Kyushu University Academic Staff Educational and Research Activities Database
List of Papers
Toshiki Tsurimoto Last modified date:2019.06.07

Professor / Molecular Life Sciences / Department of Biology / Faculty of Sciences

1. Genki Hayashida, Seijiro Shioi, Kyoko Hidaka, Ryosuke Fujikane, Masumi Hidaka, Toshiki Tsurimoto, Teruhisa Tsuzuki, Shinya Oda, Yoshimichi Nakatsu, Differential genomic destabilisation in human cells with pathogenic MSH2 mutations introduced by genome editing, Experimental Cell Research, 10.1016/j.yexcr.2019.02.020, 377, 1-2, 24-35, 2019.04, Repeat destabilisation is variously associated with human disease. In neoplastic diseases, microsatellite instability (MSI) has been regarded as simply reflecting DNA mismatch repair (MMR) deficiency. However, several discrepancies have been pointed out. Firstly, the MSI + phenotype is not uniform in human neoplasms. Established classification utilises the frequency of microsatellite changes, i.e. MSI-H (high) and -L (low), the former regarded as an authentic MMR-defective phenotype. In addition, we have observed the qualitatively distinct modes of MSI, i.e. Type A and Type B. One discrepancy we previously pointed out is that tumours occurring in MMR gene knockout mice exhibited not drastic microsatellite changes typical in MSI-H tumours (i.e. Type B mode) but minor and more subtle alterations (i.e. Type A mode). In the present study, MSH2 mutations reported in Lynch syndrome (LS) kindred have been introduced into HeLa cells using the CRISPR/Cas9 system. The established mutant clones clearly exhibited MMR-defective phenotypes with alkylating agent-tolerance and elevated mutation frequencies. Nevertheless, microsatellites were not markedly destabilised as in MSI-H tumours occurring in LS patients, and all the observed alterations were uniformly Type A, which confirms the results in mice. Our findings suggest added complexities to the molecular mechanisms underlying repeat destabilisation in human genome..
2. Ewa Kowalska, Filip Bartnicki, Ryo Fujisawa, Piotr Bonarek, Pawet Hermanowicz, Toshiki Tsurimoto, Klaudia Muszynska, Wojciech Strzalka, Inhibition of DNA replication by an anti-PCNA aptamer/PCNA complex, Nucleic Acids Research, 10.1093/nar/gkx1184, 46, 1, 25-41, 2018.01, Proliferating cell nuclear antigen (PCNA) is a multifunctional protein present in the nuclei of eukaryotic cells that plays an important role as a component of the DNA replication machinery, as well as DNA repair systems. PCNA was recently proposed as a potential non-oncogenic target for anti-cancer therapy. In this study, using the Systematic Evolution of Ligands by EXponential enrichment (SELEX) method, we developed a short DNA aptamer that binds human PCNA. In the presence of PCNA, the anti-PCNA aptamer inhibited the activity of human DNA polymerase and at nM concentrations. Moreover, PCNA protected the anti-PCNA aptamer against the exonucleolytic activity of these DNA polymerases. Investigation of the mechanism of anti-PCNA aptamer-dependent inhibition of DNA replication revealed that the aptamer did not block formation, but was a component of PCNA/DNA polymerase or complexes. Additionally, the anti-PCNA aptamer competed with the primer-template DNA for binding to the PCNA/DNA polymerase or complex. Based on the observations, a model of anti-PCNA aptamer/PCNA complex-dependent inhibition of DNA replication was proposed..
3. Masataka Tsuda, Kazuhiro Terada, Masato Ooka, Koji Kobayashi, Hiroyuki Sasanuma, Ryo Fujisawa, Toshiki Tsurimoto, Junpei Yamamoto, Shigenori Iwai, Kei Kadoda, Remi Akagawa, Shar Yin Naomi Huang, Yves Pommier, Julian E. Sale, Shunichi Takeda, Kouji Hirota, The dominant role of proofreading exonuclease activity of replicative polymerase ε in cellular tolerance to cytarabine (Ara-C), Oncotarget, 10.18632/oncotarget.16508, 8, 20, 33457-33474, 2017.01, Chemotherapeutic nucleoside analogs, such as Ara-C, 5-Fluorouracil (5-FU) and Trifluridine (FTD), are frequently incorporated into DNA by the replicative DNA polymerases. However, it remains unclear how this incorporation kills cycling cells. There are two possibilities: Nucleoside analog triphosphates inhibit the replicative DNA polymerases, and/or nucleotide analogs mis-incorporated into genomic DNA interfere with the next round of DNA synthesis as replicative DNA polymerases recognize them as template DNA lesions, arresting synthesis. To address the first possibility, we selectively disrupted the proofreading exonuclease activity of DNA polymerase ε (Polε), the leading-strand replicative polymerase in avian DT40 and human TK6 cell lines. To address the second, we disrupted RAD18, a gene involved in translesion DNA synthesis, a mechanism that relieves stalled replication. Strikingly, POLE1exo-/- cells, but not RAD18-/- cells, were hypersensitive to Ara-C, while RAD18-/- cells were hypersensitive to FTD. γH2AX focus formation following a pulse of Ara-C was immediate and did not progress into the next round of replication, while γH2AX focus formation following a pulse of 5-FU and FTD was delayed to the next round of replication. Biochemical studies indicate that human proofreading-deficient Pole-exoholoenzyme incorporates Ara-CTP, but subsequently extend from this base several times less efficiently than from intact nucleotides. Together our results suggest that Ara-C acts by blocking extension of the nascent DNA strand and is counteracted by the proofreading activity of Pole, while 5-FU and FTD are efficiently incorporated but act as replication fork blocks in the subsequent S phase, which is counteracted by translesion synthesis..
4. 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, 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..
5. Eiji Ohashi, Toshiki Tsurimoto, Functions of multiple clamp and clamp-loader complexes in eukaryotic DNA replication, Advances in Experimental Medicine and Biology, 10.1007/978-981-10-6955-0_7, 135-162, 2017.01, Proliferating cell nuclear antigen (PCNA) and replication factor C (RFC) were identified in the late 1980s as essential factors for replication of simian virus 40 DNA in human cells, by reconstitution of the reaction in vitro. Initially, they were only thought to be involved in the elongation stage of DNA replication. Subsequent studies have demonstrated that PCNA functions as more than a replication factor, through its involvement in multiple protein-protein interactions. PCNA appears as a functional hub on replicating and replicated chromosomal DNA and has an essential role in the maintenance genome integrity in proliferating cells. Eukaryotes have multiple paralogues of sliding clamp, PCNA and its loader, RFC. The PCNA paralogues, RAD9, HUS1, and RAD1 form the heterotrimeric 9-1-1 ring that is similar to the PCNA homotrimeric ring, and the 9-1-1 clamp complex is loaded onto sites of DNA damage by its specific loader RAD17-RFC. This alternative clamp-loader system transmits DNA-damage signals in genomic DNA to the checkpoint-activation network and the DNA-repair apparatus. Another two alternative loader complexes, CTF18-RFC and ELG1-RFC, have roles that are distinguishable from the role of the canonical loader, RFC. CTF18-RFC interacts with one of the replicative DNA polymerases, Polε, and loads PCNA onto leading-strand DNA, and ELG1-RFC unloads PCNA after ligation of lagging-strand DNA. In the progression of S phase, these alternative PCNA loaders maintain appropriate amounts of PCNA on the replicating sister DNAs to ensure that specific enzymes are tethered at specific chromosomal locations..
6. Hirota K, Tsuda M, Mohiuddin, Tsurimoto T, Cohen IS, Livneh Z, Kobayashi K, Narita T, Nishihara K, Murai J, Iwai S, Guilbaud G, Sale JE, Takeda S, In vivo evidence for translesion synthesis by the replicative DNA polymerase δ., Nucleic Acids Research, 10.1093/nar/gkw439., 44, 7242-7250, 2016.09, The intolerance of DNA polymerase δ (Polδ) to incorrect base pairing contributes to its extremely high accuracy during replication, but is believed to inhibit translesion synthesis (TLS). However, chicken DT40 cells lacking the POLD3 subunit of Polδ are deficient in TLS. Previous genetic and biochemical analysis showed that POLD3 may promote lesion bypass by Polδ itself independently of the translesion polymerase Polζ of which POLD3 is also a subunit. To test this hypothesis, we have inactivated Polδ proofreading in pold3 cells. This significantly restored TLS in pold3 mutants, enhancing dA incorporation opposite abasic sites. Purified proofreading-deficient human Polδ holoenzyme performs TLS of abasic sites in vitro much more efficiently than the wild type enzyme, with over 90% of TLS events resulting in dA incorporation. Furthermore, proofreading deficiency enhances the capability of Polδ to continue DNA synthesis over UV lesions both in vivo and in vitro These data support Polδ contributing to TLS in vivo and suggest that the mutagenesis resulting from loss of Polδ proofreading activity may in part be explained by enhanced lesion bypass..
7. Kawasoe Y, Tsurimoto T, Nakagawa T, Masukata H, Takahashi TS, MutSα maintains the mismatch repair capability by inhibiting PCNA unloading., E Life, 10.7554/eLife.15155.001, 5, e15155, 2016.08, Eukaryotic mismatch repair (MMR) utilizes single-strand breaks as signals to target the strand to be repaired. DNA-bound PCNA is also presumed to direct MMR. The MMR capability must be limited to a post-replicative temporal window during which the signals are available. However, both identity of the signal(s) involved in the retention of this temporal window and the mechanism that maintains the MMR capability after DNA synthesis remain unclear. Using Xenopus egg extracts, we discovered a mechanism that ensures long-term retention of the MMR capability. We show that DNA-bound PCNA induces strand-specific MMR in the absence of strand discontinuities. Strikingly, MutSα inhibited PCNA unloading through its PCNA-interacting motif, thereby extending significantly the temporal window permissive to strand-specific MMR. Our data identify DNA-bound PCNA as the signal that enables strand discrimination after the disappearance of strand discontinuities, and uncover a novel role of MutSα in the retention of the post-replicative MMR capability..
8. Yang C-C, Suzuki M, Yamakawa S, Uno S, Ishii A, Yamazaki S, Fukatsu R, Fujisawa R, Sakimura K, Tsurimoto T, Masai H, Claspin recruits Cdc7 kinase for initiation of DNA replication in human cells., NATURE COMMUNICATIONS, 10.1038/ncomms12135., 7, 12135, 2016.07, Claspin transmits replication stress signal from ATR to Chk1 effector kinase as a mediator. It also plays a role in efficient replication fork progression during normal growth. Here we have generated conditional knockout of Claspin and show that Claspin knockout mice are dead by E12.5 and Claspin knockout mouse embryonic fibroblast (MEF) cells show defect in S phase. Using the mutant cell lines, we report the crucial roles of the acidic patch (AP) near the C terminus of Claspin in initiation of DNA replication. Cdc7 kinase binds to AP and this binding is required for phosphorylation of Mcm. AP is involved also in intramolecular interaction with a N-terminal segment, masking the DNA-binding domain and a newly identified PIP motif, and Cdc7-mediated phosphorylation reduces the intramolecular interaction. Our results suggest a new role of Claspin in initiation of DNA replication during normal S phase through the recruitment of Cdc7 that facilitates phosphorylation of Mcm proteins..
9. Okimoto H, Tanaka E, Araki H, Ohashi E, Tsurimoto T, Conserved interaction of Ctf18-RFC with DNA polymerase ε is critical for maintenance of genome stability in Saccharomyces cerevisiae. , Molecular Biology Society of Japan, Genes to Cells, 10.1111/gtc.12356., 21, 482-491, 2016.05, Human Ctf18-RFC, a PCNA loader complex, interacts with DNA polymerase e (Pole) through
a structure formed by the Ctf18, Dcc1 and Ctf8 subunits. The C-terminal stretch of Ctf18,
which is highly conserved from yeast to human, is necessary to form the Pole-capturing structure.
We found that in the budding yeast Saccharomyces cerevisiae, Ctf18, Dcc1 and Ctf8 formed
the same structure through the conserved C-terminus and interacted specifically with Pole.
Thus, the specific interaction of Ctf18-RFC with Pole is a conserved feature between these
proteins. A C-terminal deletion mutant of Ctf18 (ctf18DC) exhibited the same high sensitivity to
hydroxyurea as the complete deletion strain (ctf18D) or ATPase-deficient mutant (ctf18K189A),
but was somewhat less sensitive to methyl methanesulfonate than either of them. These phenotypes
were also observed in dcc1D and ctf8D, predicted to be deficient in the interaction with
Pole. Furthermore, both plasmid loss and gross chromosomal rearrangement (GCR) rates
were increased in ctf18DC cells to the same extent as in ctf18D cells. These results indicate
that the Ctf18-RFC/Pole interaction plays a crucial role in maintaining genome stability in
budding yeast, probably through recruitment of this PCNA loader to the replication fork..
10. Hironori Kawakami (corresponding author), Ohashi E, Tsurimoto T, Tsutomu Katayama, Rapid Purification and Characterization of Mutant Origin Recognition Complexes in Saccharomyces cerevisiae., Front Microbiol. , 10.3389/fmicb.2016.00521., 7, 2016.04, Purification of the origin recognition complex (ORC) from wild-type budding yeast cells more than two decades ago opened up doors to analyze the initiation of eukaryotic chromosomal DNA replication biochemically. Although revised methods to purify ORC from overproducing cells were reported later, purification of mutant proteins using these systems still depends on time-consuming processes including genetic manipulation to construct and amplify mutant baculoviruses or yeast strains as well as several canonical protein fractionations. Here, we present a streamlined method to construct mutant overproducers, followed by purification of mutant ORCs. Use of mammalian cells co-transfected with conveniently mutagenized plasmids bearing a His tag excludes many of the construction and fractionation steps. Transfection is highly efficient. All the six subunits of ORC are overexpressed at a considerable level and isolated as a functional heterohexameric complex. Furthermore, use of mammalian cells prevents contamination of wild-type ORC from yeast cells. The method is applicable to wild-type and at least three mutant ORCs, and the resultant purified complexes show expected biochemical activities. The rapid acquisition of mutant ORCs using this system will boost systematic biochemical dissection of ORC and can be even applied to the purification of protein complexes other than ORC. .
11. Le H P, Masuda Y, Tsurimoto T, Maki S, Tsutomu Katayama, Furukohri A, Maki H, Short CCG repeat in huntingtin gene is an obstacle for replicative DNA polymerases, potentially hampering progression of replication fork, The Molecular Biology Society of Japan, Genes to Cells, 10.1111/gtc.12275. , 20, 817-833, 2015.10, Trinucleotide repeats (TNRs) are highly unstable in genomes, and their expansions are linked to human disorders. DNA replication is reported to be involved in TNR instability, but the current models are insufficient in explaining TNR expansion is induced during replication. Here, we investigated replication fork progression across huntingtin (HTT)-gene-derived frag- ments using an Escherichia coli oriC plasmid DNA replication system. We found most of the forks to travel smoothly across the HTT fragments even when the fragments had a pathologi- cal length of CAG/CTG repeats (approximately 120 repeats). A little fork stalling in the frag- ments was observed, but it occurred within a short 30-flanking region downstream of the repeats. This region contains another short TNR, (CCG/CGG)7, and the sense strand contain- ing CCG repeats appeared to impede the replicative DNA polymerase Pol III. Examining the behavior of the human leading and lagging replicative polymerases Pol epsilon (hPole) and Pol delta (hPold) on this sequence, we found hPold replicating DNA across the CCG repeats but hPole stalling at the CCG repeats even if the secondary structure is eliminated by a single- stranded binding protein. These findings offer insights into the distinct behavior of leading and lagging polymerases at CCG/CGG repeats, which may be important for understanding the process of replication arrest and genome instability at the HTT gene..
12. Hironori Kawakami (corresponding author), Ohashi E, Kanamoto S, Tsurimoto T, Tsutomu Katayama, Specific binding of eukaryotic ORC to DNA replication origins depends on highly conserved basic residues., Scientific Reports , 10.1038/srep14929., 5, 2015.10, In eukaryotes, the origin recognition complex (ORC) heterohexamer preferentially binds replication origins to trigger initiation of DNA replication. Crystallographic studies using eubacterial and archaeal ORC orthologs suggested that eukaryotic ORC may bind to origin DNA via putative winged-helix DNA-binding domains and AAA+ ATPase domains. However, the mechanisms how eukaryotic ORC recognizes origin DNA remain elusive. Here, we show in budding yeast that Lys-362 and Arg-367 residues of the largest subunit (Orc1), both outside the aforementioned domains, are crucial for specific binding of ORC to origin DNA. These basic residues, which reside in a putative disordered domain, were dispensable for interaction with ATP and non-specific DNA sequences, suggesting a specific role in recognition. Consistent with this, both residues were required for origin binding of Orc1 in vivo. A truncated Orc1 polypeptide containing these residues solely recognizes ARS sequence with low affinity and Arg-367 residue stimulates sequence specific binding mode of the polypeptide. Lys-362 and Arg-367 residues of Orc1 are highly conserved among eukaryotic ORCs, but not in eubacterial and archaeal orthologs, suggesting a eukaryote-specific mechanism underlying recognition of replication origins by ORC..
13. Takeishi M, Iwaya R, Ohashi E, Tsurimoto T, Intramolecular Binding of the Rad9 C Terminus in the Checkpoint Clamp Rad9-Hus1-Rad1 Is Closely Linked with Its DNA Binding. , JOURNAL OF BIOLOGICAL CHEMISTRY, 10.1074/jbc.M115.669002., 290, 19923-19932, 2015.08, The human checkpoint clamp Rad9-Hus1-Rad1 (9-1-1) is
loaded onto chromatin by its loader complex, Rad17-RFC, following
DNA damage. The 120-amino acid (aa) stretch of the
Rad9 C terminus (C-tail) is unstructured and projects from the
core ring structure (CRS). Recent studies showed that 9-1-1 and
CRS bind DNA independently of Rad17-RFC. The DNA-binding
affinity of mutant 9C-1-1, which lacked the Rad9 C-tail, was
much higher than that of wild-type 9-1-1, suggesting that 9-1-1
has intrinsic DNA binding activity that manifests in the absence
of the C-tail. C-tail added in trans interacted with CRS and prevented
it from binding to DNA. We narrowed down the amino
acid sequence in the C-tail necessary for CRS binding to a 15-aa
stretch harboring two conserved consecutive phenylalanine residues.
We prepared 9-1-1 mutants containing the variant C-tail
deficient for CRS binding, and we demonstrated that the mutant
form restored DNA binding as efficiently as 9C-1-1. Furthermore,
we mapped the sequence necessary for TopBP1 binding
within the same 15-aa stretch, demonstrating that TopBP1 and
CRS share the same binding region in the C-tail. Indeed, we
observed their competitive binding to the C-tail with purified
proteins. The importance of interaction between 9-1-1 and
TopBP1 for DNA damage signaling suggests that the competitive
interactions of TopBP1 and CRS with the C-tail will be crucial
for the activation mechanism..
14. Kouji Hirota, Kazunori Yoshikiyo, Guillaume Guilbaud, Tsurimoto T, Junko Murai, Masataka Tsuda, Lara G. Phillips, Takeo Narita, Kana Nishihara, Kaori Kobayashi, Kouich Yamada, Jun Nakamura, Yves Pommier, Alan Lehmann, Julian E. Sale, Shunichi Takeda, The POLD3 subunit of DNA polymerase delta can promote translesion synthesis independently of DNA polymerase zeta, NUCLEIC ACIDS RESEARCH, 10.1093/nar/gkv023, 43, 3, 1671-1683, 2015.02, The replicative DNA polymerase Polδ consists of a
catalytic subunit POLD1/p125 and three regulatory
subunits POLD2/p50, POLD3/p66 and POLD4/p12.
The ortholog of POLD3 inSaccharomyces cerevisiae,
Pol32, is required for a significant proportion of spontaneous
and UV-induced mutagenesis through its
additional role in translesion synthesis (TLS) as a
subunit of DNA polymerase δ . Remarkably, chicken
DT40 B lymphocytes deficient in POLD3 are viable
and able to replicate undamaged genomic DNA with
normal kinetics. Like its counterpart in yeast, POLD3
is required for fully effective TLS, its loss resulting
in hypersensitivity to a variety of DNA damaging
agents, a diminished ability to maintain replication
fork progression after UV irradiation and a significant
decrease in abasic site-induced mutagenesis in the
immunoglobulin loci. However, these defects appear
to be largely independent of Polδ , suggesting that
POLD3 makes a significant contribution to TLS independently
of Polδ in DT40 cells. Indeed, combining
polη, polζ and pold3 mutations results in synthetic
lethality. Additionally, we show in vitro that POLD3
promotes extension beyond an abasic by the Polδ 
holoenzyme suggesting that while POLD3 is not required
for normal replication, it may help Polδ to complete
abasic site bypass independently of canonical
TLS polymerases..
15. Eiji Ohashi, Yukimasa Takeishi, Satoshi Ueda, Tsurimoto T, Interaction between Rad9-Hus1-Rad1 and TopBP1 activates ATR-ATRIP and promotes TopBP1 recruitment to sites of UV-damage, DNA REPAIR, 10.1016/j.dnarep.2014.05.001, 21, 1-11, 2014.09, The checkpoint clamp Rad9–Hus1–Rad1 (9–1–1) interacts with TopBP1 via two casein kinase 2 (CK2)-phosphorylation sites, Ser-341 and Ser-387 in Rad9. While this interaction is known to be important forthe activation of ATR-Chk1 pathway, how the interaction contributes to their accumulation at sites of DNAdamage remains controversial. Here, we have studied the contribution of the 9–1–1/TopBP1 interaction tothe assembly and activation of checkpoint proteins at damaged DNA. UV-irradiation enhanced associationof Rad9 with chromatin and its localization to sites of DNA damage without a direct interaction withTopBP1. TopBP1, as well as RPA and Rad17 facilitated Rad9 recruitment to DNA damage sites. Similarto Rad9, TopBP1 also localized to sites of UV-induced DNA damage. The DNA damage-induced TopBP1redistribution was delayed in cells expressing a TopBP1 binding-deficient Rad9 mutant. Pharmacologicalinhibition of ATR recapitulated the delayed accumulation of TopBP1 in the cells, suggesting that ATRactivation will induce more efficient accumulation of TopBP1. Taken together, TopBP1 and Rad9 can beindependently recruited to damaged DNA. Once recruited, a direct interaction of 9–1–1/TopBP1 occursand induces ATR activation leading to further TopBP1 accumulation and amplification of the checkpointsignal. Thus, we propose a new positive feedback mechanism that is necessary for successful formationof the damage-sensing complex and DNA damage checkpoint signaling in human cells..
16. Sun Q, Juillard F, Li, S, Vazquez E DL, Tsurimoto T, Kaye K, Li L, Chen, She, Kaposi's sarcoma-associated herpesvirus LANA recruits the DNA polymerase clamp loader to mediate efficient replication and virus persistence, Proc Natl Acad Sci U S A., 10.1073/pnas.1404219111, 111, 32, 11816-11821, 2014.08, Kaposi’s sarcoma herpesvirus (KSHV) latency-associated nuclear antigen (LANA)
mediates viral episomal replication and persistence but little is known regarding the
underlying mechanisms. We find that LANA recruits replication factor C (RFC), the
DNA polymerase clamp (proliferating cell nuclear antigen (PCNA)) loader, to efficiently
drive DNA replication. Mutated LANA lacking RFC interaction was deficient for LANA
mediated DNA replication and episome persistence. RFC depletion severely impacted
LANA’s ability to replicate and maintain viral DNA in cells containing artificial KSHV
episomes or in infected cells, leading to loss of virus. LANA greatly increased PCNA
loading onto DNA in vitro and recruited RFC and PCNA to KSHV DNA in cells. These
findings suggest that PCNA loading is a rate-limiting step in DNA replication that is
incompatible with viral survival. LANA enhancement of PCNA loading permits efficient
virus replication and persistence, revealing a novel mechanism for KSHV latency..
17. Strzalka W, Bartnicki F, Pels K,, Jakubowska A, Toshiki Tsurimoto, Tanaka K, RAD5a ubiquitin ligase is involved in ubiquitination of Arabidopsis thaliana proliferating cell nuclear antigen , Journal of Experimental Botany, 64, 4, 859-869, 2013.01, The proliferating cell nuclear antigen (PCNA) is post-translationally modified by ubiquitin in yeast and mammalian cells. It is widely accepted that in yeast mono- and polyubiquitinated PCNA is involved in distinct pathways of DNA postreplication repair. This study showed an interaction between plant ubiquitin and PCNA in the plant cell. Using different approaches, it was demonstrated that Arabidopsis RAD5a ubiquitin ligase is involved in the post-translational modification of plant PCNA. A detailed analysis of the properties of selected Arabidopsis ubiquitin-conjugating enzymes (AtUBC) has shown that a plant homologue of yeast RAD6 (AtUBC2) is sufficient to monoubiquitinate AtPCNA in the absence of ubiquitin ligase. Using different combinations of selected AtUBC proteins together with AtRAD5a, it was demonstrated that plants have potential to use different pathways to ubiquitinate PCNA. The analysis of Arabidopsis PCNA1 and PCNA2 did not demonstrate substantial differences in the ubiquitination pattern between these two proteins. The major ubiquitination target of Arabidopsis PCNA, conserved in eukaryotes, is lysine 164. Taken together, the presented results clearly demonstrate the involvement of Arabidopsis UBC and RAD5a proteins in the ubiquitination of plant PCNA at lysine 164. The data show the complexity of the plant ubiquitination system and open new questions about its regulation in the plant cell..
18. Nakao S, Zhang S, Vaara M, Syväoja JE, Lee MY, Toshiki Tsurimoto, Karran P, Oda S, Efficient long DNA gap-filling in a mammalian cell-free system: a potential new in vitro DNA replication assay., Biochimie. , 95, 2, 320-328, 95(2):320-8., 2013.02, In vitro assay of mammalian DNA replication has been variously approached. Using gapped circular duplex substrates containing a 500-base single-stranded DNA region, we have constructed a mammalian cell-free system in which physiological DNA replication may be reproduced. Reaction of the gapped plasmid substrate with crude extracts of human HeLaS3 cells induces efficient DNA synthesis in vitro. The induced synthesis was strongly inhibited by aphidicolin and completely depended on dNTP added to the system. In cell extracts in which PCNA was depleted step-wise by immunoprecipitation, DNA synthesis was accordingly reduced. These data suggest that replicative DNA polymerases, particularly pol delta, may chiefly function in this system. Furthermore, DNA synthesis is made quantifiable in this system, which enables us to evaluate the efficiency of DNA replication induced. Our system sensitively and quantitatively detected the reduction of the DNA replication efficiency in the DNA substrates damaged by oxidation or UV cross-linking and in the presence of a potent chain terminator, ara-CTP. The quantitative assessment of mammalian DNA replication may provide various advantages not only in basic research but also in drug development..
19. Ueda S, Takeishi Y, Eiji Ohashi, Toshiki Tsurimoto, Two serine phosphorylation sites in the C-terminus of Rad9 are critical for 9-1-1 binding to TopBP1 and activation of the DNA damage checkpoint response in HeLa cells., Genes to Cells, 17, 807-816, 2012.10, A heteromeric proliferating cell nuclear antigen-like ring complex 9-1-1 is comprised of Rad9, Hus1 and Rad1. When assembled, 9-1-1 binds to TopBP1 and activates the ATR-Chk1 checkpoint pathway. This binding in vitro depends on the phosphorylation of Ser-341 and Ser-387 in Rad9 and is reduced to 70% and 20% by an alanine substitution for Ser-341 (S341A) and Ser-387 (S387A), respectively, and to background level by their simultaneous substitution (2A). Here, we show the importance of phosphorylation of these two serine residues in vivo. siRNA-mediated knockdown of Rad9 in HeLa cells impaired UV-induced phosphorylation of checkpoint kinase, Chk1, and conferred hypersensitivity to UV irradiation and to methyl methane sulfonate or hydroxyurea treatments. Either siRNA-resistant wild-type Rad9 (Rad9R(r) ) or Rad9R(r) harboring the S341A substitution restored the phosphorylation of Chk1 and damage sensitivity, whereas Rad9R(r) harboring S387A or 2A did not. However, high expression of S387A restored Chk1 phosphorylation and partially suppressed the hypersensitivity. Thus, the affinity of Rad9 to TopBP1 correlates with the activation of the cellular DNA damage response and survival after DNA damage in HeLa cells, and phosphorylation of Ser-341 and Ser-387 of Rad9 is critical for full activation of the checkpoint response to DNA damage..
20. Moriyama K, Yoshizawa-Sugata N, Obuse C, Tsurimoto T, Masai H., EBNA1-dependent recruitment of Orc on OriP of Epstein-Barr Virus with purified proteins: Stimulation by Cdc6 through its direct interaction with EBNA1., J. Biol. Chem. , 287, 28, 23977-94, 2012.07, Origin recognition complex (Orc) plays an essential role in directing assembly of prereplicative complex at selective sites on chromosomes. However, Orc from vertebrates is reported to bind to DNA in a sequence-nonspecific manner, and it is still unclear how it selects specific genomic loci and how Cdc6, another conserved AAA(+) factor known to interact with Orc, participates in this process. Replication from oriP, the latent origin of Epstein-Barr virus, provides an excellent model system for the study of initiation on the host chromosomes because it is known to depend on prereplicative complex factors, including Orc and Mcm. Here, we show that Orc is recruited selectively at the essential dyad symmetry element in nuclear extracts in a manner dependent on EBNA1, which specifically binds to dyad symmetry. With purified proteins, EBNA1 can recruit both Cdc6 and Orc independently on a DNA containing EBNA1 binding sites, and Cdc6 facilitates the Orc recruitment by EBNA1. Purified Cdc6 directly binds to EBNA1, whereas association of Orc with EBNA1 requires the presence of the oriP DNA. Nuclease protection assays suggest that Orc associates with DNA segments on both sides adjacent to the EBNA1 binding sites and that this process is stimulated by the presence of Cdc6. Thus, EBNA1 can direct localized assembly of Orc in a process that is facilitated by Cdc6. The possibility of similar modes of recruitment of Orc/Cdc6 at the human chromosomal origins will be discussed..
21. Sugiyama T, Chino M, Tsurimoto T, Nozaki N, Ishimi Y, Interaction of heliquinomycin with single-stranded DNA inhibits MCM4/6/7 helicase, J. Biochem, 39, 129-137, 2012.02.
22. Hayashi-Takanaka Y, Yamagata K, Wakayama T, Stasevich TJ, Kainuma T, Tsurimoto T, Tachibana M, Shinkai Y, Kurumizaka H, Nozaki N, Kimura H., Tracking epigenetic histone modifications in single cells using Fab-based live endogenous modification labeling, Nucleic Acids Research, 39, 6475–6488, 2011.05.
23. Kanamori M, Seki M, Yoshimura A, Tsurimoto T, Tada S, Enomoto T., Werner interacting protein 1 promotes binding of Werner protein to template-primer DNA, Biol. Pharm. Bull. , 34, 1314—1318, 2011.03.
24. Narita T, Tsurimoto T, Yamamoto J, Nishihara K, Ogawa K, Ohashi E, Evans T, Iwai S, Takeda S, and Hirota K., Human replicative DNA polymerase delta can bypass T-T (6-4) ultraviolet photoproducts on template strands, Genes to Cells, 15, 1228-1239, 2010.12.
25. Murakami T, Takano R, Takeo S, Taniguchi R, Ogawa K, Ohashi E, and Tsurimoto T., Stable interaction between the human PCNA loader complex Ctf18-RFC and DNA polymerase epsilon is mediated by the cohesion specific subunits, Ctf18, Dcc1 and Ctf8. , J. Biol. Chem, 285, 34608-34615 , 2010.09.
26. Takeishi, Y., Eiji Ohashi, E., Ogawa, K., Masai, H., Obuse, C., Tsurimoto, T., Casein Kinase 2-dependent phosphorylation of human Rad9 mediates the interaction between human Rad9-Hus1-Rad1 complex and TopBP1, Genes to Cells, 15, 761-771 , 2010.07.
27. Yoshimura, A., Seki, Kanamori, M., Takeishi, S., Tsurimoto, T., Tada, S., Enomoto, T. , Physical and functional interaction between WRNIP1 and RAD18. , Genes Genet. Syst., 84, 171-178, 2009.03.
28. Nishitani H, Shiomi Y, Iida H, Michishita M, Takami,T, Tsurimoto T. , CDK inhibitor p21 is degraded by a PCNA coupled Cul4-DDB1Cdt2 pathway during S phase and after UV irradiation, J Biol Chem., 283, 29045-29052, 2008.07.
29. Tomida J, Masuda Y, Hiroaki H, Ishikawa T, Song I, Tsurimoto T, Tateishi S, Shiomi T, Kamei Y, Kim J, Kamiya K, Vaziri C, Ohmori H, Todo T., DNA damage induced ubiquitylation of RFC2 subunit of RFC complex. , J Biol Chem., 283, 9071-9079, 2008.02.
30. Tsuji, Y.,Watanabe, K., Araki, K., Shinohara, M., Yamagata, Y., Tsurimoto, T., Hanaoka, F., Yamamura, K., Yamaizumi, M., and Tateishi, S., Recognition of forked and single-stranded DNA structures by human RAD18 complexed with RAD6B protein triggers its recruitment to stalled replication forks., Genes Cells , 13, 343-354, 2008.02.
31. Masuda Y, Suzuki M, Piao J, Gu Y, Tsurimoto T, Kamiya K., Dynamics of human replication factors in the elongation phase of DNA replication., Nucleic Acids Res., 35, 6904-6916, 2007.10.
32. 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.
33. Nishitani H, Sugimoto N, Roukos V, Nakanishi Y, Saijo M, Obuse C, Tsurimoto T, Nakayama KI, Nakayama K, Fujita M, Lygerou Z, Nishimoto T, Two E3 ubiquitin ligases, SCF-Skp2 and DDB1-Cul4, target human Cdt1 for proteolysis., EMBO J., 25, 1126-1136, 2006.08.
34. Watanabe, K., Tateishi, S., Kawasuji, M., Tsurimoto, T., Inoue, H., Yamaizumi M., Rad18 guides polη to replication stalling sites through physical interaction and PCNA monoubiquitination., EMBO J., 10.1038/sj.emboj.7600383, 23, 19, 3886-3896, 23, 3886-3896, 2004.11.
35. Shiomi, Y., Shinozaki, A. Sugimoto, K., Usukura, J., Obuse C. Tsurimoto, T., Reconstituted human Chl12-RFC complex functions as a second PCNA loader., Genes Cells, 9, 279-290, 2004.01.
36. Ohta, S., Tatsumi, Y., Fujita, M., Tsurimoto, T., Obuse, C., The ORC1 cycle in human cells: II. Dynamic changes in the human ORC complex during the cell cycle., J. Biol. Chem., 278, 41535-41540, 2003.01.
37. Tatsumi, Y., Ohta, S., Kimura, H., Tsurimoto, T., Obuse, C., The ORC1 cycle in human cells: I. Cell cycle-regulated oscillation of human ORC1., J. Biol. Chem., 278, 41528-41534, 2003.01.
38. Tadokoro, R., Fujita, M., Miura, H., Shirahige, K., Yoshikawa, H., Tsurimoto, T., Obuse, C., Scheduled Conversion of Replication Complex Architecture at Replication Origins of S. cerevisiae during the cell cycle., J. Biol. Chem., 277, 15881-15889, 2002.01.
39. Iida, T., Suetake, I., Tajima, S., Morioka, H., Ohta, S., Obuse, C., Tsurimoto, T, PCNA clamp facilitates action of DNA cytosine methyltransferase 1 on hemimetylated DNA., Genes Cells, 7, 997-1007, 2002.01.
40. Shiomi, Y., Shinozaki, A. Nakada, D. Sugimoto, K., Usukura, J., Obuse C. Tsurimoto, T., Clamp and clamp loader structures of human checkpoint protein complexes, Rad9-1-1 and Rad17-RFC., Genes Cells, 7, 861-868, 2002.01.
41. 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.
42. Shikata, K., Ohta, S., Yamada, K., Obuse, C., Yoshikawa H., Tsurimoto, T., The human homologue of fission yeast cdc27, p66, is a component of active human DNA polymerase d., J. Biochem., 129, 699 - 708, 2001.01.
43. Ohki, R. Tsurimoto, T. Ishikawa, F., In vitro reconstitution of the end replication problem., Mol Cel. Biol., 21, 5753-5766., 2001.01.
44. Ishino, Y. Tsurimoto, T. Ishino, S. Cann, I. K. O., Functional interaction of an archaeal sliding clamp with mammalian clamp loader and DNA polymerase d., Genes Cells., 6, 699-706., 2001.01.
45. Tatsumi Y, Tsurimoto T, Shirahige K, Yoshikawa H, Obuse C., Association of human origin recognition complex 1 with chromatin DNA and nuclease-resistant nuclear structures., J Biol Chem., 275, 5904-5910, 2000.01.
46. 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.
47. Hohmura, K., Itokazu, Y., Yoshimura, S., Mizuguchi, G., Masamura, Y., Takeyasu, K., Shiomi, Y., Tsurimoto, T., Nishijima, H., Akita S., Nakamura Y., AFM with carbon nanotube probe resolves the subunit organization of protein complexes., J Electron. Microsc., 49, 415-21, 2000.01.
48. Fujita M, Hori Y, Shirahige K, Tsurimoto T, Yoshikawa H, Obuse C., Cell cycle dependent topological changes of chromosomal replication origins in Saccharomyces cerevisiae., Genes Cells., 3, 737-49., 1999.01.
49. Kobayashi H, Sato K, Komatsu Y, Morioka H, Stewart JD, Tsurimoto T, Ohtsuka E., Effects of a high-affinity antibody fragment on DNA polymerase reactions near a (6-4) photoproduct site., Photochem Photobiol., 69, 226-230, 1999.01.
50. Shirahige K, Hori Y, Shiraishi K, Yamashita M, Takahashi K, Obuse C, Tsurimoto T, Yoshikawa H., Regulation of DNA-replication origins during cell-cycle progression., Nature, 395, 618-621, 1998.01.
51. Oku T, Ikeda S, Sasaki H, Fukuda K, Morioka H, Ohtsuka E, Yoshikawa H, Tsurimoto T., Functional sites of human PCNA which interact with p21 (Cip1/Waf1), DNA polymerase delta and replication factor C., Genes Cells, 3, 357-369, 1998.01.