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Mitsuhiro Yanagida

Publications and source records attributed to Mitsuhiro Yanagida.

At least 37 records · Page 2Linked to original sources
PubMed2004

Cti1/C1D interacts with condensin SMC hinge and supports the DNA repair function of condensin.

Condensin is a conserved five-subunit complex containing two SMC (structural maintenance of chromosomes) and three non-SMC subunits and plays a major role in mitotic chromosome condensation. Condensin also acts in interphase and is required for DNA repair and replication checkpoint control. We attempted to study the function of the condensin in greater detail by means of the isolation of interacting proteins with the two-hybrid system. Using the hinge domain of Cut3/SMC4 as bait, we found one Cut three-interacting (Cti) 14-kDa nuclear protein, Cti1. GST pull-down assay and immunoprecipitation supported physical interaction between Cti1 and condensin. Cti1 is similar to human C1D, which associates tightly with genomic DNA and functions to activate DNA protein kinase. SpC1D is essential for viability. The null mutant could germinate but arrest after replication, indicating that it is required for interphase growth. Importantly, an elevated dosage of spC1D suppressed the temperature, UV irradiation, and hydroxyurea sensitivity of the mutant of Cnd2, a non-SMC subunit of condensin. Upon exposure to hydroxyurea, spC1D accumulated on the nuclear chromatin, and the fraction of spC1D that was chromatin-bound increased. Cti1 is the first example of the protein that interacts with the hinge domain of SMC. Cti1 may have a supporting role for the DNA repair function of condensin.

Adenosine Triphosphatases↗
PubMed2004

Heads and tails.

Explore the source record for details and available documents.

Bacteriophage T4↗
PubMed2004

Suppression of a mitotic mutant by tRNA-Ala anticodon mutations that produce a dominant defect in late mitosis.

Cold-sensitive dominant mutants scn1 and scn2 of Schizosaccharomyces pombe were isolated by their ability to suppress temperature-sensitive cut9-665 defective in an essential subunit (human Apc6/budding yeast Cdc16 ortholog) of anaphase promoting complex/cyclosome (APC/C). APC/C mutants were defective in metaphase/anaphase transition, whereas single scn mutants showed the delay in anaphase spindle elongation at 20 degrees C. The scn mutants lost viability because of chromosome missegregation, and were sensitive to a tubulin poison. To understand the scn phenotypes, mutant genes were identified. Surprisingly, scn1 and scn2 have the same substitution in the anticodon of two different tRNA-Ala (UGC) genes. UGC was altered to UGU so that the binding of the tRNA-Ala to the ACA Thr codon in mRNA became possible. As cut9-665 contained an Ala535Thr substitution, wild-type Cut9 protein was probably produced in scn mutants. Indeed, plasmid carrying tRNA-Ala (UGU) conferred cold-sensitivity to wild-type and suppressed cut9-665 in a dominant fashion. The previously identified scn1(+) (renamed as scn3(+)) turned out to be a high copy suppressor for scn1 and scn2. These are the first tRNA mutants that cause a mitotic defect.

Anaphase↗
PubMed2004

Regulation of checkpoint kinases through dynamic interaction with Crb2.

ATR/Rad3-like kinases promote the DNA damage checkpoint through regulating Chk1 that restrains the activation of cyclin-dependent kinases. In fission yeast, Crb2, a BRCT-domain protein that is similar to vertebrate 53BP1, plays a crucial role in establishing this checkpoint. We report here that Crb2 regulates DNA damage checkpoint through temporal and dynamic interactions with Rad3, Chk1 and replication factor Cut5. The active complex formation between Chk1 and Crb2 is regulated by Rad3 and became maximal during the checkpoint arrest. Chk1 activation seems to need two steps of interaction changes: the loss of Rad3-Chk1 and Rad3-Crb2 interactions, and the association between hyperphosphorylated forms of Chk1 and Crb2. Chk1 is the major checkpoint kinase for the arrest of DNA polymerase mutants. The in vitro assay of Chk1 showed that its activation requires the presence of Crb2 BRCT. Hyperphosphorylation of Crb2 is also dependent on its intact BRCT. Finally, we show direct interaction between Rad3 and Crb2, which is inhibitory to Rad3 activity. Hence, Crb2 is the first to interact with both Rad3 and Chk1 kinases.

Cell Cycle↗
PubMed2004

An interactive gene network for securin-separase, condensin, cohesin, Dis1/Mtc1 and histones constructed by mass transformation.

The small genome of fission yeast Schizosaccharomyces pombe contains 4824 predicted genes and gene disruption suggests that approximately 850 are essential for viability. To obtain information on interactions among genes required for chromosome segregation, an approach called Strategy B was taken using mass transformation of the 1015 temperature-sensitive (ts) mutants that were made by random mutagenesis and transformed by plasmids carrying the genes for securin, separase, condensin, cohesin, kinetochore microtubule-binding proteins Dis1/Mtc1 or histones. Mutant strains whose phenotypes were either suppressed or inhibited by plasmids were selected. Each plasmid interacted positively or negatively with the average 14 strains. Identification of the mutant gene products by cloning revealed many hitherto unknown interactions. The interactive networks of segregation therefore may consist of genes with a variety of functions. For example, separase/Cut1 interacts with Cdc48/p97/VCP, which stabilizes securin and separase. Surprisingly, S. pombe cdc48 mutants displayed the mitotic phenotype highly similar to separase/cut1 mutants. This approach also provides a novel way of mutant isolation, resulting in two histone H2B strains and a cohesion mutant with a new phenotype.

Adenosine Triphosphatases↗
PubMed2003

Condensin but not cohesin SMC heterodimer induces DNA reannealing through protein-protein assembly.

Condensin and cohesin are chromosomal protein complexes required for chromosome condensation and sister chromatid cohesion, respectively. They commonly contain the SMC (structural maintenance of chromosomes) subunits consisting of a long coiled-coil with the terminal globular domains and the central hinge. Condensin and cohesin holo-complexes contain three and two non-SMC subunits, respectively. In this study, DNA interaction with cohesin and condensin complexes purified from fission yeast was investigated. The DNA reannealing activity is strong for condensin SMC heterodimer but weak for holo-condensin, whereas no annealing activity is found for cohesin heterodimer SMC and Rad21-bound heterotrimer complexes. One set of globular domains of the same condensin SMC is essential for the DNA reannealing activity. In addition, the coiled-coil and hinge region of another SMC are needed. Atomic force microscopy discloses the molecular events of DNA reannealing. SMC assembly that occurs on reannealing DNA seems to be a necessary intermediary step. SMC is eliminated from the completed double-stranded DNA. The ability of heterodimeric SMC to reanneal DNA may be regulated in vivo possibly through the non-SMC heterotrimeric complex.

Adenosine Triphosphatases↗
PubMed2003

The role of Ppe1/PP6 phosphatase for equal chromosome segregation in fission yeast kinetochore.

Mis12 is a kinetochore protein essential for equal chromosome segregation and is evolutionarily conserved from yeast to human. In this study, we report the isolation and characterization of suppressors of the mis12 mutant in fission yeast. Our results indicate that Mis12 is negatively regulated by a highly conserved protein phosphatase Ppe1 (scSit4/dmPPV/hPP6) or its bound partner Ekc1 (scSAP), and it is positively regulated by a counteracting kinase Gsk3. Mass spectrometry analysis shows that at least two sites in Mis12 are phosphorylated. This mechanism of suppression occurs at the level of localization recovery of Mis12 to the kinetochore chromatin. Consistently, Mis12 and a subpopulation of Ppe1/Ekc1 were found to behave like non-histone-type chromatin-associating proteins in the chromatin fractionation assay. Mutant analysis of Ppe1 and Ekc1 revealed that they are important for faithful chromosome segregation, as the mutants exhibited unequal chromosome segregation similar to mis12 in the presence of a low concentration of tubulin poison. Ppe1/PP6 directly or indirectly modulates kinetochore chromatin protein Mis12 to ensure progression into normal anaphase.

Amino Acid Sequence↗
PubMed2003

The kic1 kinase of schizosaccharomyces pombe is a CLK/STY orthologue that regulates cell-cell separation.

The CLK/STY kinases are a family of dual-specificity protein kinases implicated in the regulation of cellular growth and differentiation. Some of the kinases in the family are shown to phosphorylate serine-arginine-rich splicing factors and to regulate pre-mRNA splicing. However, the actual cellular mechanism that regulates cell growth, differentiation, and development by CLK/STY remains unclear. Here we show that a functionally conserved CLK/STY kinase exists in Schizosaccharomyces pombe, and this orthologue, called Kic1, regulates the cell surface and septum formation as well as a late step in cytokinesis. The Kic1 protein is modified in vivo, likely by phosphorylation, suggesting that it can be involved in a control cascade. In addition, kic1(+) together with dsk1(+), which encodes a related SR-specific protein kinase, constitutes a critical in vivo function for cell growth. The results provide the first in vivo evidence for the functional conservation of the CLK/STY family through evolution from fission yeast to mammals. Furthermore, since cell division and cell-cell interaction are fundamental for the differentiation and development of an organism, the novel cellular role of kic1(+) revealed from this study offers a clue to the understanding of its counterparts in higher eukaryotes.

Animals↗
PubMed2003

Human centromere chromatin protein hMis12, essential for equal segregation, is independent of CENP-A loading pathway.

Kinetochores are the chromosomal sites for spindle interaction and play a vital role for chromosome segregation. The composition of kinetochore proteins and their cellular roles are, however, poorly understood in higher eukaryotes. We identified a novel kinetochore protein family conserved from yeast to human that is essential for equal chromosome segregation. The human homologue hMis12 of yeast spMis12/scMtw1 retains conserved sequence features and locates at the kinetochore region indistinguishable from CENP-A, a centromeric histone variant. RNA interference (RNAi) analysis of HeLa cells shows that the reduced hMis12 results in misaligned metaphase chromosomes, lagging anaphase chromosomes, and interphase micronuclei without mitotic delay, while CENP-A is located at kinetochores. Further, the metaphase spindle length is abnormally extended. Spindle checkpoint protein hMad2 temporally localizes at kinetochores at early mitotic stages after RNAi. The RNAi deficiency of CENP-A leads to a similar mitotic phenotype, but the kinetochore signals of other kinetochore proteins, hMis6 and CENP-C, are greatly diminished. RNAi for hMis6, like that of a kinetochore kinesin CENP-E, induces mitotic arrest. Kinetochore localization of hMis12 is unaffected by CENP-A RNAi, demonstrating an independent pathway of CENP-A in human kinetochores.

Amino Acid Sequence↗
PubMed2003

A cell cycle-regulated GATA factor promotes centromeric localization of CENP-A in fission yeast.

CENP-A, the centromere-specific histone H3 variant, plays a crucial role in organizing kinetochore chromatin for precise chromosome segregation. We have isolated Ams2, a Daxx-like motif-containing GATA factor, and histone H4, as multicopy suppressors of cnp1-1, an S. pombe CENP-A mutant. While depletion of Ams2 results in the reduction of CENP-A binding to the centromere and chromosome missegregation, increasing its dosage restores association of a CENP-A mutant protein with centromeres. Conversely, overexpression of CENP-A or histone H4 suppresses an ams2 disruptant. The intracellular amount of Ams2 thus affects centromeric nucleosomal constituents. Ams2 is abundant in S phase and associates with chromatin, including the central centromeres through binding to GATA-core sequences. Ams2 is thus a cell cycle-regulated GATA factor that is required for centromere function.

Amino Acid Motifs↗
PubMed2003

A brute force postgenome approach to identify temperature-sensitive mutations that negatively interact with separase and securin plasmids.

BACKGROUND: The fission yeast Schizosaccharomyces pombe separase/Cut1 and securin/Cut2 are required for anaphase-specific activation of proteolysis that leads to proper sister chromatid separation. We intended to identify ts (temperature sensitive) strains whose growth was inhibited by multicopy plasmid pCUT1 or pCUT2 at the permissive temperature. RESULTS: After a one-by-one transformation of 1015 randomly isolated ts strains, 18 transformants that retarded in colony formation at the permissive or semipermissive temperature were isolated. Six of them, in the absence of pCUT1 or pCUT2, produced mitotic phenotypes with condensed chromosomes at the restrictive temperature. Gene cloning established that these mutants were defective in either the subunits (Cut9, Cut23, Cut20 or Apc10) of APC (anaphase promoting complex)/cyclosome or Cut8, a regulator for 26S proteasome localization. The inhibitory effect of separase against APC/cyclosome mutations was abolished when the catalytic site mutation C1730A was introduced and overproduced, indicating that inhibition needs an active separase. Securin/Cut2 overproduction also caused a negative effect on these mutants. Surprisingly, the phenotypes of cut9 and cut23 in the presence of pCUT1 or pCUT2 were not the mitotic arrest, and they were strikingly different depending on pCUT1 or pCUT2. CONCLUSIONS: This study shows the functional link between separase/Cut1 and APC/cyclosome in a separase activity-dependent manner. The negative effect of active separase overproduction on APC/cyclosome mutations is possibly due to the direct inhibition of APC/cyclosome. In addition, the manner of the inhibition by high copy securin and separase plasmids were quite different each other and did not result in the mitotic block.

Anaphase-Promoting Complex-Cyclosome↗
PubMed2002

Two budding yeast RAD4 homologs in fission yeast play different roles in the repair of UV-induced DNA damage.

We have identified two fission yeast homologs of budding yeast Rad4 and human xeroderma pigmentosum complementation group C (XP-C) correcting protein, designated Rhp4A and Rhp4B. Here we show that the rhp4 genes encode NER factors that are required for UV-induced DNA damage repair in fission yeast. The rhp4A-deficient cells but not the rhp4B-deficient cells are sensitive to UV irradiation. However, the disruption of both rhp4A and rhp4B resulted in UV sensitivity that was greater than that of the rhp4A-deficient cells, revealing that Rhp4B plays a role in DNA repair on its own. Fission yeast has two pathways to repair photolesions on DNA, namely, nucleotide excision repair (NER) and UV-damaged DNA endonuclease-dependent excision repair (UVER). Studies with the NER-deficient rad13 and the UVER-deficient (Delta)uvde mutants showed the two rhp4 genes are involved in NER and not UVER. Assessment of the ability of the various mutants to remove cyclobutane pyrimidine dimers (CPDs) from the rbp2 gene locus indicated that Rhp4A is involved in the preferential repair of lesions on the transcribed DNA strand and plays the major role in fission yeast NER. Rhp4B in contrast acts as an accessory protein in non-transcribed strand (NTS) repair.

Cell Cycle Proteins↗
PubMed2002

Cnd2 has dual roles in mitotic condensation and interphase.

Chromosome condensation requires condensin, which comprises five subunits. Two of these subunits--both being structural maintenance of chromosome (SMC) proteins-are coiled-coils with globular terminal domains that interact with ATP and DNA. The remaining three, non-SMC subunits also have essential, albeit undefined, roles in condensation. Here we report that Cnd2 (ref. 6), a non-SMC subunit of fission yeast similar to Drosophila Barren and the budding yeast protein Brn1 (refs 8, 9), is required for both interphase and mitotic condensation. In cnd2-1 mutants, ultraviolet-induced DNA damage is not repaired, and cells arrested by hydroxyurea do not recover. A definitive defect of interphase is abolishment of Cds1 (a checkpoint kinase) activation in the presence of hydroxyurea in both cnd2-1 mutant cells and in cells where other condensin subunits have been genetically disrupted. In the absence of hydroxyurea, a G2 checkpoint delay occurred in cnd2-1 mutants in a manner dependent on Cds1 and ATM-like Rad3, but not Chk1 (refs 10-13), before the mitotic condensation defect. Furthermore, cnd2-1 was synthetic-lethal with mutations of excision repair, RecQ helicase and DNA replication enzymes. These interphase and mitotic defects provide insight into the mechanistic role of non-SMC subunits that interact with the globular SMC domains in the heteropentameric holocomplex.

Cell Cycle Proteins↗
PubMed2002

Control of localization of a spindle checkpoint protein, Mad2, in fission yeast.

To ensure accurate chromosome segregation, the spindle checkpoint delays the onset of sister chromatid separation when the spindle is not attached to a kinetochore. Mad2, a component of the checkpoint, targets fission yeast Slp1/budding yeast Cdc20/human p55CDC and prevents it from promoting proteolysis, which is a prerequisite to sister chromatid separation. The protein is localized to unattached kinetochores in higher eukaryotes, and it is thought to be required for activation of the checkpoint as well. In this study, Mad2 and its target Slp1 were visualized in a tractable organism, fission yeast Schizosaccharomyces pombe. When cells were arrested at a prometaphase-like stage, the Mad2-Slp1 complex was stable and the two proteins were colocalized to unattached kinetochores. When the spindle attachment was completed, the complex was no longer detectable and only Mad2 was found associated to the spindle. These results would suggest that unattached kinetochores provide sites for assembly of the Mad2-Slp1 complex. During interphase, Mad2 was localized to the nuclear periphery as well as to the chromatin domain. This localization was abolished in a yeast strain lacking Mad1, a protein that physically interacts with Mad2. Mad1 may anchor Mad2 to the nuclear membrane and regulate its entry into the nucleus.

Animals↗
PubMed2002

Condensin architecture and interaction with DNA: regulatory non-SMC subunits bind to the head of SMC heterodimer.

Condensin and cohesin are two protein complexes that act as the central mediators of chromosome condensation and sister chromatid cohesion, respectively. The basic underlying mechanism of action of these complexes remained enigmatic. Direct visualization of condensin and cohesin was expected to provide hints to their mechanisms. They are composed of heterodimers of distinct structural maintenance of chromosome (SMC) proteins and other non-SMC subunits. Here, we report the first observation of the architecture of condensin and its interaction with DNA by atomic force microscopy (AFM). The purified condensin SMC heterodimer shows a head-tail structure with a single head composed of globular domains and a tail with the coiled-coil region. Unexpectedly, the condensin non-SMC trimers associate with the head of SMC heterodimers, producing a larger head with the tail. The heteropentamer is bound to DNA in a distributive fashion, whereas condensin SMC heterodimers interact with DNA as aggregates within a large DNA-protein assembly. Thus, non-SMC trimers may regulate the ATPase activity of condensin by directly interacting with the globular domains of SMC heterodimer and alter the mode of DNA interaction. A model for the action of heteropentamer is presented.

Adenosine Triphosphatases↗
PubMed2002

Requirement of chromatid cohesion proteins rad21/scc1 and mis4/scc2 for normal spindle-kinetochore interaction in fission yeast.

BACKGROUND: Proteins conserved from yeast to human hold two sister chromatids together. The failure to form cohesion in the S phase results in premature separation of chromatids in G2/M. Mitotic kinetochores free from microtubules or the lack of tension are known to activate spindle checkpoint. RESULTS: The loss of chromatid cohesion in fission yeast mutants (mis4-242 and rad21-K1) leads to the activation of Mad2- and Bub1-dependent checkpoint, possibly due to a diminished microtubule-kinetochore interaction. Bub1, a checkpoint kinase, localizes briefly at early mitotic kinetochores in wild-type, whereas the cohesion mutation greatly increases the duration of kinetochore localization. Bub1 is bound to the central centromere region of mitotic cells. These cohesion mutants are hypersensitive to a tubulin poison and are synthetic lethal with dis1 and bir1/cut17, which are defective in microtubule-kinetochore interaction. The formation of specialized centromere chromatin containing CENP-A does not require cohesion. Dominant-negative noncleavable Rad21 fails to activate checkpoint but blocks sister chromatid separation and full spindle elongation in anaphase. CONCLUSIONS: Mis4 and Rad21 (budding yeast Scc2 and Scc1 homologs, respectively) act in establishing the normal spindle-kinetochore interaction in early mitosis and inhibit sister chromatid separation until the cleavage of Rad21 in anaphase. Checkpoint directly or indirectly monitors the states of cohesion in early mitosis. Full spindle extension occurs with unequal nuclear division in cohesion mutants in the absence of Mad2.

Cell Cycle↗
PubMed2002

The model unicellular eukaryote, Schizosaccharomyces pombe.

The fission yeast Schizosaccharomyces pombe has long been a model organism for studies of eukaryotic cells, winning renown especially for studies of the cell cycle. Now that its genome has been sequenced, S. pombe is ready to assume its rightful place in the pantheon of small eukaryotic giants.

Eukaryotic Cells↗