|Toshiki Tsurimoto||Last modified date：2018.06.14|
Professor / Integrative Biology / Department of Biology / Faculty of Sciences
|Toshiki Tsurimoto||Last modified date：2018.06.14|
|1.||＠Eiji Ohashi, ＃Naoya Iwata, ＃Kensuke Tatsukawa, ＠Toshiki Tsurimoto, Intramolecular binding of Rad9 C-terminal tail to Rad9-Hus1-Rad1 core ring structure regulates the binding of DNA damage response proteins, Cold Spring Harbor Laboratory Meeting “Eukaryotic DNA Replication & Genome Maintenance”, 2017.09, .|
|2.||＠Tsurimoto T, ＃Fujisawa R, ＠Ohashi E, CTF18-RFC complexed with DNA polymerase ε actively loads PCNA to maintain the efficient DNA synthesis, Cold Spring Harbor Laboratory Meeting “Eukaryotic DNA Replication & Genome Maintenance”, 2017.09, .|
|3.||＃Fujisawa R, ＠Ohashi E, ＠Hirota K, ＠Tsurimoto T, Ctf18-RFC bound to Polε loads PCNA efficiently., The 10th 3R Symposium, 2016.11.|
|4.||Ohashi E, Iwata N, Tsurimoto T, Intramolecular folding of Rad9 C-terminal tail in the Rad9-Hus1-Rad1 checkpoint clamp regulates its interaction with DNA damage response proteins., The 10th 3R Symposium, 2016.11.|
|5.||＃Fujisawa R, ＠Eiji Ohashi, ＠Toshiki Tsurimoto, Functional significance of the interaction between the second PCNA loader Ctf18-RFC and Polε, CSHL Workshop: Eukaryotic DNA Replication & Genome Maintenance., 2015.09, Alternative PCNA loader Ctf18-RFC is involved in sister chromatid cohesion and S-phase checkpoint in eukaryotes. It loads PCNA similarly as the replicative PCNA loader RFC, although these they play distinct roles in vivo. Recently, we demonstrated that Ctf18-RFC specifically interacts with DNA polymerases ε (Polε) in human cells. However, the functional significance of their interaction remained to be explored.
Here, we report that the interaction is crucial for PCNA loading by Ctf18-RFC. We quantified PCNA loading by Ctf18-RFC using gapped-DNA beads, and demonstrated that the interaction of Polε to Ctf18-RFC augmented its PCNA loading significantly. Since PCNA loading by Ctf18-RFC was intrinsically salt sensitive, it appeared to be active at physiological condition only when Ctf18-RFC was in a complex with Polε. Our pull-down experiments with various structured oligo-DNA demonstrated that the augmented PCNA loading by the Ctf18-RFC/Polε complex occurred at 3’ primer/template junction. Indeed, under this condition, Ctf18-RFC alone could not bind to 3’-end of primer stably but in the presence of Polε, they assembled there cooperatively. Furthermore, photo-reactive crosslinker experiments demonstrated that Polε mainly occupied the 3’-end under this condition. Thus, Ctf18-RFC is recruited to its functional DNA sites through the interaction with Polε at the 3’-end, quite differently from the case of RFC, in which it can access to 3’-end by itself..
|6.||＠Eiji Ohashi, ＃Takeishi M, ＠Toshiki Tsurimoto, Multiple roles of Rad9 C-tail in the checkpoint clamp Rad9-Hus1-Rad1., CSHL Workshop: Eukaryotic DNA Replication & Genome Maintenance., 2015.09, Rad9-Hus1-Rad1 (9-1-1) checkpoint clamp is a ring-shaped heterotrimeric complex
that is loaded by Rad17-RFC clamp loader upon DNA damage and functions as a damage
sensor. Unlike Rad1 and Hus1, Rad9 has an intrinsically disordered region at its Cterminus
(C-tail) projecting from the 9-1-1 core ring structure (CRS). We previously
showed that the phosphorylation of Ser-341 and Ser-387 at the C-tail of Rad9 by casein
kinase 2 (CK2) is required for the interaction between 9-1-1 and TopBP1 and for
subsequent activation of ATR dependent checkpoint pathway. We further demonstrated
that the interaction indirectly promotes TopBP1 accumulation at DNA damage sites
through ATR activation.
Recently, it was shown that 9-1-1 binds to DNA independently of Rad17-RFC. We
found that 9ΔC-1-1 lacking Rad9 C-tail corresponding to CRS bound to DNA more
efficiently than wild-type 9-1-1. Furthermore, we showed that the C-tail binds directly to
CRS and that the intramolecular binding suppresses the DNA binding of 9-1-1.
Interestingly, the sequence in C-tail necessary for 9ΔC-1-1 binding was partly coincident
with that for TopBP1 binding, and 9ΔC-1-1 and TopBP1 competed with each other for the
binding to C-tail. Thus, by interacting with CRS, Rad9 C-tail may regulate both the DNA
binding activity of 9-1-1 and the binding mode of 9-1-1 to multiple binding partners
|7.||The C-terminal of Rad9 associates to the core ring structure of the checkpoint clamp, Rad9-Hus1-Rad1, and regulates its DNA binding activity..|
|8.||Human Ctf18-RFC forms holocomplex with Polε and load PCNA functionally.|
|9.||Intra‒ and intermolecular crosstalk of the origin recognition
complex in budding yeast.
|10.||The ATR activation through the triangular interactions among factors in ATR-Chk1 DNA damage checkpoint pathway: ATR-ATRIP, Rad9-Hus1-Rad1, and TopBP1.|
|11.||Fujisawa R, Eiji Ohashi, Toshiki Tsurimoto, Interaction of Ctf18-RFC with DNA polymerase ε specifies their functional sites for the active PCNA loading., The 9th 3R Symposium, 2014.11, In eukaryotes, PCNA clamp is loaded onto DNA either by RFC or Ctf18-RFC, though they are functionally distinguishable in vivo. For example, RFC is required for DNA synthesis during DNA replication and repair, and Ctf18-RFC is for establishment of chromosome cohesion and S-phase checkpoint. Thus, there should be a mechanism to distinguish their functional sites. Recently, we demonstrated that Ctf18-RFC forms a stable complex with Pol ε. To investigate functional significance of their interaction, we analyzed PCNA loading by Ctf18-RFC with a gapped plasmid DNA. First, we demonstrated that Polε augmented PCNA loading activity specifically by Ctf18-RFC, retaining the structure to interact with Polε. We further demonstrated that Polε p261N, harboring the exonuclase and DNA polymerase catalytic domains, is sufficient for the interaction and the stimulation of PCNA loading. One of the differences of PCNA loading between RFC and Ctf18-RFC are their salt sensitivity. RFC is active at high salt condition higher than 0.1 M NaCl, while Ctf18-RFC alone is almost inactive at this condition. We observed that in the presence of p261N, Ctf18-RFC exhibited sufficient PCNA loading activity even at the high salt condition. Thus, Ctf18-RFC will be active only in a complex with Polε at a physiological salt condition. We suggest that this mechanism will specify the site for active PCNA loading by Ctf18-RFC on replicating DNA..|
|12.||Eiji Ohashi, Yukimasa Takeishi, Toshiki Tsurimoto, The ATR activation through the accumulation of the checkpoint factors and their interactions at the site of DNA damage., The 9th 3R Symposium, 2014.11, ATR-Chk1 pathway is a major DNA damage checkpoint pathway that responds to exposed single-stranded DNA (ssDNA). Upon DNA damage, ATR and its downstream kinase, Chk1 phosphorylate dozens of proteins and control DNA repair, cell cycle and apoptosis. To activate this pathway, not only protein accumulation at the DNA damage sites but also their physical interactions are important. A mediator, TopBP1 binds to both ATR-ATRIP and Rad9-Hus1-Rad1 (9-1-1) complexes to activate ATR. We previously showed that TopBP1 interacts with 9-1-1 through CK2-mediated phosphorylation of Ser-341 and Ser-387 at the C-terminal tail of Rad9 (Refs 1, 2). While their interaction is important for ATR activation, how the actual mechanism by their accumulation at the sites of DNA damage remains to be elucidated.
Here, we have studied significance of their interaction for assembly and activation of checkpoint proteins at damaged DNA. Rad9 accumulated at damage sites depending on TopBP1 but not on their direct interaction. Though their interaction resulted in ATR activation and contributed the efficient redistribution of TopBP1 upon DNA damage, the localization of Rad9 itself was not affected by ATR activation, suggesting that TopBP1 and Rad9 can be recruited to the damaged sites independently. Finally, we found that Rad9 interacts with ATR/ATRIP irrespective of binding to TopBP1 in human cells. Taken together, the 9-1-1/TopBP1 interaction may not contribute to initial recruitment of these checkpoint factors to the damaged DNA nor to bridging 9-1-1 and ATR-ATRIP complexes. But once they associate together after their recruitment at damaged sites, some structure will be induced in the complex to activate ATR, leading to further accumulation of TopBP1 at the sites. This positive feedback mechanism would be important for successful formation of the damage-sensing complex and DNA damage checkpoint signaling in human cells.
|13.||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.
|14.||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.
|15.||Eiji Ohashi, Yukimasa Takeishi, Satoshi Ueda, Toshiki Tsurimoto, Interaction of Rad9-Hus1-Rad1 with TopBP1 through CK2 sites in Rad9 is required for ATR-dependent checkpoint activation but not for their accumulation at DNA damage site, Cold Spring Harbor Lab. Eukaryotic DNA Replication & Genome Maintenanceワークショップ, 2013.09, The checkpoint clamp Rad9-Hus1-Rad1 (9-1-1) is a heterotrimeric PCNA-like complex that is loaded onto damaged DNA by Rad17-RFC clamp loader in a manner dependent on RPA. 9-1-1 interacts with the ATR-activator, TopBP1 through two casein kinase 2 (CK2)-phosphorylation sites, Ser-341 and Ser-387 in Rad9. Although the phospho-dependent interaction is essential for ATR-dependent checkpoint activation, neither the phosphorylation nor the interaction is damage inducible.
Here, we have studied the contribution of 9-1-1/TopBP1 interactions to the assembly and activation of checkpoint proteins at damaged DNA. UV-irradiation enhanced association of Rad9 with chromatin and its localization to sites of DNA damage in a manner that was independent of its CK2-mediated phosphorylation. TopBP1 as well as RPA and Rad17 facilitated DNA damage-dependent Rad9 recruitment. Similar to Rad9, TopBP1 also localized to sites of UV-induced DNA damage. However, the DNA damage-induced TopBP1 redistribution was delayed in cells expressing TopBP1 binding-deficient Rad9. Pharmacological inhibition of ATR recapitulated the delayed accumulation of TopBP1 in the cells but did not affect the accumulation of Rad9.
Thus, our results suggest that TopBP1 and Rad9 are recruited to damaged DNA independently. Once recruited, 9-1-1/TopBP1 interaction will contribute to generating a complex that is competent for ATR activation. In turn, the activated ATR facilitates further accumulation of TopBP1 and amplifies the checkpoint signal. This positive feedback mechanism will be necessary for successful DNA damage response in human cells.
|16.||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.
|17.||The iteraction between Rad9 and TopBP1 indirectly promotes TopBP1 accumulation at the sites of DNA damage through ATR activation.|
|18.||Function of the holocomplex between DNA polymerase e and PCNA loader Ctf18-RFC.|
|19.||Molecular crosstalk between DNA synthesis and chromosome cohesion apparatuses.|
|20.||Direct interaction of 9-1-1 with TopBP1 in human cells is not required for their initial accumulation at DNA damage sites but for subsequent activation of the DNA damage checkpoint.|
|21.||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.
|22.||The second PCNA loader Ctf18-RFC interacts with DNA polymerase ε in human cells..|
|23.||Analysis of intrinsic DNA binding activity of 9-1-1 complex.|
|24.||Roles of the 9-1-1/TopBP1 interaction for DNA damage responses in human cells..|
|25.||Analyses of interactions between TopBP1 and PCNA-Rad9Cter fusion protein.|
|26.||THE PHOSPHORYLATION OF RAD9 AT SER-341 AND SER-387 BY CK2 PROMOTES THE INTERACTION BETWEEN 9-1-1 AND TOPBP1..|
|27.||INTERACTION OF LOADER COMPLEXES WITH MULTIPLE REPLICATION PROTEINS IN HUMAN CELLS.|
|28.||Molecular interactions and reconstitutions of DNA replication reactions.|
|29.||Functional network of clamp-loader proteins in chromosomal DNA replication in human cells.|
|30.||CK2-phosphorylation dependent interaction between 9-1-1 and TopBP1.|
|31.||Functional Significance of specific interaction between loader complexes and MCM in human cells.|
|32.||Analyses of specific interactions between human loader complexes and MCM.|
|33.||Roles of the phosphorylation of Rad9 at Ser-341 and Ser-387 by CK2 in vitro.|
|34.||Roles of the Rad9 phosphorylation by CK2 in vivo.|
|35.||Multiple interaction networks of replication proteins in human cells..|
|36.||Studies on molecular interactions between clamp loader complexes and MCM subcomplexes.|
|37.||Studies on functional domains of human DNA polymerase epsilon catalytic subunit p261.|
|38.||Studies on molecular interactions between clamp loader complexes and MCM subcomplexes.|
|39.||Stimulation DNA polymerase activity with ubiquitianted PCNA.|
|40.||Studies on molecular interactions between clamp loader complexes and DNA replication proteins.|
|41.||Analyses on interaction between loader complexes and replication proteins in human cells.|
|42.||STUDIES ON MOLECULAR INTERACTIONS BETWEEN LOADER COMPLEXES AND REPLICATION FORK COMPONENTS.|
|43.||Studies on molecular interactions of clamp loader complexes with DNA polymerase e .|
|44.||Studies on molecular interactions of clamp loader complexes with DNA polymerase e and RPA.|
|45.||Studies on molecular interactions between clamp loader complexes and DNA replication proteins.|
|46.||Stimulation DNA polymerase activity with ubiquitianted PCNA.|
|47.||Analysis of human 9-1-1 complex interacting proteins.|
|48.||Functions of WRNIP1 (WRN helicase interaction protein1) as a novel regulatory factor for DNA polymerase delta.|
|49.||Studies on functional switch of PCNA: Specificity factors for two PCNA loaders, RFC and Chl12-RFC.|
|50.||Preparation of ubiquitinated PCNA and its functional analysis.|
|51.||Studies on specificity of PCNA loader proteins.|
|52.||Specific clamp-clamp loader systems, which mediate communications between DNA replication and repair reactions.|
|53.||Functions of human RFC family proteins for maintenance of replication fork activity.|