Publications by authors named "Takashi Tsuyama"

11 Publications

  • Page 1 of 1

N-terminal region of RecQ4 inhibits non-homologous end joining and chromatin association of the Ku heterodimer in Xenopus egg extracts.

Gene 2021 Jun 15;787:145647. Epub 2021 Apr 15.

Department of Molecular Biology, Faculty of Pharmaceutical Sciences, Toho University, Funabashi-shi, Chiba 274-8510, Japan. Electronic address:

RecQ4, a member of the RecQ helicase family, is required for the maintenance of genome integrity. RecQ4 has been shown to promote the following two DNA double-strand break (DSB) repair pathways: non-homologous end joining (NHEJ) and homologous recombination (HR). However, its molecular function has not been fully elucidated. In the present study, we aimed to investigate the role of RecQ4 in NHEJ using Xenopus egg extracts. The N-terminal 598 amino acid region of Xenopus RecQ4 (N598), which lacks a central helicase domain and a downstream C-terminal region, was added to the extracts and its effect on the joining of DNA ends was analyzed. We found that N598 inhibited the joining of linearized DNA ends in the extracts. In addition, N598 inhibited DSB-induced chromatin binding of Ku70, which is essential for NHEJ, while the DSB-induced chromatin binding of the HR-associated proteins, replication protein A (RPA) and Rad51, increased upon the addition of N598. These results suggest that RecQ4 possibly influences the choice of the DSB repair pathway by influencing the association of the Ku heterodimer with the DNA ends.
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http://dx.doi.org/10.1016/j.gene.2021.145647DOI Listing
June 2021

Mutant analysis of Cdt1's function in suppressing nascent strand elongation during DNA replication in Xenopus egg extracts.

Biochem Biophys Res Commun 2017 09 8;490(4):1375-1380. Epub 2017 Jul 8.

Department of Molecular Biology, Faculty of Pharmaceutical Sciences, Toho University, Funabashi-shi, Chiba 274-8510, Japan. Electronic address:

The initiation of DNA replication is strictly regulated by multiple mechanisms to ensure precise duplication of chromosomes. In higher eukaryotes, activity of the Cdt1 protein is temporally regulated during the cell cycle, and deregulation of Cdt1 induces DNA re-replication. In previous studies, we showed that excess Cdt1 inhibits DNA replication by suppressing progression of replication forks in Xenopus egg extracts. Here, we investigated the functional regions of Cdt1 that are required for the inhibition of DNA replication. We constructed a series of N-terminally or C-terminally deleted mutants of Cdt1 and examined their inhibitory effects on DNA replication in Xenopus egg extracts. Our results showed that the region spanning amino acids (a. a.) 255-620 is required for efficient inhibition of DNA replication, and that, within this region, a. a. 255-289 have a critical role in inhibition. Moreover, one of the Cdt1 mutants, Cdt1 R285A, was compromised with respect to the licensing activity but still inhibited DNA replication. This result suggests that Cdt1 has an unforeseen function in the negative regulation of DNA replication, and that this function is located within a molecular region that is distinct from those required for the licensing activity.
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http://dx.doi.org/10.1016/j.bbrc.2017.07.034DOI Listing
September 2017

Excess Cdt1 inhibits nascent strand elongation by repressing the progression of replication forks in Xenopus egg extracts.

Biochem Biophys Res Commun 2016 Feb 8;470(2):405-410. Epub 2016 Jan 8.

Faculty of Pharmaceutical Sciences, Teikyo Heisei University, Nakano-ku, Tokyo, Japan; Department of Molecular Biology, Faculty of Pharmaceutical Sciences, Toho University, Funabashi-shi, Chiba, Japan. Electronic address:

Cdt1 is a protein essential for initiation of DNA replication; it recruits MCM helicase, a core component of the replicative DNA helicase, onto replication origins. In our previous study, we showed that addition of excess Cdt1 inhibits nascent strand elongation during DNA replication in Xenopus egg extracts. In the present study, we investigated the mechanism behind the inhibitory effect of Cdt1. We found that addition of recombinant Cdt1 inhibited nascent DNA synthesis in a reinitiation-independent manner. To identify the mechanism by which Cdt1 inhibits nascent strand elongation, the effect of Cdt1 on loading of Mcm4 and Rpa70 onto chromatin was examined. The results showed that Cdt1 suppressed the excessive Rpa70 binding caused by extensive, aphidicolin-induced DNA unwinding; this unwinding occurs between stalled DNA polymerases and advancing replication forks. These findings suggested that excess Cdt1 suppressed the progression of replication forks.
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http://dx.doi.org/10.1016/j.bbrc.2016.01.028DOI Listing
February 2016

Structure of the Cdt1 C-terminal domain: conservation of the winged helix fold in replication licensing factors.

Protein Sci 2009 Nov;18(11):2252-64

The Magnetic Resonance Team, Korea Basic Science Institute, 804-1 Yangchung-Ri, Ochang, Chungbuk 363-883, South Korea.

In eukaryotic replication licensing, Cdt1 plays a key role by recruiting the MCM2-7 complex onto the origin of chromosome. The C-terminal domain of mouse Cdt1 (mCdt1C), the most conserved region in Cdt1, is essential for licensing and directly interacts with the MCM2-7 complex. We have determined the structures of mCdt1CS (mCdt1C_small; residues 452 to 557) and mCdt1CL (mCdt1C_large; residues 420 to 557) using X-ray crystallography and solution NMR spectroscopy, respectively. While the N-terminal 31 residues of mCdt1CL form a flexible loop with a short helix near the middle, the rest of mCdt1C folds into a winged helix structure. Together with the middle domain of mouse Cdt1 (mCdt1M, residues 172-368), this study reveals that Cdt1 is formed with a tandem repeat of the winged helix domain. The winged helix fold is also conserved in other licensing factors including archaeal ORC and Cdc6, which supports an idea that these replication initiators may have evolved from a common ancestor. Based on the structure of mCdt1C, in conjunction with the biochemical analysis, we propose a binding site for the MCM complex within the mCdt1C.
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http://dx.doi.org/10.1002/pro.236DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2788280PMC
November 2009

Repression of nascent strand elongation by deregulated Cdt1 during DNA replication in Xenopus egg extracts.

Mol Biol Cell 2009 Feb 8;20(3):937-47. Epub 2008 Dec 8.

Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi, 980-8578, Japan.

Excess Cdt1 reportedly induces rereplication of chromatin in cultured cells and Xenopus egg extracts, suggesting that the regulation of Cdt1 activity by cell cycle-dependent proteolysis and expression of the Cdt1 inhibitor geminin is crucial for the inhibition of chromosomal overreplication between S phase and metaphase. We analyzed the consequences of excess Cdt1 for DNA replication and found that increased Cdt1 activity inhibited the elongation of nascent strands in Xenopus egg extracts. In Cdt1-supplemented extracts, overreplication was remarkably induced by the further addition of the Cdt1-binding domain of geminin (Gem79-130), which lacks licensing inhibitor activity. Further analyses indicated that fully active geminin, as well as Gem79-130, restored nascent strand elongation in Cdt1-supplemented extracts even after the Cdt1-induced stalling of replication fork elongation had been established. Our results demonstrate an unforeseen, negative role for Cdt1 in elongation and suggest that its function in the control of replication should be redefined. We propose a novel surveillance mechanism in which Cdt1 blocks nascent chain elongation after detecting illegitimate activation of the licensing system.
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http://dx.doi.org/10.1091/mbc.e08-06-0613DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2633378PMC
February 2009

Possible involvement of RecQL4 in the repair of double-strand DNA breaks in Xenopus egg extracts.

Biochim Biophys Acta 2007 Apr 18;1773(4):556-64. Epub 2007 Jan 18.

Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi 980-8578, Japan.

Mutations in RecQL4 are a causative factor in Rothmund-Thomson syndrome, a human autosomal recessive disorder characterized by premature aging. To study the role of RecQL4, we employed a cell-free experimental system consisting of Xenopus egg extracts. RecQL4 loading onto chromatin was observed regardless of the presence or absence of EcoRI. However, in the absence of EcoRI, RecQL4 loading was suppressed by geminin, an inhibitor of pre-replicative complex formation, while in the presence of EcoRI, it was not affected. These results suggest that under the former condition, RecQL4-loading depended on DNA replication, while under the latter, the interaction occurred in response to double-stranded DNA breaks (DSBs) induced by EcoRI. DSB-induced RecQL4 loading depended on the function of the ataxia-telangiectasia mutated protein, DNA-dependent protein kinase (DNA-PK), and replication protein A, while there were only minor changes in DNA replication-associated RecQL4 loading upon suppression of these proteins. Furthermore, analyses using a chromatin-immunoprecipitation assay and quantification of gammaH2AX after induction of DSBs suggested that RecQL4 is loaded adjacent to Ku heterodimer-binding sites on damaged chromatin, and functions in the repair of DSBs.
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http://dx.doi.org/10.1016/j.bbamcr.2007.01.005DOI Listing
April 2007

Chromatin loading of Smc5/6 is induced by DNA replication but not by DNA double-strand breaks.

Biochem Biophys Res Commun 2006 Dec 2;351(4):935-9. Epub 2006 Nov 2.

Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi 980-8578, Japan.

Smc6, a member of the structural maintenance of chromosomes (SMC) family of proteins, forms a complex with related Smc5. Genetic analyses of yeast have demonstrated the involvement of Smc6 in DNA repair and checkpoint responses. In this study, we investigated the role of the Smc5/6 complex in higher eukaryotes by analyzing its behavior in Xenopus laevis egg extracts. Smc5/6 was loaded onto chromatin during DNA replication in a manner dependent on the initiation of DNA synthesis, and it dissociated from chromatin during mitosis. Moreover, the induction of DNA double-strand breaks following replication did not significantly affect the amount of chromatin-associated Smc6. These findings suggest that the Smc5/6 complex is regulated during the cell cycle, presumably in anticipation of DNA damage that may arise during replication.
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http://dx.doi.org/10.1016/j.bbrc.2006.10.133DOI Listing
December 2006

Analyses of the interaction of WRNIP1 with Werner syndrome protein (WRN) in vitro and in the cell.

DNA Repair (Amst) 2006 Jul;5(7):816-28

Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba 6-3, Aramaki, Sendai 980-8578, Japan

Werner was originally identified as a protein that interacts with the product of the Werner syndrome (WS) gene, WRN. To examine the function of the WRNIP1/WRN complex in cells, we generated knock-out cell lines that were deficient in either WRN (WRN(-/-)), WRNIP1 (WRNIP10(-/-/-)), or both (WRNIP1(-/-/-)/WRN(-/-)), using a chicken B lymphocyte cell line, DT40. WRNIP1(-/-/-)/WRN(-/-) DT40 cells grew at a similar rate as wild-type cells, but the rate of spontaneous sister-chromatid exchange was augmented compared to that of either of the single mutant cell lines. Moreover, while WRNIP1(-/-/-) and WRN(-/-) cells were moderately sensitive to camptothecin (CPT), double mutant cells showed a synergistic increase in CPT sensitivity. This suggested that WRNIP1 and WRN do not always function cooperatively to repair DNA lesions. The lack of a discernable functional interaction between WRNIP1 and WRN prompted us to reevaluate the nature of the physical interaction between these proteins. We found that MBP-tagged WRNIP1 interacted directly with WRN, and that the interaction was enhanced by the addition of ATP. Mutations in the Walker A motifs of the two proteins revealed that WRNIP1, but not WRN, must bind ATP before an efficient interaction can occur.
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http://dx.doi.org/10.1016/j.dnarep.2006.04.006DOI Listing
July 2006

Caenorhabditis elegans geminin homologue participates in cell cycle regulation and germ line development.

J Biol Chem 2005 May 4;280(20):19689-94. Epub 2005 Apr 4.

Cellular Physiology Laboratory, Discovery Research Institute, RIKEN, Wako, Saitama, Japan.

Cdt1 is an essential component for the assembly of a pre-replicative complex. Cdt1 activity is inhibited by geminin, which also participates in neural development and embryonic differentiation in many eukaryotes. Although Cdt1 homologues have been identified in organisms ranging from yeast to human, geminin homologues had not been described for Caenorhabditis elegans and fungi. Here, we identify the C. elegans geminin, GMN-1. Biochemical analysis reveals that GMN-1 associates with C. elegans CDT-1, the Hox protein NOB-1, and the Six protein CEH-32. GMN-1 inhibits not only the interaction between mouse Cdt1 and Mcm6 but also licensing activity in Xenopus egg extracts. RNA interference-mediated reduction of GMN-1 is associated with enlarged germ nuclei with aberrant nucleolar morphology, severely impaired gametogenesis, and chromosome bridging in intestinal cells. We conclude that the Cdt1-geminin system is conserved throughout metazoans and that geminin has evolved in these taxa to regulate proliferation and differentiation by directly interacting with Cdt1 and homeobox proteins.
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http://dx.doi.org/10.1074/jbc.C500070200DOI Listing
May 2005

Licensing for DNA replication requires a strict sequential assembly of Cdc6 and Cdt1 onto chromatin in Xenopus egg extracts.

Nucleic Acids Res 2005 1;33(2):765-75. Epub 2005 Feb 1.

Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University Aoba-ku, Sendai, Miyagi 980-8578, Japan.

Replication origins are licensed for a single initiation event by the loading of Mcm2-7 proteins during late mitosis and G1. Sequential associations of origin recognition complex, Cdc6 and Mcm2-7 are essential for completion of the licensing. Although Cdt1 also binds to the chromatin when the licensing reaction takes place, whether the binding is a requirement for Cdt1 to function is unclear. To analyze the relevance of the chromatin association of Cdt1, we carried out chromatin transfer experiments using either immunodepleted Xenopus egg extracts or purified proteins. Licensing assay and immunoblotting analyses indicated that Cdt1 could only license DNA replication and load Mcm2-7 onto DNA when it binds to chromatin that has already associated with Cdc6. These results provide evidence supporting that Cdc6 and Cdt1 must bind to chromatin in a strict order for DNA licensing to occur.
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http://dx.doi.org/10.1093/nar/gki226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC548366PMC
February 2005

Focus-formation of replication protein A, activation of checkpoint system and DNA repair synthesis induced by DNA double-strand breaks in Xenopus egg extract.

J Cell Sci 2002 Aug;115(Pt 15):3159-69

Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba-ku, Sendai, Miyagi 980-8578, Japan.

The response to DNA damage was analyzed using a cell-free system consisting of Xenopus egg extract and demembranated sperm nuclei. In the absence of DNA-damaging agents, detergent-resistant accumulation of replication protein A appeared in nuclei after a 30 minute incubation, and a considerable portion of the replication protein A signals disappeared during a further 30 minute incubation. Similar replication protein A accumulation was observed in the nuclei after a 30 minute incubation in the extract containing camptothecin, whereas a further 30 minute incubation generated discrete replication protein A foci. The addition of camptothecin also induced formation of gamma-H2AX foci, which have been previously shown to localize at sites of DSBs. Analysis of the time course of DNA replication and results obtained using geminin, an inhibitor of licensing for DNA replication, suggest that the discrete replication protein A foci formed in response to camptothecin-induced DNA damage occur in a DNA-replication-dependent manner. When the nuclei were incubated in the extract containing EcoRI, discrete replication protein A foci were observed at 30 minutes as well as at 60 and 90 minutes after incubation, and the focus-formation of replication protein A was not sensitive to geminin. DNA replication was almost completely inhibited in the presence of EcoRI and the inhibition was sensitive to caffeine, an inhibitor of ataxia telangiectasia mutated protein (ATM) and ATM- and Rad3-related protein (ATR). However, the focus-formation of replication protein A in the presence of EcoRI was not influenced by caffeine treatment. EcoRI-induced incorporation of biotin-dUTP into chromatin was observed following geminin-mediated inhibition of DNA replication, suggesting that the incorporation was the result of DNA repair. The biotin-dUTP signal co-localized with replication protein A foci and was not significantly suppressed or stimulated by the addition of caffeine.
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August 2002
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