Kyushu University Academic Staff Educational and Research Activities Database
List of Papers
Takahashi Tatsuro Last modified date:2018.06.21

Associate Professor / Integrative Biology / Department of Biology / Faculty of Sciences


Papers
1. Riki Terui, Koji Nagao, Yoshitaka Kawasoe, Kanae Taki, Torahiko L. Higashi, Seiji Tanaka, Takuro Nakagawa, Chikashi Obuse, Hisao Masukata, Tatsuro S. Takahashi, Nucleosomes around a mismatched base pair are excluded via an Msh2-dependent reaction with the aid of SNF2 family ATPase Smarcad1., Genes & development, 10.1101/gad.310995.117, 32, 806-821, 2018.06, [URL], Post-replicative correction of replication errors by the mismatch repair (MMR) system is critical for suppression of mutations. Although the MMR system may need to handle nucleosomes at the site of chromatin replication, how MMR occurs in the chromatin environment remains unclear. Here, we show that nucleosomes are excluded from a >1-kb region surrounding a mismatched base pair in Xenopus egg extracts. The exclusion was dependent on the Msh2-Msh6 mismatch recognition complex but not the Mlh1-containing MutL homologs and counteracts both the HIRA- and CAF-1 (chromatin assembly factor 1)-mediated chromatin assembly pathways. We further found that the Smarcad1 chromatin remodeling ATPase is recruited to mismatch-carrying DNA in an Msh2-dependent but Mlh1-independent manner to assist nucleosome exclusion and that Smarcad1 facilitates the repair of mismatches when nucleosomes are preassembled on DNA. In budding yeast, deletion of FUN30, the homolog of Smarcad1, showed a synergistic increase of spontaneous mutations in combination with MSH6 or MSH3 deletion but no significant increase with MSH2 deletion. Genetic analyses also suggested that the function of Fun30 in MMR is to counteract CAF-1. Our study uncovers that the eukaryotic MMR system has an ability to exclude local nucleosomes and identifies Smarcad1/Fun30 as an accessory factor for the MMR reaction..
2. Niyo Kato, Yoshitaka Kawasoe, Hannah Williams, Elena Coates, Upasana Roy, Yuqian Shi, Lorena S. Beese, Orlando D. Schärer, Hong Yan, Max E. Gottesman, Tatsuro Takahashi, Jean Gautier, Sensing and Processing of DNA Interstrand Crosslinks by the Mismatch Repair Pathway, Cell Reports, 10.1016/j.celrep.2017.10.032, 21, 5, 1375-1385, 2017.10, DNA interstrand crosslinks (ICLs) that are repaired in non-dividing cells must be recognized independently of replication-associated DNA unwinding. Using cell-free extracts from Xenopus eggs that support neither replication nor transcription, we establish that ICLs are recognized and processed by the mismatch repair (MMR) machinery. We find that ICL repair requires MutSα (MSH2–MSH6) and the mismatch recognition FXE motif in MSH6, strongly suggesting that MutSα functions as an ICL sensor. MutSα recruits MutLα and EXO1 to ICL lesions, and the catalytic activity of both these nucleases is essential for ICL repair. As anticipated for a DNA unwinding-independent recognition process, we demonstrate that least distorting ICLs fail to be recognized and repaired by the MMR machinery. This establishes that ICL structure is a critical determinant of repair efficiency outside of DNA replication. Kato et al. identify a mechanism of ICL recognition that operates independently of DNA replication and transcription. In the absence of these processes, ICLs are recognized and repaired by the MMR machinery. MutSα is critical for ICL recognition, while MutLα and EXO1 contribute to key downstream nucleolytic steps during ICL repair..
3. Faria Zafar, Akiko K. Okita, Atsushi T. Onaka, Jie Su, Yasuhiro Katahira, Jun Ichi Nakayama, Tatsuro Takahashi, Hisao Masukata, Takuro Nakagawa, Regulation of mitotic recombination between DNA repeats in centromeres, Nucleic Acids Research, 10.1093/nar/gkx763, 45, 19, 11222-11235, 2017.01, Centromeres that are essential for faithful segregation of chromosomes consist of unique DNA repeats in many eukaryotes. Although recombination is under-represented around centromeres during meiosis, little is known about recombination between centromere repeats in mitotic cells. Here, we compared spontaneous recombination that occurs between ade6B/ade6X inverted repeats integrated at centromere 1 (cen1) or at a non-centromeric ura4 locus in fission yeast. Remarkably, distinct mechanisms of homologous recombination (HR) were observed in centromere and non-centromere regions. Rad51-dependent HR that requires Rad51, Rad54 and Rad52 was predominant in the centromere, whereas Rad51-independent HR that requires Rad52 also occurred in the arm region. Crossovers between inverted repeats (i.e. inversions) were underrepresented in the centromere as compared to the arm region. While heterochromatin was dispensable, Mhf1/CENP-S, Mhf2/CENP-X histone-fold proteins and Fml1/FANCM helicase were required to suppress crossovers. Furthermore, Mhf1 and Fml1 were found to prevent gross chromosomal rearrangements mediated by centromere repeats. These data uncovered the regulation of mitotic recombination between DNA repeats in centromeres and its physiological role in maintaining genome integrity..
4. Yoshitaka Kawasoe, Toshiki Tsurimoto, Takuro Nakagawa, Hisao Masukata, Tatsuro Takahashi, MutSα maintains the mismatch repair capability by inhibiting PCNA unloading, eLife, 10.7554/eLife.15155, 5, 2016JULY, 2016.07, 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..
5. Catherine Johnson, Vamsi K. Gali, Tatsuro Takahashi, Takashi Kubota, PCNA Retention on DNA into G2/M Phase Causes Genome Instability in Cells Lacking Elg1, Cell Reports, 10.1016/j.celrep.2016.06.030, 16, 3, 684-695, 2016.07, Loss of the genome maintenance factor Elg1 causes serious genome instability that leads to cancer, but the underlying mechanism is unknown. Elg1 forms the major subunit of a replication factor C-like complex, Elg1-RLC, which unloads the ring-shaped polymerase clamp PCNA from DNA during replication. Here, we show that prolonged retention of PCNA on DNA into G2/M phase is the major cause of genome instability in elg1Δ yeast. Overexpression-induced accumulation of PCNA on DNA causes genome instability. Conversely, disassembly-prone PCNA mutants that relieve PCNA accumulation rescue the genome instability of elg1Δ cells. Covalent modifications to the retained PCNA make only a minor contribution to elg1Δ genome instability. By engineering cell-cycle-regulated ELG1 alleles, we show that abnormal accumulation of PCNA on DNA during S phase causes moderate genome instability and its retention through G2/M phase exacerbates genome instability. Our results reveal that PCNA unloading by Elg1-RLC is critical for genome maintenance..
6. Atsushi T. Onaka, Naoko Toyofuku, Takahiro Inoue, Akiko K. Okita, Minami Sagawa, Jie Su, Takeshi Shitanda, Rei Matsuyama, Faria Zafar, Tatsuro Takahashi, Hisao Masukata, Takuro Nakagawa, Rad51 and Rad54 promote noncrossover recombination between centromere repeats on the same chromatid to prevent isochromosome formation, Nucleic Acids Research, 10.1093/nar/gkw874, 44, 22, 10744-10757, 2016.01, Centromeres consist of DNA repeats inmany eukaryotes. Non-allelic homologous recombination (HR) between them can result in gross chromosomal rearrangements (GCRs). In fission yeast, Rad51 suppresses isochromosome formation that occurs between inverted repeats in the centromere. However, how the HR enzyme prevents homology-mediated GCRs remains unclear. Here, we provide evidence that Rad51 with the aid of the Swi/Snf-type motor protein Rad54 promotes non-crossover recombination between centromere repeats to prevent isochromosome formation. Mutations in Rad51 and Rad54 epistatically increased the rates of isochromosome formation and chromosome loss. In sharp contrast, these mutations decreased gene conversion between inverted repeats in the centromere. Remarkably, analysis of recombinant DNAs revealed that rad51 and rad54 increase the proportion of crossovers. In the absence of Rad51, deletion of the structure-specific endonuclease Mus81 decreased both crossovers and isochromosomes, while the cdc27/pol32-D1 mutation, which impairs break-induced replication, did not. We propose that Rad51 and Rad54 promote non-crossover recombination between centromere repeats on the same chromatid, thereby suppressing crossover between non-allelic repeats on sister chromatids that leads to chromosomal rearrangements. Furthermore, we found that Rad51 and Rad54 are required for gene silencing in centromeres, suggesting that HR also plays a role in the structure and function of centromeres..
7. Yosuke Sanuki, Yumiko Kubota, Masato T. Kanemaki, Tatsuro Takahashi, Satoru Mimura, Haruhiko Takisawa, RecQ4 promotes the conversion of the pre-initiation complex at a site-specific origin for DNA unwinding in xenopus egg extracts, Cell Cycle, 10.1080/15384101.2015.1007003, 14, 7, 1010-1023, 2015.04, Eukaryotic DNA replication is initiated through stepwise assembly of evolutionarily conserved replication proteins onto replication origins, but how the origin DNA is unwound during the assembly process remains elusive. Here, we established a site-specific origin on a plasmid DNA, using in vitro replication systems derived from Xenopus egg extracts. We found that the pre-replicative complex (pre-RC) was preferentially assembled in the vicinity of GAL4 DNA-binding sites of the plasmid, depending on the binding of Cdc6 fused with a GAL4 DNA-binding domain in Cdc6-depleted extracts. Subsequent addition of nucleoplasmic S-phase extracts to the GAL4-dependent pre-RC promoted initiation of DNA replication from the origin, and components of the pre-initiation complex (pre-IC) and the replisome were recruited to the origin concomitant with origin unwinding. In this replication system, RecQ4 is dispensable for both recruitment of Cdc45 onto the origin and stable binding of Cdc45 and GINS to the pre-RC assembled plasmid. However, both origin binding of DNA polymerase a and unwinding of DNA were diminished upon depletion of RecQ4 from the extracts. These results suggest that RecQ4 plays an important role in the conversion of pre-ICs into active replisomes requiring the unwinding of origin DNA in vertebrates..
8. Keishi Shintomi, Tatsuro Takahashi, Tatsuya Hirano, Reconstitution of mitotic chromatids with a minimum set of purified factors, Nature Cell Biology, 10.1038/ncb3187, 17, 8, 1014-1023, 2015.01, The assembly of mitotic chromosomes, each composed of a pair of rod-shaped chromatids, is an essential prerequisite for accurate transmission of the genome during cell division. It remains poorly understood, however, how this fundamental process might be achieved and regulated in the cell. Here we report an in vitro system in which mitotic chromatids can be reconstituted by mixing a simple substrate with only six purified factors: core histones, three histone chaperones (nucleoplasmin, Nap1 and FACT), topoisomerase II (topo II) and condensin I. We find that octameric nucleosomes containing the embryonic variant H2A.X-F are highly susceptible to FACT and function as the most productive substrate for subsequent actions of topo II and condensin I. Cdk1 phosphorylation of condensin I is the sole mitosis-specific modification required for chromatid reconstitution. This experimental system will enhance our understanding of the mechanisms of action of individual factors and their cooperation during this process..
9. Tamara J. Slenn, Benjamin Morris, Courtney G. Havens, Robert M. Freeman, Tatsuro Takahashi, Johannes C. Walter, Thymine DNA glycosylase is a CRL4Cdt2 substrate, Journal of Biological Chemistry, 10.1074/jbc.M114.574194, 289, 33, 23043-23055, 2014.08, The E3 ubiquitin ligase CRL4Cdt2 targets proteins for destruction in S phase and after DNA damage by coupling ubiquitylation to DNA-bound proliferating cell nuclear antigen (PCNA). Coupling to PCNA involves a PCNA-interacting peptide (PIP) degron motif in the substrate that recruits CRL4Cdt2 while binding to PCNA. In vertebrates, CRL4Cdt2 promotes degradation of proteins whose presence in S phase is deleterious, including Cdt1, Set8, and p21. Here, we show that CRL4Cdt2 targets thymine DNA glycosylase (TDG), a base excision repair enzyme that is involved in DNA demethylation. TDG contains a conserved and nearly perfect match to the PIP degron consensus. TDG is ubiquitylated and destroyed in a PCNA-, Cdt2-, and PIP degron-dependent manner during DNA repair in Xenopus egg extract. The protein can also be destroyed during DNA replication in this system. During Xenopus development, TDG first accumulates during gastrulation, and its expression is down-regulated by CRL4Cdt2. Our results expand the group of vertebrate CRL4Cdt2 substrates to include a bona fide DNA repair enzyme..
10. Atsuhiro Shimada, Yoshitaka Kawasoe, Yoshito Hata, Tatsuro Takahashi, Ryoji Masui, Seiki Kuramitsu, Kenji Fukui, MutS stimulates the endonuclease activity of MutL in an ATP-hydrolysis-dependent manner, FEBS Journal, 10.1111/febs.12344, 280, 14, 3467-3479, 2013.07, In the initial steps of DNA mismatch repair, MutS recognizes a mismatched base and recruits the latent endonuclease MutL onto the mismatch-containing DNA in concert with other proteins. MutL then cleaves the error-containing strand to introduce an entry point for the downstream excision reaction. Because MutL has no intrinsic ability to recognize a mismatch and discriminate between newly synthesized and template strands, the endonuclease activity of MutL is strictly regulated by ATP-binding in order to avoid nonspecific degradation of the genomic DNA. However, the activation mechanism for its endonuclease activity remains unclear. In this study, we found that the coexistence of a mismatch, ATP and MutS unlocks the ATP-binding-dependent suppression of MutL endonuclease activity. Interestingly, ATPase-deficient mutants of MutS were unable to activate MutL. Furthermore, wild-type MutS activated ATPase-deficient mutants of MutL less efficiently than wild-type MutL. We concluded that ATP hydrolysis by MutS and MutL is involved in the mismatch-dependent activation of MutL endonuclease activity. MutS and MutL participate in the initial steps in DNA mismatch repair. The role of their ATP hydrolysis in the repair pathway has been barely understood..
11. Atsutoshi Tazumi, Masayoshi Fukuura, Ryuichiro Nakato, Ami Kishimoto, Tomokazu Takenaka, Shiho Ogawa, Ji Hoon Song, Tatsuro Takahashi, Takuro Nakagawa, Katsuhiko Shirahige, Hisao Masukata, Telomere-binding protein Taz1 controls global replication timing through its localization near late replication origins in fission yeast, Genes and Development, 10.1101/gad.194282.112, 26, 18, 2050-2062, 2012.09, In eukaryotes, the replication of chromosome DNA is coordinated by a replication timing program that temporally regulates the firing of individual replication origins. However, the molecular mechanism underlying the program remains elusive. Here, we report that the telomere-binding protein Taz1 plays a crucial role in the control of replication timing in fission yeast. A DNA element located proximal to a late origin in the chromosome arm represses initiation from the origin in early S phase. Systematic deletion and substitution experiments demonstrated that two tandem telomeric repeats are essential for this repression. The telomeric repeats recruit Taz1, a counterpart of human TRF1 and TRF2, to the locus. Genome-wide analysis revealed that Taz1 regulates about half of chromosomal late origins, including those in subtelomeres. The Taz1-mediated mechanism prevents Dbf4-dependent kinase (DDK)-dependent Sld3 loading onto the origins. Our results demonstrate that the replication timing program in fission yeast uses the internal telomeric repeats and binding of Taz1..
12. Tetsuya Handa, Mai Kanke, Tatsuro Takahashi, Takuro Nakagawa, Hisao Masukata, DNA polymerization-independent functions of DNA polymerase epsilon in assembly and progression of the replisome in fission yeast., Molecular Biology of the Cell, 23, 16, 3240-3253, 2012.08, DNA polymerase epsilon (Pol ε) synthesizes the leading strands, following the CMG (Cdc45, Mcm2-7, and GINS [Go-Ichi-Nii-San]) helicase that translocates on the leading-strand template at eukaryotic replication forks. Although Pol ε is essential for the viability of fission and budding yeasts, the N-terminal polymerase domain of the catalytic subunit, Cdc20/Pol2, is dispensable for viability, leaving the following question: what is the essential role(s) of Pol ε? In this study, we investigated the essential roles of Pol ε using a temperature-sensitive mutant and a recently developed protein-depletion (off-aid) system in fission yeast. In cdc20-ct1 cells carrying mutations in the C-terminal domain of Cdc20, the CMG components, RPA, Pol α, and Pol δ were loaded onto replication origins, but Cdc45 did not translocate from the origins, suggesting that Pol ε is required for CMG helicase progression. In contrast, depletion of Cdc20 abolished the loading of GINS and Cdc45 onto origins, indicating that Pol ε is essential for assembly of the CMG complex. These results demonstrate that Pol ε plays essential roles in both the assembly and progression of CMG helicase..
13. Vladimir P. Bermudez, Andrea Farina, Torahiko L. Higashi, Fang Du, Inger Tappin, Tatsuro Takahashi, Jerard Hurwitz, In vitro loading of human cohesin on DNA by the human Scc2-Scc4 loader complex, Proceedings of the National Academy of Sciences of the United States of America, 10.1073/pnas.1206840109, 109, 24, 9366-9371, 2012.06, The loading of cohesin onto chromatin requires the heterodimeric complex sister chromatid cohesion (Scc)2 and Scc4 (Scc2/4), which is highly conserved in all species. Here, we describe the purification of the human (h)-Scc2/4 and show that it interacts with hcohesin and the heterodimeric Smc1-Smc3 complex but not with the Smc1 or Smc3 subunit alone. We demonstrate that both h-Scc2/4 and h-cohesin are loaded onto dsDNA containing the prereplication complex (pre-RC) generated in vitro by Xenopus high-speed soluble extracts. The addition of geminin, which blocks pre-RC formation, prevents the loading of Scc2/4 and cohesin. Xenopus extracts depleted of endogenous Scc2/4 with specific antibodies, although able to form pre-RCs, did not support cohesin loading unless supplemented with purified h-Scc2/4. The results presented here indicate that the Xenopus or h-Scc2/4 complex supports the loading of Xenopus and/or h-cohesin onto pre-RCs formed by Xenopus high-speed extracts. We show that cohesin loaded onto pre-RCs either by h-Scc2/4 and/or the Xenopus complex was dissociated from chromatin by low salt extraction, similar to cohesin loaded onto chromatin in G1 by HeLa cells in vivo. Replication of cohesin-loaded DNA, both in vitro and in vivo, markedly increased the stability of cohesin associated with DNA. Collectively, these in vitro findings partly recapitulate the in vivo pathway by which sister chromatids are linked together, leading to cohesion..
14. Torahiko L. Higashi, Megumi Ikeda, Hiroshi Tanaka, Takuro Nakagawa, Masashige Bando, Katsuhiko Shirahige, Yumiko Kubota, Haruhiko Takisawa, Hisao Masukata, Tatsuro Takahashi, The prereplication complex recruits XEco2 to chromatin to promote cohesin acetylation in Xenopus egg extracts, Current Biology, 10.1016/j.cub.2012.04.013, 22, 11, 977-988, 2012.06, Background: Sister chromatids are held together by the ring-shaped cohesin complex, which is loaded onto chromosomes before DNA replication. Cohesion between sister chromosomes is established during DNA replication, and it requires acetylation of the Smc3 subunit of cohesin by evolutionally conserved cohesin acetyltransferases (CoATs). However, how CoATs are recruited to chromatin and how cohesin acetylation is regulated remain unclear. Results: We found that cohesin acetylation requires pre-RC-dependent chromatin loading of cohesin, but surprisingly, it is independent of DNA synthesis in Xenopus egg extracts. Immunodepletion experiments revealed that XEco2 is the CoAT responsible for Smc3 acetylation and sister chromatid cohesion. Recruitment of XEco2 onto chromatin was dependent on pre-RC assembly but was independent of cohesin loading and DNA synthesis. Two short N-terminal motifs, PBM-A and PBM-B, which are conserved among vertebrate Esco2/XEco2 homologs, were collectively essential for pre-RC-dependent chromatin association of XEco2, cohesin acetylation, and subsequent sister chromatid cohesion. The conserved PCNA-interacting protein box in XEco2 was largely dispensable for Smc3 acetylation but was partially required for cohesion. Interaction of acetylated cohesin with DNA was stabilized against salt-wash treatments after DNA replication. Conclusions: Our results demonstrate that pre-RC formation regulates chromatin association of XEco2 in Xenopus egg extracts. We propose that this reaction is critical to acetylate cohesin, whose DNA binding is subsequently stabilized by DNA replication..
15. Mai Kanke, Yukako Kodama, Tatsuro Takahashi, Takuro Nakagawa, Hisao Masukata, Mcm10 plays an essential role in origin DNA unwinding after loading of the CMG components, EMBO Journal, 10.1038/emboj.2012.68, 31, 9, 2182-2194, 2012.05, The CMG complex composed of Mcm2-7, Cdc45 and GINS is postulated to be the eukaryotic replicative DNA helicase, whose activation requires sequential recruitment of replication proteins onto Mcm2-7. Current models suggest that Mcm10 is involved in assembly of the CMG complex, and in tethering of DNA polymerase α at replication forks. Here, we report that Mcm10 is required for origin DNA unwinding after association of the CMG components with replication origins in fission yeast. A combination of promoter shut-off and the auxin-inducible protein degradation (off-aid) system efficiently depleted cellular Mcm10 to <0.5% of the wild-type level. Depletion of Mcm10 did not affect origin loading of Mcm2-7, Cdc45 or GINS, but impaired recruitment of RPA and DNA polymerases. Mutations in a conserved zinc finger of Mcm10 abolished RPA loading after recruitment of Mcm10. These results show that Mcm10, together with the CMG components, plays a novel essential role in origin DNA unwinding through its zinc-finger function..
16. Kentaro Maki, Takahiro Inoue, Atsushi Onaka, Hiroko Hashizume, Naoko Somete, Yuko Kobayashi, Shigefumi Murakami, Chikako Shigaki, Tatsuro Takahashi, Hisao Masukata, Takuro Nakagawa, Abundance of prereplicative complexes (Pre-RCs) facilitates recombinational repair under replication stress in fission yeast, Journal of Biological Chemistry, 10.1074/jbc.M111.285619, 286, 48, 41701-41710, 2011.12, Mcm2-7 complexes are loaded onto chromatin with the aid of Cdt1 and Cdc18/Cdc6 and form prereplicative complexes (pre- RCs) at multiple sites on each chromosome. Pre-RCs are essential for DNA replication and surviving replication stress. However, the mechanism by which pre-RCs contribute to surviving replication stress is largely unknown. Here, we isolated the fission yeast mcm6-S1 mutant that was hypersensitive to methyl methanesulfonate (MMS) and camptothecin (CPT), both of which cause forks to collapse. The mcm6-S1 mutation impaired the interaction with Cdt1 and decreased the binding of minichromosome maintenance (MCM) proteins to replication origins. Overexpression of Cdt1 restored MCM binding and suppressed the sensitivity to MMS and CPT, suggesting that the Cdt1-Mcm6 interaction is important for the assembly of pre-RCs and the repair of collapsed forks. MMS-induced Chk1 phosphorylation and Rad22/Rad52 focus formation occurred normally, whereas cells containing Rhp54/Rad54 foci, which are involved inDNAstrand exchange and dissociation of the joint molecules, were increased. Remarkably, G 1 phase extension through deletion of an S phase cyclin, Cig2, as well as Cdt1 overexpression restored pre-RC assembly and suppressed Rhp54 accumulation. A cdc18 mutation also caused hypersensitivity to MMS and CPT and accumulation of Rhp54 foci. These data suggest that an abundance of pre-RCs facilitates a late step in the recombinational repair of collapsed forks in the following S phase..
17. Masayoshi Fukuura, Koji Nagao, Chikashi Obuse, Tatsuro Takahashi, Takuro Nakagawa, Hisao Masukata, CDK promotes interactions of Sld3 and Drc1 with Cut5 for initiation of DNA replication in fission yeast, Molecular Biology of the Cell, 10.1091/mbc.E10-12-0995, 22, 14, 2620-2633, 2011.07, Cyclin-dependent kinase (CDK) plays essential roles in the initiation of DNA replication in eukaryotes. Although interactions of CDK-phosphorylated Sld2/Drc1 and Sld3 with Dpb11 have been shown to be essential in budding yeast, it is not known whether the mechanism is conserved. In this study, we investigated how CDK promotes the assembly of replication proteins onto replication origins in fission yeast. Phosphorylation of Sld3 was found to be dependent on CDK in S phase. Alanine substitutions at CDK sites decreased the interaction with Cut5/Dpb11 at the N-terminal BRCT motifs and decreased the loading of Cut5 onto replication origins. This defect was suppressed by overexpression of drc1 +. Phosphorylation of a conserved CDK site, Thr-111, in Drc1 was critical for interaction with Cut5 at the C-terminal BRCT motifs and was required for loading of Cut5. In a yeast three-hybrid assay, Sld3, Cut5, and Drc1 were found to form a ternary complex dependent on the CDK sites of Sld3 and Drc1, and Drc1-Cut5 binding enhanced the Sld3-Cut5 interaction. These results show that the mechanism of CDK-dependent loading of Cut5 is conserved in fission yeast in a manner similar to that elucidated in budding yeast..
18. Mai Kanke, Kohei Nishimura, Masato Kanemaki, Tatsuo Kakimoto, Tatsuro Takahashi, Takuro Nakagawa, Hisao Masukata, Auxin-inducible protein depletion system in fission yeast, BMC Cell Biology, 10.1186/1471-2121-12-8, 12, 2011.02, Background: Inducible inactivation of a protein is a powerful approach for analysis of its function within cells. Fission yeast is a useful model for studying the fundamental mechanisms such as chromosome maintenance and cell cycle. However, previously published strategies for protein-depletion are successful only for some proteins in some specific conditions and still do not achieve efficient depletion to cause acute phenotypes such as immediate cell cycle arrest. The aim of this work was to construct a useful and powerful protein-depletion system in Shizosaccaromyces pombe.Results: We constructed an auxin-inducible degron (AID) system, which utilizes auxin-dependent poly-ubiquitination of Aux/IAA proteins by SCFTIR1 in plants, in fission yeast. Although expression of a plant F-box protein, TIR1, decreased Mcm4-aid, a component of the MCM complex essential for DNA replication tagged with Aux/IAA peptide, depletion did not result in an evident growth defect. We successfully improved degradation efficiency of Mcm4-aid by fusion of TIR1 with fission yeast Skp1, a conserved F-box-interacting component of SCF (improved-AID system; i-AID), and the cells showed severe defect in growth. The i-AID system induced degradation of Mcm4-aid in the chromatin-bound MCM complex as well as those in soluble fractions. The i-AID system in conjunction with transcription repression (off-AID system), we achieved more efficient depletion of other proteins including Pol1 and Cdc45, causing early S phase arrest.Conclusion: Improvement of the AID system allowed us to construct conditional null mutants of S. pombe. We propose that the off-AID system is the powerful method for in vivo protein-depletion in fission yeast..
19. Ronald Lebofsky, Tatsuro Takahashi, Johannes C. Walter, DNA replication in nucleus-free xenopus egg extracts, DNA Replication Methods and Protocols, 10.1007/978-1-60327-815-7_13, 521, 229-252, 2009.12, Extracts derived from Xenopus laevis eggs represent a powerful cell-free system to study eukaryotic DNA replication. A variation of the system allows for DNA replication not only in a cell-free environment, but also in the absence of a nucleus. In this nucleus-free system, DNA templates are licensed with High-Speed Supernatant (HSS) and then replicated with a concentrated NucleoPlasmic Extract (NPE). This method has the advantage of allowing replication of small plasmids with desired modifications and manipulation of the nuclear environment. This chapter describes the protocols needed to prepare HSS and NPE and how these extracts are used to study DNA replication..
20. Makoto T. Hayashi, Tatsuro Takahashi, Takuro Nakagawa, Jun Ichi Nakayama, Hisao Masukata, The heterochromatin protein Swi6/HP1 activates replication origins at the pericentromeric region and silent mating-type locus, Nature Cell Biology, 10.1038/ncb1845, 11, 3, 357-362, 2009.02, Heterochromatin is a structurally compacted region of chromosomes in which transcription and recombination are inactivated. DNA replication is temporally regulated in heterochromatin, but the molecular mechanism for regulation has not been elucidated. Among heterochromatin loci in Schizosaccharomyces pombe, the pericentromeric region and the silent mating-type (mat) locus replicate in early S phase, whereas the sub-telomeric region does not, suggesting complex mechanisms for regulation of replication in heterochromatic regions. Here, we show that Swi6, an S. pombe counterpart of heterochromatin protein 1 (HP1), is required for early replication of the pericentromeric region and the mat locus. Origin-loading of Sld3, which depends on Dfp1/Dbf4-dependent kinase Cdc7 (DDK), is stimulated by Swi6. An HP1-binding motif within Dfp1 is required for interaction with Swi6 in vitro and for early replication of the pericentromeric region and mat locus. Tethering of Dfp1 to the pericentromeric region and mat locus in swi6-deficient cells restores early replication of these loci. Our results show that a heterochromatic protein positively regulates initiation of replication in silenced chromatin by interacting with an essential kinase..
21. Ken Ichi Nakamura, Aya Okamoto, Yuki Katou, Chie Yadani, Takeshi Shitanda, Chitrada Kaweeteerawat, Tatsuro Takahashi, Takehiko Itoh, Katsuhiko Shirahige, Hisao Masukata, Takuro Nakagawa, Rad51 suppresses gross chromosomal rearrangement at centromere in Schizosaccharomyces pombe, EMBO Journal, 10.1038/emboj.2008.215, 27, 22, 3036-3046, 2008.11, Centromere that plays a pivotal role in chromosome segregation is composed of repetitive elements in many eukaryotes. Although chromosomal regions containing repeats are the hotspots of rearrangements, little is known about the stability of centromere repeats. Here, by using a minichromosome that has a complete set of centromere sequences, we have developed a fission yeast system to detect gross chromosomal rearrangements (GCRs) that occur spontaneously. Southern and comprehensive genome hybridization analyses of rearranged chromosomes show two types of GCRs: translocation between homologous chromosomes and formation of isochromosomes in which a chromosome arm is replaced by a copy of the other. Remarkably, all the examined isochromosomes contain the breakpoint in centromere repeats, showing that isochromosomes are produced by centromere rearrangement. Mutations in the Rad3 checkpoint kinase increase both types of GCRs. In contrast, the deletion of Rad51 recombinase preferentially elevates isochromosome formation. Chromatin immunoprecipitation analysis shows that Rad51 localizes at centromere around S phase. These data suggest that Rad51 suppresses rearrangements of centromere repeats that result in isochromosome formation..
22. Tatsuro Takahashi, Abhijit Basu, Vladimir Bermudez, Jerard Hurwitz, Johannes C. Walter, Cdc7-Drf1 kinase links chromosome cohesion to the initiation of DNA replication in Xenopus egg extracts, Genes and Development, 10.1101/gad.1683308, 22, 14, 1894-1905, 2008.07, To establish functional cohesion between replicated sister chromatids, cohesin is recruited to chromatin before S phase. Cohesin is loaded onto chromosomes in the G1 phase by the Scc2-Scc4 complex, but little is known about how Scc2-Scc4 itself is recruited to chromatin. Using Xenopus egg extracts as a vertebrate model system, we showed previously that the chromatin association of Scc2 and cohesin is dependent on the prior establishment of prereplication complexes (pre-RCs) at origins of replication. Here, we report that Scc2-Scc4 exists in a stable complex with the Cdc7-Drf1 protein kinase (DDK), which is known to bind pre-RCs and activate them for DNA replication. Immunodepletion of DDK from Xenopus egg extracts impairs chromatin association of Scc2-Scc4, a defect that is reversed by wild-type, but not catalytically inactive DDK. A complex of Scc4 and the N terminus of Scc2 is sufficient for chromatin loading of Scc2-Scc4, but not for cohesin recruitment. These results show that DDK is required to tether Scc2-Scc4 to pre-RCs, and they underscore the intimate link between early steps in DNA replication and cohesion..
23. Makoto Hayashi, Yuki Katou, Takehiko Itoh, Atsutoshi Tazumi, Yoshiki Yamada, Tatsuro Takahashi, Takuro Nakagawa, Katsuhiko Shirahige, Hisao Masukata, Erratum
Genome-wide localization of pre-RC sites and identification of replication origins in fission yeast (The EMBO Journal (2007) 26 (1327-1339) DOI: 10.1038/sj.emboj.7601585), EMBO Journal, 10.1038/sj.emboj.7601708, 26, 11, 2821, 2007.06.
24. Makoto Hayashi, Yuki Katou, Takehiko Itoh, Mitsutoshi Tazumi, Yoshiki Yamada, Tatsuro Takahashi, Takuro Nakagawa, Katsuhiko Shirahige, Hisao Masukata, Genome-wide localization of pre-RC sites and identification of replication origins in fission yeast, EMBO Journal, 10.1038/sj.emboj.7601585, 26, 5, 1327-1339, 2007.03, DNA replication of eukaryotic chromosomes initiates at a number of discrete loci, called replication origins. Distribution and regulation of origins are important for complete duplication of the genome. Here, we determined locations of Orc1 and Mcm6, components of pre-replicative complex (pre-RC), on the whole genome of Schizosaccharomyces pombe using a high-resolution tiling array. Pre-RC sites were identified in 460 intergenic regions, where Orc1 and Mcm6 colocalized. By mapping of 5-bromo-2′-deoxyuridine (BrdU)-incorporated DNA in the presence of hydroxyurea (HU), 307 pre-RC sites were identified as early-firing origins. In contrast, 153 pre-RC sites without BrdU incorporation were considered to be late and/or inefficient origins. Inactivation of replication checkpoint by Cds1 deletion resulted in BrdU incorporation with HU specifically at the late origins. Early and late origins tend to distribute separately in large chromosome regions. Interestingly, pericentromeric heterochromatin and the silent mating-type locus replicated in the presence of HU, whereas the inner centromere or subtelomeric heterochromatin did not. Notably, MCM did not bind to inner centromeres where origin recognition complex was located. Thus, replication is differentially regulated in chromosome domains..
25. Tatsuro Takahashi, Johannes C. Walter, Cdc7-Drf1 is a developmentally regulated protein kinase required for the initiation of vertebrate DNA replication, Genes and Development, 10.1101/gad.1339805, 19, 19, 2295-2300, 2005.10, Cdc7, a protein kinase required for the initiation of eukaryotic DNA replication, is activated by a regulatory subunit, Dbf4. A second activator of Cdc7 called Drf1 exists in vertebrates, but its function is unknown. Here, we report that in Xenopus egg extracts, Cdc7-Drf1 is far more abundant than Cdc7-Dbf4, and removal of Drf1 but not Dbf4 severely inhibits phosphorylation of Mcm4 and DNA replication. After gastrulation, when the cell cycle acquires somatic characteristics, Drf1 levels decline sharply and Cdc7-Dbf4 becomes the more abundant kinase. These results identify Drf1 as a developmentally regulated, essential activator of Cdc7 in Xenopus..
26. Tatsuro Takahashi, Pannyun Yiu, Michael F. Chou, Steven Gygi, Johannes C. Walter, Recruitment of Xenopus Scc2 and cohesin to chromatin requires the pre-replication complex, Nature Cell Biology, 10.1038/ncb1177, 6, 10, 991-996, 2004.10, Cohesin is a multi-subunit, ring-shaped protein complex that holds sister chromatids together from the time of their synthesis in S phase until they are segregated in anaphase. In yeast, the loading of cohesin onto chromosomes requires the Scc2 protein. In vertebrates, cohesins first bind to chromosomes as cells exit mitosis, but the mechanism is unknown. Concurrent with cohesin binding, pre-replication complexes (pre-RCs) are assembled at origins of DNA replication through the sequential loading of the initiation factors ORC, Cdc6, Cdt1 and MCM2-7 (the 'licensing' reaction). In S phase, the protein kinase Cdk2 activates pre-RCs, causing origin unwinding and DNA replication. Here, we use Xenopus egg extracts to show that the recruitment of cohesins to chromosomes requires fully licensed chromatin and is dependent on ORC, Cdc6, Cdt1 and MCM2-7, but is independent of Cdk2. We further show that Xenopus Scc2 is required for cohesin loading and that binding of XScc2 to chromatin is MCM2-7 dependent. Our results define a novel pre-RC-dependent pathway for cohesin recruitment to chromosomes in a vertebrate model system..
27. Tatsuro Takahashi, Eri Ohara, Hideo Nishitani, Hisao Masukata, Multiple ORC-binding sites are required for efficient MCM loading and origin firing in fission yeast, EMBO Journal, 10.1093/emboj/cdg079, 22, 4, 964-974, 2003.02, In most eukaryotes, replication origins are composed of long chromosome regions, and the exact sequences required for origin recognition complex (ORC) and minichromosome maintenance (MCM) complex association remain elusive. Here, we show that two stretches of adenine/thymine residues are collectively essential for a fission yeast chromosomal origin. Chromatin immunoprecipitation assays revealed that the ORC subunits are located within a 1 kb region of ori2004. Analyses of deletion derivatives of ori2004 showed that adenine stretches are required for ORC binding in vivo. Synergistic interaction between ORC and adenine stretches was observed. On the other hand, MCM subunits were localized preferentially to a region near the initiation site, which is distant from adenine stretches. This association was dependent on adenine stretches and stimulated by a non-adenine element. Our results suggest that association of multiple ORC molecules with a replication origin is required for efficient MCM loading and origin firing in fission yeast..
28. Tatsuro Takahashi, Hisao Masukata, Interaction of fission yeast ORC with essential adenine/thymine stretches in replication origins, Genes to Cells, 10.1046/j.1365-2443.2001.00468.x, 6, 10, 837-849, 2001, Background: Eukaryotic DNA replication is initiated from distinct regions on the chromosome. However, the mechanism for recognition of replication origins is not known for most eukaryotes. In fission yeast, replication origins are isolated as autonomously replicating sequences (ARSs). Multiple adenine/thymine clusters are essential for replication, but no short consensus sequences are found. In this paper, we examined the interaction of adenine/thymine clusters with the replication initiation factor ORC. Results: The SpOrc1 or SpOrc2 immunoprecipitates (IPs) containing at least four subunits of SpORC, interacted with the ars2004 fragment, which is derived from a predominant replication origin on the chromosome. SpORC-IPs preferentially interacted with two regions of the ars2004, which consist of consecutive adenines and AAAAT repeats and are essential for ARS activity. The nucleotide sequences required for the interaction with SpORC-IPs correspond closely to those necessary for in vivo ARS activity. Conclusion: Our results suggest that the SpORC interacts with adenine/thymine stretches, which have been shown to be the most important component in the fission yeast replication origin. The presence of multiple SpORC-binding sites, with certain sequence variations, is characteristic for the fission yeast replication origins..
29. Yuya Ogawa, Tatsuro Takahashi, Hisao Masukata, Association of fission yeast Orp1 and Mcm6 proteins with chromosomal replication origins, Molecular and Cellular Biology, 10.1128/MCB.19.10.7228, 19, 10, 7228-7236, 1999.01, We have previously shown that replication of fission yeast chromosomes is initiated in distinct regions. Analyses of autonomous replicating sequences have suggested that regions required for replication are very different from those in budding yeast. Here, we present evidence that fission yeast replication origins are specifically associated with proteins that participate in initiation of replication. Most Orp1p, a putative subunit of the fission yeast origin recognition complex (ORC), was found to be associated with chromatin-enriched insoluble components throughout the cell cycle. In contrast, the minichromosome maintenance (Mcm) proteins, SpMcm2p and SpMcm6p, encoded by the nda1+/cdc19+ and mis5+ genes, respectively, were associated with chromatin DNA only during the G1 and S phases. Immunostaining of spread nuclei showed SpMcm6p to be localized at discrete foci on chromatin during the G1 and S phases. A chromatin immunoprecipitation assay demonstrated that Orp1p was preferentially localized at the ars2004 and ars3002 origins of the chromosome throughout the cell cycle, while SpMcm6p was associated with these origins only in the G1 and S phases. Both Orp1p and SpMcm6p were associated with a 1-kb region that contains elements required for autonomous replication of ars2004. The results suggest that the fission yeast ORC specifically interacts with chromosomal replication origins and that Mcm proteins are loaded onto the origins to play a role in initiation of replication..
30. Tatsuro Takahashi, H. Masukata, Initiation-protein complex for eukaryotic DNA replication, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme, 44, 12 Suppl, 1804-1812, 1999.