Masahiro Uritani

Competition between the Rad50 Complex and the Ku Heterodimer Reveals a Role for Exo1 in Processing Double-Strand Breaks but Not Telomeres

ABSTRACT The Mre11-Rad50-Nbs1(Xrs2) complex and the Ku70-Ku80 heterodimer are thought to compete with each other for binding to DNA ends. To investigate the mechanism underlying this competition, we analyzed both DNA damage sensitivity and telomere overhangs in Schizosaccharomyces pombe rad50-d, rad50-d pku70-d, rad50-d exo1-d, and pku70-d rad50-d exo1-d cells. We found that rad50 exo1 double mutants are more methyl methanesulfonate (MMS) sensitive than the respective single mutants. The MMS sensitivity of rad50-d cells was suppressed by concomitant deletion of pku70+ . However, the MMS sensitivity of the rad50 exo1 double mutant was not suppressed by the deletion of pku70+ . The G-rich overhang at telomere ends in taz1-d cells disappeared upon deletion of rad50+ , but the overhang reappeared following concomitant deletion of pku70+ . Our data suggest that the Rad50 complex can process DSB ends and telomere ends in the presence of the Ku heterodimer. However, the Ku heterodimer inhibits processing of DSB ends and telomere ends by alternative nucleases in the absence of the Rad50-Rad32 protein complex. While we have identified Exo1 as the alternative nuclease targeting DNA break sites, the identity of the nuclease acting on the telomere ends remains elusive.

Fission yeast Tor2 links nitrogen signals to cell proliferation and acts downstream of the Rheb GTPase.

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 (tor2ts) mutants. Remarkably, we have found that tor2ts 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, tor1Δtor2ts 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.

Fission yeast Dna2 is required for generation of the telomeric single-strand overhang

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.

TOR regulates late steps of ribosome maturation in the nucleoplasm via Nog1 in response to nutrients.

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.

A starvation-specific serine protease gene, isp6+, is involved in both autophagy and sexual development in Schizosaccharomyces pombe.

Schizosaccharomyces pombe isp6+ gene encodes a vacuolar serine protease, which is specifically induced during nitrogen starvation. An isp6-disruption mutant, isp6Δ, 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 isp6Δ were apparently aberrant. isp6Δ was infertile under nitrogen source-free conditions with poor expression of ste11+, a gene critical for sexual development. A protein kinase A-disruption mutant, pka1Δ, is prone to sexual development because expression of ste11+ is derepressed. However, isp6Δpka1Δ still showed defects in ste11+ expression and sexual development under nitrogen source-free conditions. isp6Δ and isp6Δpka1Δ 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, isp6Δpat1-114 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.

Purification and properties of trehalose-synthesizing enzyme from Pseudomonas sp. F1

The trehalose-synthesizing enzyme, which catalyzes the conversion of maltose to trehalose by intramolecular transglucosylation, was purified from a bacterium, Pseudomonas sp. F1. Its molecular mass was estimated to be 250 kDa by gel filtration and 67 kDa by SDS-polyacrylamide gel electrophoresis, and its pI was 5.8. The native enzyme may consist of 4 subunits. The enzyme was active on maltose and trehalose among saccharides tested as substrates. The N-terminal amino acid of the enzyme was threonine.

APC/C-Cdh1-dependent anaphase and telophase progression during mitotic slippage

BackgroundThe 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.ResultsHere 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.ConclusionsThe 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.

Involvement of a CCAAT-binding Complex in the Expression of a Nitrogen-Starvation-Specific Gene, isp6 +, in Schizosaccharomyces pombe

The fission yeast gene isp6 + is needed in nitrogen-starvation response but its transcriptional regulation has been unclear. isp6 + was repressed under nutrient conditions, in which cAMP-dependent protein kinase A, the stress-activated protein kinase cascade, and the CCAAT-binding complex were concerned. The CCAAT-binding complex also was involved in the induction of isp6 + during nitrogen starvation.

A monoclonal antibody specific for carboxy‐terminal region of yeast translation elongation factor‐3 inhibits ribosome‐activated ATPase activity but not intrinsic ATPase activity

Elongation factor‐3 (EF‐3) is an essential translation elongation factor specific to yeasts and fungi. When EF‐3 interacts with yeast ribosomes, its ATPase activity, which is indispensable for the function of EF‐3 in translation, is drastically enhanced. In this study, a monoclonal antibody specific for the carboxy‐terminal region inhibited the ribosome‐activated ATPase activity of EF‐3, while it did not inhibit the intrinsic ATPase activity of EF‐3. The results suggest that the carboxy‐terminal region of EF‐3 is involved in the interaction with yeast ribosomes. The monoclonal antibody also inhibited poly(U)‐directed poly(Phe) synthesis, which indicates that the carboxy‐terminal region is important for EF‐3 to express its function in the polypeptide elongation cycle.

Schizosaccharomyces pombe taf1+ is required for nitrogen starvation-induced sexual development and for entering the dormant G0 state

Abstract Environmental change, such as nutritional starvation, induces physiological and morphological alterations that enable fission yeast cells to survive. We isolated a novel gene, taf1+, required for the response to nitrogen starvation in the fission yeast Schizosaccharomyces pombe. taf1 disruptants could not mate upon nitrogen starvation, but could upon carbon starvation. taf1 disruptants had a defect in inducing ste11+ expression under nitrogen starvation conditions. Furthermore, they lost viability quickly in nitrogen-depleted medium. Unlike wild-type cells, starved taf1− cells had nuclear chromatin that were flat and adhered to the cell periphery. These results indicate that taf1+ is required for nitrogen starvation-induced sexual development and entering the dormant G0 state.