Kyushu University Academic Staff Educational and Research Activities Database
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Toshiki Tsurimoto Last modified date:2018.06.14

Professor / Integrative Biology
Department of Biology
Faculty of Sciences


Graduate School
Undergraduate School
Other Organization


Homepage
http://seibutsu.biology.kyushu-u.ac.jp/~chromosome/
The Homepage of the Research Team for Chromosomal DNA Replication in Department of Biology, Faculty of Sciences, Kyushu University .
Academic Degree
PhD in Science
Country of degree conferring institution (Overseas)
No
Field of Specialization
Molecular Biology, Biochemistry
Total Priod of education and research career in the foreign country
03years03months
Outline Activities
I have been studying molecular mechanism of DNA replication in human cells focusing on its functional links with cellular factors involved in maintenance of the genome integrity during cell proliferation.
Through these researches, I offered basic knowledge for molecular biology and advanced technology for studies on protein functions to undergraduate- and graduate- students.
Since my research subjects also have tight relation with mechanism of cancer development, it can be expected to provide a new idea to develop anti-cancer drug. Therefore, I have actively advanced collaborative researches with cancer researchers.
Research
Research Interests
  • Molecular mechanism of eukaryotic chromosomal DNA replication and its regulation
    keyword : replication/cell cycle/ DNA polymearse/ clamp/ clamp loader/ AAA+ ATPase
    1991.07~2020.03I have been studying molecular mechanism of DNA replication in human cells focusing on its functional links with cellular factors involved in maintenance of the genome integrity during cell proliferation..
Academic Activities
Reports
1. Ohashi E, Tsurimoto T, Functions of multiple clamp and clamp-loader complexes in eukaryotic DNA replication, Adv Exp Med and Biol., 10.1007/978-981-10-6955-0_7, 2017.08.
2. Tsurimoto T., PCNA interacting proteins., Proliferating Cell Nuclear Antigen (PCNA). ed. Hoyun Lee, Research Signpost, Kerala, India, 2006.09.
3. Tsurimoto, T., PCNA and RFC proteins in Genomic Stability., DNA Replication and Human Disease ed. DePamphilis, M. L, Cold Spring Harbor Press NY, 2006.09.
Papers
1. Ryo Fujisawa, Eiji Ohashi, Kouji Hirota, Toshiki Tsurimoto, Human CTF18-RFC clamp-loader complexed with non-synthesising DNA polymerase ϵ efficiently loads the PCNA sliding clamp, Nucleic Acids Research, https://doi.org/10.1093/nar/gkx096, 45, 8, 4550-4563, 2017.01, The alternative proliferating-cell nuclear antigen (PCNA)-loader CTF18-RFC forms a stable complex with DNA polymerase ϵ (Polϵ). We observed that, under near-physiological conditions, CTF18- RFC alone loaded PCNA inefficiently, but loaded it efficiently when complexed with Polϵ. During efficient PCNA loading, CTF18-RFC and Polϵ assembled at a 3ϵ primer-template junction cooperatively, and directed PCNA to the loading site. Site-specific photo-crosslinking of directly interacting proteins at the primer-template junction showed similar cooperative binding, in which the catalytic N-terminal portion of Polϵ acted as the major docking protein. In the PCNA-loading intermediate with ATPαS, binding of CTF18 to the DNA structures increased, suggesting transient access of CTF18-RFC to the primer terminus. Polϵ placed in DNA synthesis mode using a substrate DNA with a deoxidised 3ϵ primer end did not stimulate PCNA loading, suggesting that DNA synthesis and PCNA loading are mutually exclusive at the 3ϵ primer-template junction. Furthermore, PCNA and CTF18-RFC-Polϵ complex engaged in stable trimeric assembly on the template DNA and synthesised DNA efficiently. Thus, CTF18-RFC appears to be involved in leading-strand DNA synthesis through its interaction with Polϵ, and can load PCNA onto DNA when Polϵ is not in DNA synthesis mode to restore DNA synthesis..
2. Shiomi, Y., Masutani, C., Hanaoka, F., Kimura H. and Tsurimoto, T. , A second PCNA loader complex, Ctf18-RFC, stimulates DNA polymerase η activity. , J. Biol. Chem., 282, 20906-20914 , 2007.07.
3. Ohta, S., Shiomi, Y., Sugimoto, K., Obuse, C., Tsurimoto, T., A proteomics approach to identify PCNA binding proteins in human cell lysates: identification of the human CHL12/RFCs2-5 complex as a novel PCNA binding protein., J. Biol. Chem., 277, 40362-40367, 2002.01.
4. Shiomi, Y., Usukura, J., Masamura, Y., Takeyasu, K., Nakayama, Y., Obuse, C., Yoshikawa, H. Tsurimoto, T., ATP-dependent structural change of the eukaryotic clamp loader protein, RFC., Proc. Natl. Acad. Sci. USA, 97, 14127-14132, 2000.01.
Presentations
1. Toshiki Tsurimoto, Fujisawa R, Eiji Ohashi, Seiji Tanaka, Hiroyuki Araki, Sun Q, Kaye K, Novel mechanisms of PCNA loader complexes to specify their functions. , The 9th 3R Symposium, 2014.11, Eukaryotes have two active PCNA loader complexes, RFC and Ctf18-RFC, necessary for DNA replication and maintenance of genome integrity. It has been demonstrated that their PCNA loadings have distinct roles, for example, DNA synthesis for replication and repair by RFC and chromosome cohesion and DNA damage response by Ctf18-RFC. However, the mechanism to specify their functions mainly remains to be elucidated.
We have worked on the interaction of Ctf18-RFC and DNA polymerase ε (Pol ε). The C-terminal of Ctf18 is highly conserved from yeast to human and essential for the interaction with Pol ε. The C-terminal deletion of Ctf18 in yeast resulted in hypersensitivity to DNA damage by MMS and to inhibition of fork progression by HU and increased loss-rate of ARS-plasmids as similar as the CTF18 deletion. Thus, most of Ctf18-RFC functions will be mediated through its interaction with Pol ε. Indeed, we demonstrated that binding of Pol ε to Ctf18-RFC stimulated its PCNA loading activity and made its intrinsic salt-sensitive PCNA loading active even at a high salt condition. These results indicated that function of Ctf18-RFC would be specified by its binding partner, Pol ε .
The second example of the mechanism came from the interaction of RFC with Kaposi’s sarcoma-associated herpes virus (KSHV) latency-associated nuclear antigen (LANA), required for the viral DNA replication and persistence. We demonstrated that RFC specifically bound to LANA and was crucial for maintenance of the viral episomes. Furthermore, LANA recruited RFC to KSHV DNA and enhanced PCNA loading in vitro. Thus, the interaction will direct the host replication machinery to the viral genome.
These results indicated that a specific loader-target interaction is one of the mechanisms to specify functional sited for loader complexes.
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2. Hiroko Okimoto, Seiji Tanaka, Hiroyuki Araki, Eiji Ohashi, Toshiki Tsurimoto, Roles of loader proteins for coupling between replication and chromosome maintenance, 新学術領域研究「ゲノム普遍的制御」主催、International Conference"Coupling of replication, repair and transcription, and their common mechanism of chromatin remodeling"公開シンポジウム, 2014.02, Leading strand synthesis DNA polymerase, Pol ε and chromosome cohesion PCNA loader Ctf18-RFC form a stable complex in human cells. This holocomplex will be involved in functional collaboration between replication fork progression and various chromosome maintenance steps including chromosome cohesion. We demonstrated that Pol ε interacted Ctf18-RFC through the trimeric assembly of the C-terminal of Ctf18 bound with two cohesion specific subunits, Dcc1 and Ctf8. Furthermore, the interaction occurred through the N-terminal catalytic domain of Pol ε p261 subunit and suppressed the DNA synthesis activity partially. We also demonstrated that the complex formation resulted in stimulation of PCNA loading by Ctf18-RFC. These results suggest that the leading DNA synthesis mode will be regulated through dynamics of this novel holocomplex formation.
The Ctf18 C terminal is highly conserved from budding yeast to human. To address whether this Pol ε/Ctf18-RFC holocomplex structure would be also conserved in budding yeast, we expressed yCtf18 C-terminal along with yDcc1 and yCtf8 by an efficient expression system with human 293T cell. We demonstrated that the yCtf18 C-terminal also formed the same assembly as human one and bound specifically with purified yPol ε. Based on this result, we studied phenotypes of the yCtf18 C-terminal deletion (ctf18ΔC) in budding yeast and observed its high sensitivity to DNA damaging reagents, plasmid loss rate and GCR (Gross Chromosomal Rearrangement) similarly as ctf18 deletion mutant. Therefore, the holocomplex formed through the C-terminal of yCtf18 is an essential structure for yCtf18-RFC function to maintain the genome stability in budding yeast.
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3. Hiroko Okimoto, Seiji Tanaka, Hiroyuki Araki, Eiji Ohashi, Toshiki Tsurimoto, Functions of the C-terminal of the chromosome cohesion PCNA loader, Ctf18-RFC, Cold Spring Harbor Lab. Eukaryotic DNA Replication & Genome Maintenanceワークショップ, 2013.09, Chromosome cohesion is established during DNA replication through functional coordination between DNA synthesis and cohesion apparatuses. We have worked on the interaction of the chromosome cohesion PCNA loader, Ctf18-RFC and DNA polymerase epsilon (Pol e) in human cells. We demonstrated that the C-terminal of Ctf18 formed the trimeric assembly with Dcc1 and Ctf8 that interacts specifically with Pol e, suggesting its importance for the link between DNA synthesis and cohesion reactions. The interaction occurred through the N-terminal catalytic domain of Pol e p261 subunit and modulated the DNA synthesis activity. Furthermore, the assembly structure and its interaction with Pol e were highly conserved from human to budding yeast.
To study cellular functions of the assembly in Ctf18, we employed genetic analyses with budding yeast. Deletion of CTF18 resulted in hypersensitivity to DNA damage by MMS and to inhibition of fork progression by HU and increased loss-rate of ARS-plasmids. Analyses of its C-terminal deletion mutant demonstrated that the structure in yeast Ctf18 was highly involved in progression of DNA replication and faithful minichromosome segregation. These results suggest that the complex of Ctf18-RFC and Pol e will be integrated in the replication fork and crucial for its link with the cohesion apparatus.
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4. Function of the holocomplex between DNA polymerase e and PCNA loader Ctf18-RFC.
5. Molecular crosstalk between DNA synthesis and chromosome cohesion apparatuses.
6. 釣本 敏樹, Okimoto H, Tanaka S, Araki H, 大橋 英治, Two functional structures of the chromosome cohesion PCNA loader, Ctf18-RFC , 第8回3Rシンポジウム, 2012.11, Chromosome cohesion and DNA replication tightly couples. We have worked on an alternate RFC complex, Ctf18-RFC, which functions as a second PCNA loader and plays a key role for establishment of cohesion. It consists of two functional structures, the assembly of ATPase subunits necessary for PCNA loading and a complex of cohesion specific subunits, Ctf18, Dcc1 and Ctf8. Even with its obvious involvement for coupling of replication and cohesion, little is known regarding the mechanism. We identified novel interactions of human RFC complexes with several DNA polymerases, such as Pol η, δ and ε. The assembly of ATPase subunits mainly mediated these interactions. Interestingly, Ctf18-RFC and Pol ε have an additive interaction, which is more stable than other cases and occurred through the assembly of cohesion specific subunits. It bound to the N-terminal catalytic domain of Pol ε and exhibited inhibitory function to its DNA synthesis. Thus, their specific interaction will function to coordinate progression of the DNA fork during cohesion establishment.
To study significance of the interaction in cells, we employed genetic analyses with budding yeast. Deletion of CTF18 resulted in hypersensitivity to DNA damage by MMS and inhibition of fork progression by HU. Analyses of its mutants demonstrated that yeast Ctf18 was also functionally separable into two structures, as their defects could be distinguishable by sensitivities to these treatments. These results suggest that Ctf18-RFC will be an integrated part of the replication fork and connect the cohesion apparatus with the fork through at least two distinct mechanisms.
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7. The second PCNA loader Ctf18-RFC interacts with DNA polymerase ε in human cells..
8. Analysis of intrinsic DNA binding activity of 9-1-1 complex.
9. Roles of the 9-1-1/TopBP1 interaction for DNA damage responses in human cells..
10. Studies on molecular interactions between clamp loader complexes and MCM subcomplexes.
11. Studies on functional domains of human DNA polymerase epsilon catalytic subunit p261.
12. Studies on molecular interactions between clamp loader complexes and MCM subcomplexes.
13. Stimulation DNA polymerase activity with ubiquitianted PCNA.
14. Studies on molecular interactions between clamp loader complexes and DNA replication proteins.
15. Analyses on interaction between loader complexes and replication proteins in human cells.
16. STUDIES ON MOLECULAR INTERACTIONS BETWEEN LOADER COMPLEXES AND REPLICATION FORK COMPONENTS.
17. Studies on molecular interactions of clamp loader complexes with DNA polymerase e .
18. Studies on molecular interactions of clamp loader complexes with DNA polymerase e and RPA.
19. Studies on molecular interactions between clamp loader complexes and DNA replication proteins.
20. Stimulation DNA polymerase activity with ubiquitianted PCNA.
Educational
Other Educational Activities
  • 2018.03.
  • 2009.03.
  • 2008.08.
  • 2008.03.
  • 2007.03.
  • 2007.07.