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ABSTRACT: Pot1 binds to single-stranded telomere overhangs and protects chromosome ends. RecQ helicases regulate homologous recombination at multiple stages including resection, strand displacement and resolution. Double mutants between fission yeast pot1 and the RecQ helicase rqh1 are synthetically lethal but the mechanism is not fully understood. Here, we show that the synthetic lethality of pot1 rqh1 double mutants is due to inappropriate homologous recombination as it is suppressed by deletion of rad51(+). The expression of Rad51 in the pot1 rqh1 rad51 triple mutant, which has circular chromosomes, is lethal. Reduction of expression of Rqh1 in a pot1 disruptant with circular chromosomes caused chromosome mis-segregation and this defect was partially suppressed by deletion of rad51(+). Taken together, our results suggest that Rqh1 is required for the maintenance of circular chromosomes when homologous recombination is active. Crossovers between circular monomeric chromosomes generate dimers that cannot segregate properly in E. coli. We propose that Rqh1 inhibits crossovers between circular monomeric chromosomes to suppress the generation of circular dimers.
Molecular and cellular biology 01/2013; · 6.06 Impact Factor
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ABSTRACT: In the fission yeast Schizosaccharomyces pombe, deletion of trt1(+) causes gradual telomere shortening, while deletion of pot1(+) causes rapid telomere loss. The double mutant between pot1 and RecQ helicase rqh1 is synthetically lethal. We found that the trt1 rqh1 double mutant was not synthetically lethal. The chromosome end fragments in both the trt1Δ rqh1Δ and the trt1Δ rqh1-hd (helicase dead) double mutants did not enter a pulsed-field electrophoresis gel. Both the trt1Δ rqh1Δ and the trt1Δ rqh1-hd double mutants were sensitive to the anti-microtubule drug thiabendazole. Moreover, the trt1Δ rqh1-hd double mutant displayed RPA foci on the chromosome bridge at high frequency in M phase cells. These phenotypes are very similar to that of the pot1Δ rqh1-hd double mutant, in which recombination intermediates accumulate at the chromosme ends in the M phase. These results suggest that the entangled chromosome ends, most likely recombination intermediates, are present in the M phase in the trt1Δ rqh1-hd double mutant.
Bioscience Biotechnology and Biochemistry 02/2012; 76(2):264-9. · 1.28 Impact Factor
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ABSTRACT: The spindle assembly checkpoint (SAC) inhibits anaphase progression in the presence of insufficient kinetochore-microtubule attachments, but cells can eventually override mitotic arrest by a process known as mitotic slippage or adaptation. This is a problem for cancer chemotherapy using microtubule poisons.
Here we describe mitotic slippage in yeast bub2Δ mutant cells that are defective in the repression of precocious telophase onset (mitotic exit). Precocious activation of anaphase promoting complex/cyclosome (APC/C)-Cdh1 caused mitotic slippage in the presence of nocodazole, while the SAC was still active. APC/C-Cdh1, but not APC/C-Cdc20, triggered anaphase progression (securin degradation, separase-mediated cohesin cleavage, sister-chromatid separation and chromosome missegregation), in addition to telophase onset (mitotic exit), during mitotic slippage. This demonstrates that an inhibitory system not only of APC/C-Cdc20 but also of APC/C-Cdh1 is critical for accurate chromosome segregation in the presence of insufficient kinetochore-microtubule attachments.
The sequential activation of APC/C-Cdc20 to APC/C-Cdh1 during mitosis is central to accurate mitosis. Precocious activation of APC/C-Cdh1 in metaphase (pre-anaphase) causes mitotic slippage in SAC-activated cells. For the prevention of mitotic slippage, concomitant inhibition of APC/C-Cdh1 may be effective for tumor therapy with mitotic spindle poisons in humans.
Cell Division 02/2012; 7:4. · 3.00 Impact Factor
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Katsunori Takahashi,
Ryota Imano,
Tatsuya Kibe,
Hiroyuki Seimiya,
Yukiko Muramatsu,
Naoki Kawabata,
Genki Tanaka,
Yoshitake Matsumoto,
Taisuke Hiromoto,
Yuka Koizumi,
Norihiko Nakazawa,
Mitsuhiro Yanagida,
Masashi Yukawa,
Eiko Tsuchiya, Masaru Ueno
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ABSTRACT: Pot1 is a single-stranded telomere-binding protein that is conserved from fission yeast to mammals. Deletion of Schizosaccharomyces pombe pot1(+) causes immediate telomere loss. S. pombe Rqh1 is a homolog of the human RecQ helicase WRN, which plays essential roles in the maintenance of genomic stability. Here, we demonstrate that a pot1Δ rqh1-hd (helicase-dead) double mutant maintains telomeres that are dependent on Rad51-mediated homologous recombination. Interestingly, the pot1Δ rqh1-hd double mutant displays a "cut" (cell untimely torn) phenotype and is sensitive to the antimicrotubule drug thiabendazole (TBZ). Moreover, the chromosome ends of the double mutant do not enter the pulsed-field electrophoresis gel. These results suggest that the entangled chromosome ends in the pot1Δ rqh1-hd double mutant inhibit chromosome segregation, signifying that Pot1 and Rqh1 are required for efficient chromosome segregation. We also found that POT1 knockdown, WRN-deficient human cells are sensitive to the antimicrotubule drug vinblastine, implying that some of the functions of S. pombe Pot1 and Rqh1 may be conserved in their respective human counterparts POT1 and WRN.
Molecular and cellular biology 02/2011; 31(3):495-506. · 6.06 Impact Factor
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ABSTRACT: Replication protein A (RPA) binds to single-stranded DNA generated during DNA replication and other processes. The roles of RPA in telomere maintenance have been demonstrated in yeasts, but not in telomerase-positive human cells. In this study, we found that expression of mutant RPA70 in human cells caused telomere shortening, suggesting that RPA is required for telomere-length regulation in human cancer cells.
Bioscience Biotechnology and Biochemistry 02/2010; 74(2):382-5. · 1.28 Impact Factor
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ABSTRACT: The mechanisms of eukaryotic cell-cycle regulation are closely linked to cellular tumorigenesis. Compounds that affect the cell cycle are good candidates for developing anti-tumor drugs. We developed a screening method for cell-cycle blockers using a Saccharomyces cerevisiae cdc2-1 rad9Delta strain that can detect the activity of substances by cell growth. We performed screening on culture broth of various microbes, and identified five compounds, borrelidin, mycophenolic acid, UCS15A, copiamycin analog, and fredericamycin A, that were known to possess anti-tumor activity. These results indicate that this screening method is effective as a first-screening system for anti-tumor agents.
Bioscience Biotechnology and Biochemistry 02/2010; 74(2):411-4. · 1.28 Impact Factor
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Masaru Ueno
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ABSTRACT: Telomeres are protected and maintained by telomere-specific proteins. In addition, proteins involved in DNA repair, such as Mre11-Rad50-Nbs1 complex, RPA, and RecQ helicase, are also involved in telomere maintenance. In this review, the roles of these proteins in telomere maintenance and their functional relationships to the telomere-specific proteins will be discussed.
Bioscience Biotechnology and Biochemistry 01/2010; 74(1):1-6. · 1.28 Impact Factor
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ABSTRACT: Cholesterol (ergosterol in yeast) in conjunction with sphingolipids forms tight-packing microdomains, 'lipid rafts,' which are thought to be critical for intracellular protein sorting in eukaryotic cells. When the activity of Erg9 involved in the first step of ergosterol biogenesis, but not that of Erg6 involved in a late step, is compromised, vacuolar degradation of the tryptophan permease Tat2 is promoted. It is unknown whether this difference simply reflects the difference between the inhibition of early and late steps. Here, it is shown that the deletion in ERG2, which encodes sterol C8-C7 isomerase (the next enzymatic step after Erg6), promotes the vacuolar degradation of Tat2. It suggests that the accumulation of specific sterol intermediates may alter lipid raft structures, promoting Tat2 degradation. The erg2Delta-mediated Tat2 degradation required Tat2 ubiquitination. Lipid raft association of Tat2 is compromised in erg2Delta cells. The erg2Delta mutation showed a synthetic growth defect with the trp1 mutation, indicating that Tat2 sorting is preferentially compromised in these mutants. Consistent with this notion, the raft-associated protein Pma1 was associated with detergent-resistant membranes and sorted to the plasma membrane. This study suggests the potential for the pharmacological control of cellular nutrient uptake in humans by regulating enzymes involved in cholesterol biogenesis.
FEMS Microbiology Letters 08/2009; 298(2):218-27. · 2.04 Impact Factor
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ABSTRACT: In eukaryotes, the hypoacetylated state of histone N-terminal lysines at many gene-promoters, which is created by histone deacetylases (HDACs), is changed to the hyperacetylated state by the function of histone acetyltransferases (HATs) upon transcription activation. Although much insight has been obtained to date as to how modification of the histone tail regulates gene expression, little is known about how the transition between the unmodified and modified states takes place. In Saccharomyces cerevisiae, the HDAC complex containing Rpd3 (Rpd3L) represses the transcription of several sets of genes through the URS1 cis-element. We found that the histone H3 acetylation level at the URS1 of seven genes (INO1, CAT2, ACS1, YAT1, RIM4, CRC1, and SIP4) was elevated in the presence of Rpd3/HDAC in growth in acetate-containing medium (YPA), suggesting that a mechanism that regulates HDAC activity is present in this organism. The biological significance of this phenomenon is discussed below.
Bioscience Biotechnology and Biochemistry 03/2009; 73(2):378-84. · 1.28 Impact Factor
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Masaru Ueno
Seikagaku. The Journal of Japanese Biochemical Society 10/2007; 79(9):868-71. · 0.04 Impact Factor
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ABSTRACT: Ergosterol is the yeast functional equivalent of cholesterol in mammalian cells. Deletion of the ERG6 gene, which encodes an enzyme catalyzing a late step of ergosterol biosynthesis, impedes targeting of the tryptophan permease Tat2p to the plasma membrane, but does not promote vacuolar degradation. It is unknown whether similar features appear when other steps of ergosterol biogenesis are inhibited. We show herein that the ergosterol biosynthesis inhibitor zaragozic acid (ZA) evoked massive vacuolar degradation of Tat2p, accompanied by a decrease in tryptophan uptake. ZA inhibits squalene synthetase (SQS, EC 2.5.1.21), which catalyzes the first committed step in the formation of cholesterol/ergosterol. The degradation of Tat2p was dependent on the Rsp5p-mediated ubiquitination of Tat2p and was not suppressed by deletions of VPS1, VPS27, VPS45 or PEP12. We will discuss ZA-mediated Tat2p degradation in the context of lipid rafts.
Biochimica et Biophysica Acta 08/2007; 1768(7):1681-90. · 4.66 Impact Factor
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ABSTRACT: The telomere complex must allow nucleases and helicases to process chromosome ends to make them substrates for telomerase, while preventing these same activities from disrupting chromosome end-protection. Replication protein A (RPA) binds to single-stranded DNA and is required for DNA replication, recombination, repair, and telomere maintenance. In fission yeast, the telomere binding protein Taz1 protects telomeres and negatively regulates telomerase. Here, we show that taz1-d rad11-D223Y double mutants lose their telomeric DNA, indicating that RPA (Rad11) and Taz1 are synergistically required to prevent telomere loss. Telomere loss in the taz1-d rad11-D223Y double mutants was suppressed by additional mutation of the helicase domain in a RecQ helicase (Rqh1), or by overexpression of Pot1, a single-strand telomere binding protein that is essential for protection of chromosome ends. From our results, we propose that in the absence of Taz1 and functional RPA, Pot1 cannot function properly and the helicase activity of Rqh1 promotes telomere loss. Our results suggest that controlling the activity of Rqh1 at telomeres is critical for the prevention of genomic instability.
Molecular Biology of the Cell 07/2007; 18(6):2378-87. · 4.94 Impact Factor
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ABSTRACT: The target of rapamycin (Tor) plays a pivotal role in cell growth and metabolism. Yeast contains two related proteins, Tor1 and Tor2. In fission yeast, Tor1 is dispensable for normal growth but is involved in amino acid uptake and cell survival under various stress conditions. In contrast, Tor2 is essential for cell proliferation; however, its physiological function remains unknown. Here we characterize the roles of fission yeast Tor2 by creating temperature sensitive (tor2(ts)) mutants. Remarkably, we have found that tor2(ts) mimics nitrogen starvation responses, because the mutant displays a number of phenotypes that are normally induced only on nitrogen deprivation. These include G1 cell-cycle arrest with a small cell size, induction of autophagy and commitment to sexual differentiation. By contrast, tor1Deltator2(ts) double mutant cells show distinct phenotypes, as the cells cease division with normal cell size in the absence of G1 arrest. Tor2 physically interacts with the conserved Rhb1/GTPase. Intriguingly, over-expression of rhb1(+) or deletion of Rhb1-GAP-encoding tsc2(+) is capable of rescuing stress-sensitive phenotypes of the tor1 mutant, implying that Tor1 and Tor2 also share functions in cell survival under adverse environment. We propose that Tor1 and Tor2 are involved in both corroborative and independent roles in nutrient sensing and stress response pathways.
Genes to Cells 01/2007; 11(12):1367-79. · 2.68 Impact Factor
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Yoshimi Honma,
Aiko Kitamura,
Ryo Shioda,
Hironori Maruyama,
Kanako Ozaki,
Yoko Oda,
Thierry Mini,
Paul Jenö,
Yasushi Maki,
Kazuyoshi Yonezawa,
Ed Hurt, Masaru Ueno,
Masahiro Uritani,
Michael N Hall,
Takashi Ushimaru
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ABSTRACT: The protein kinase TOR (target of rapamycin) controls several steps of ribosome biogenesis, including gene expression of rRNA and ribosomal proteins, and processing of the 35S rRNA precursor, in the budding yeast Saccharomyces cerevisiae. Here we show that TOR also regulates late stages of ribosome maturation in the nucleoplasm via the nuclear GTP-binding protein Nog1. Nog1 formed a complex that included 60S ribosomal proteins and pre-ribosomal proteins Nop7 and Rlp24. The Nog1 complex shuttled between the nucleolus and the nucleoplasm for ribosome biogenesis, but it was tethered to the nucleolus by both nutrient depletion and TOR inactivation, causing cessation of the late stages of ribosome biogenesis. Furthermore, after this, Nog1 and Nop7 proteins were lost, leading to complete cessation of ribosome maturation. Thus, the Nog1 complex is a critical regulator of ribosome biogenesis mediated by TOR. This is the first description of a physiological regulation of nucleolus-to-nucleoplasm translocation of pre-ribosome complexes.
The EMBO Journal 09/2006; 25(16):3832-42. · 9.20 Impact Factor
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ABSTRACT: Schizosaccharomyces pombe isp6(+) gene encodes a vacuolar serine protease, which is specifically induced during nitrogen starvation. An isp6-disruption mutant, isp6Delta, grew normally under normal conditions but was defective in large-scale protein degradation during nitrogen starvation, a hallmark of autophagy. Vacuoles are the organelles for such drastic protein degradation but those of isp6Delta were apparently aberrant. isp6Delta was infertile under nitrogen source-free conditions with poor expression of ste11(+), a gene critical for sexual development. A protein kinase A-disruption mutant, pka1Delta, is prone to sexual development because expression of ste11(+) is derepressed. However, isp6Deltapka1Delta still showed defects in ste11(+) expression and sexual development under nitrogen source-free conditions. isp6Delta and isp6Deltapka1Delta were able to initiate sexual development to produce spores when only a small amount of a nitrogen source was present. Pat1 protein kinase negatively controls meiosis, and a temperature-sensitive mutant of pat1, pat1-114, initiates meiosis irrespective of ploidy at the restrictive temperature. However, isp6Deltapat1-114 did not start meiosis under nitrogen source-free conditions even at the restrictive temperature. These observations suggest that isp6(+) contributes to sexual development by providing a nitrogen source through autophagy.
Current Genetics 07/2006; 49(6):403-13. · 2.56 Impact Factor
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ABSTRACT: It has been suggested that the Schizosaccharomyces pombe Rad50 (Rad50-Rad32-Nbs1) complex is required for the resection of the C-rich strand at telomere ends in taz1-d cells. However, the nuclease-deficient Rad32-D25A mutant can still resect the C-rich strand, suggesting the existence of a nuclease that resects the C-rich strand. Here, we demonstrate that a taz1-d dna2-2C double mutant lost the G-rich overhang at a semipermissive temperature. The amount of G-rich overhang in S phase in the dna2-C2 mutant was lower than that in wild-type cells at the semipermissive temperature. Dna2 bound to telomere DNA in a chromatin immunoprecipitation assay. Moreover, telomere length decreased with each generation after shift of the dna2-2C mutant to the semipermissive temperature. These results suggest that Dna2 is involved in the generation of G-rich overhangs in both wild-type cells and taz1-d cells. The dna2-C2 mutant was not gamma ray sensitive at the semipermissive temperature, suggesting that the ability to process double-strand break (DSB) ends was not affected in the dna2-C2 mutant. Our results reveal that DSB ends and telomere ends are processed by different mechanisms.
Molecular and Cellular Biology 12/2004; 24(21):9557-67. · 5.53 Impact Factor
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ABSTRACT: Sequence-specific protein-DNA interaction is critical for many important cellular processes such as transcription, DNA replication, and chromosome segregation. Identification of additional proteins that bind to DNA in a sequence-specific manner will contribute to the understanding of the mechanism of molecular recognition between protein and DNA. We found that the ATP phosphoribosyl transferase His1, which catalyzes the first step in histidine biosynthesis, is bound to both single- and double-stranded telomeric DNA. Competition experiments revealed that His1 is bound to a fission yeast telomeric DNA in a sequence-specific manner. Previously identified sequence-specific telomere-binding proteins contain Myb domain. In contrast, Schizosaccharomyces pombe His1 does not contain Myb domain. These findings indicate that His1 has a novel DNA-recognition domain.
Chemistry & Biodiversity 10/2004; 1(9):1344-53. · 1.80 Impact Factor
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ABSTRACT: The actin-related proteins (Arps), which are subdivided into at least eight subfamilies, are conserved from yeast to humans. A member of the Arp6 subfamily in Drosophila, Arp4/Arp6, co-localizes with heterochromatin protein 1 (HP1) in pericentric heterochromatin. Fission yeast Schizosaccharomyces pombe possesses both an HP1 homolog and an Arp6 homolog. However, the function of S.pombe Arp6 has not been characterized yet. We found that deletion of arp6(+) impaired telomere silencing, but did not affect centromere silencing. Chromatin immunoprecipitation assays revealed that Arp6 bound to the telomere region. However, unlike Drosophila Arp4/Arp6, S.pombe Arp6 was distributed throughout nuclei. The binding of Arp6 to telomere DNA was not affected by deletion of swi6(+). Moreover, the binding of Swi6 to telomere ends was not affected by deletion of arp6(+). These results suggest that Arp6 and Swi6 function independently at telomere ends. We propose that the Arp6-mediated repression mechanism works side by side with Swi6-based telomere silencing in S.pombe.
Nucleic Acids Research 02/2004; 32(2):736-41. · 8.03 Impact Factor
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ABSTRACT: Replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein involved in DNA replication, recombination and repair. In Saccharomyces cerevisiae, several mutants in the RFA1 gene encoding the large subunit of RPA have been isolated and one of the mutants with a missense allele, rfa1-D228Y, shows a synergistic reduction in telomere length when combined with a yku70 mutation. So far, only one mutant allele of the rad11(+) gene encoding the large subunit of RPA has been reported in Schizosaccharomyces pombe. To study the role of S.pombe RPA in DNA repair and possibly in telomere maintenance, we constructed a rad11-D223Y mutant, which corresponds to the S.cerevisiae rfa1-D228Y mutant. rad11-D223Y cells were methylmethane sulfonate, hydroxyurea, UV and gamma-ray sensitive, suggesting that rad11-D223Y cells have a defect in DNA repair activity. Unlike the S.cerevisiae rfa1-D228Y mutation, the rad11-D223Y mutation itself caused telomere shortening. Moreover, Rad11-Myc bound to telomere in a ChIP assay. These results strongly suggest that RPA is directly involved in telomere maintenance.
Nucleic Acids Research 01/2004; 31(24):7141-9. · 8.03 Impact Factor
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ABSTRACT: The Schizosaccharomyces pombe Ku70-Ku80 heterodimer is required for telomere length regulation. Lack of pku70+ results in telomere shortening and striking rearrangements of telomere-associated sequences. We found that the rearrangements of telomere-associated sequences in pku80+ mutants are Rhp51 dependent, but not Rad50 dependent. Rhp51 bound to telomere ends when the Ku heterodimer was not present at telomere ends. We also found that the single-stranded G-rich tails increased in S phase in wild-type strains, while deletion of pku70+ increased the single-stranded overhang in both G2 and S phase. Based on these observations, we propose that Rhp51 binds to the G-rich overhang and promotes homologous pairing between two different telomere ends in the absence of Ku heterodimer. Moreover, pku80 rhp51 double mutants showed a significantly reduced telomere hybridization signal. Our results suggest that, although Ku heterodimer sequesters Rhp51 from telomere ends to inhibit homologous recombination activity, Rhp51 plays important roles for the maintenance of telomere ends in the absence of the Ku heterodimer.
Nucleic Acids Research 10/2003; 31(17):5054-63. · 8.03 Impact Factor
