected a drastic conversion of the DNA from a bent form to a straight form, in addition to the dynamic motions of replication factors in the switching process. |
| 6. |
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, European Microscopy Congress
, 3, 57-58, Vol3, 57-58, 2016.12. |
| 7. |
Aramaki S, Mayanagi K, Jin M, Aoyama K, Yasunaga T, Filopodia formation by crosslinking of F-actin with fascin in two different binding manners, Cytoskeleton, 10.1002/cm.21309, 73(7):365-74, 2016.06. |
| 8. |
Takekawa N, Terahara N, Kato T, Gohara M, Mayanagi K, Hijikata A, Onoue Y, Kojima S, Shirai T, Namba K, Homma M, The tetrameric MotA complex as the core of the flagellar motor stator from hyperthermophilic bacterium, Scientific Reports, 10.1038/srep31526, 6:31526, 2016.08. |
| 9. |
Nakae S, Hijikata A, Tsuji T, Yonezawa K, Kouyama KI, Mayanagi K, Ishino S, Ishino Y, Shirai T, Structure of the EndoMS-DNA Complex as Mismatch Restriction Endonuclease, Structure, ２４, 1960-1971, 24, 1960-1971, 2016.07, Archaeal NucS nuclease was thought to degrade the single-stranded region of branched DNA, which contains flapped and splayed DNA. However, recent findings indicated that EndoMS, the orthologous enzyme of NucS, specifically cleaves double-stranded DNA (dsDNA) containing mismatched bases. In this study, we determined the structure of the EndoMS-DNA complex. The complex structure of the EndoMS dimer with dsDNA unexpectedly revealed that the mismatched bases were flipped out into binding sites, and the overall architecture most resembled that of restriction enzymes. The structure of the apo form was similar to the reported structure of Pyrococcus abyssi NucS, indicating that movement of the C-terminal domain from the resting state was required for activity. In addition, a model of the EndoMS-PCNA-DNA complex was preliminarily verified with electron microscopy. The structures strongly support the idea that EndoMS acts in a mismatch repair pathway.. |
| 10. |
Mayanagi K, Kiyonari S, Nishida H, Saito M, Kohda D, Ishino Y, Shirai T, Morikawa K, , Architecture of the DNA polymerase B-proliferating cell nuclear antigen (PCNA)-DNA ternary complex. , PNAS, 108, 5, 1845-1849, (Corresponding Author), 2011.02. |
| 11. |
Hirokazu. Nishida, Kouta. Mayanagi, Shinichi. Kiyonari, Yuichi. Sato, Takuji. Oyama, Yoshizumi. Ishino, Kosuke. Morikawa, Structural determinant for switching between the polymerase and exonuclease modes in the PCNA-replicative DNA polymerase complex, PNAS, 106, 20693-20698, 2009.11. |
| 12. |
Kouta Mayanagi, Shinichi Kiyonari, Mihoko Saito, Tsuyoshi Shirai, Yoshizumi Ishino, Kosuke Morikawa, Mechanism of replication machinery assembly as revealed by the DNA ligase-PCNA-DNA complex architecture, PNAS, 106, 4647-4652, (Corresponding Author), 2009.03. |
| 13. |
Yasuto Murayama, Yumiko Kurokawa, Kouta Mayanagi, and Hiroshi Iwasaki, Formation and branch migration of Holliday junctions mediated by eukaryotic recombinases, Nature, 451, 7181, 1018-1021, 2008.02. |
| 14. |
Yoshie Fujiwara, Kouta Mayanagi, and Kosuke Morikawa, Functional significance of octameric RuvA for a branch migration complex from Thermus thermophilus., BBRC, 366, 2, 426-431, 2007.12. |
| 15. |
Kouta Mayanagi, Yoshie Fujiwara, Tomoko Miyata, and Kosuke Morikawa, Electron microscopic single particle analysis of a tetrameric RuvA/RuvB/Holliday junction DNA complex, BBRC, 365, 273-278, (Corresponding Author), 2007.11. |
| 16. |
Tomoko Miyata, Hirofumi Suzuki, Takuji Oyama, Kouta Mayanagi, Yoshizumi Ishino, and Kosuke Morikawa, Open clamp structure in the clamp-loading complex visualized by electron microscopic image analysis. , PNAS, 10.1073/pnas.0506447102, 102, 39, 13795-13800, 2005.09. |
| 17. |
Tomoko Miyata, Takuji Oyama, Kouta Mayanagi, Sonoko Ishino, Yoshizumi Ishino, and Kosuke Morikawa, The Clamp-loading Complex for Processive DNA Replication, Nature Struct. & Molec. Biol. , 10.1038/nsmb788, 11, 7, 632-636, 2004.06. |
| 18. |
Kyoko Matoba, Kouta Mayanagi, Syo Nakasu, Akihiko Kikuchi, and Kosuke Morikawa, Three-dimensional electron microscopy of the reverse gyrase from Sulfolobus tokodaii, BBRC, 297, 4, 749-755, 2002.10. |
| 19. |
Naoko Kajimura, Matsuyo Yamazaki, Kosuke Morikawa, Akio Yamazaki, and Kouta Mayanagi, Three-dimensional structure of non-activated cGMP phosphodiesterase 6 and comparison of its image with those of activated forms, J. Struct. Biol., 139, 1, 27-38, (Corresponding Author), 2002.07. |
| 20. |
Mayanagi, K., Miyata, T., Oyama, T., Ishino, Y., and Morikawa, K, Three-dimensional electron microscopy of the clamp loader small subunit from Pyrococcus furiosus, J. Struct. Biol., 134, 1, 35-45, (Corresponding Author), 2001.04. |
| 21. |
Miyata, T., Yamada, K., Iwasaki, H., Shinagawa, H., Morikawa, K., and Mayanagi. K, Two different oligomeric states of the RuvB branch migration motor protein as revealed by electron microscopy, J. Struct. Biol., 131, 2, 83-89, (Corresponding Author), 2000.08. |
| 22. |
Mayanagi, K., Ishikawa, T., Toyoshima, C., Inoue, Y., and Nakazato, K, Three-dimensional electron microscopy of the photosystem II core complex, J. Struct. Biol. , 123, 3, 211-224, 1998.11. |
