Checkpoint independence of most DNA replication origins in fission yeast

<p>Abstract</p> <p>Background</p> <p>In budding yeast, the replication checkpoint slows progress through S phase by inhibiting replication origin firing. In mammals, the replication checkpoint inhibits both origin firing and replication fork movement. To find out which strategy is employed in the fission yeast, <it>Schizosaccharomyces pombe</it>, we used microarrays to investigate the use of origins by wild-type and checkpoint-mutant strains in the presence of hydroxyurea (HU), which limits the pool of deoxyribonucleoside triphosphates (dNTPs) and activates the replication checkpoint. The checkpoint-mutant cells carried deletions either of <it>rad3 </it>(which encodes the fission yeast homologue of ATR) or <it>cds1 </it>(which encodes the fission yeast homologue of Chk2).</p> <p>Results</p> <p>Our microarray results proved to be largely consistent with those independently obtained and recently published by three other laboratories. However, we were able to reconcile differences between the previous studies regarding the extent to which fission yeast replication origins are affected by the replication checkpoint. We found (consistent with the three previous studies after appropriate interpretation) that, in surprising contrast to budding yeast, most fission yeast origins, including both early- and late-firing origins, are not significantly affected by checkpoint mutations during replication in the presence of HU. A few origins (~3%) behaved like those in budding yeast: they replicated earlier in the checkpoint mutants than in wild type. These were located primarily in the heterochromatic subtelomeric regions of chromosomes 1 and 2. Indeed, the subtelomeric regions defined by the strongest checkpoint restraint correspond precisely to previously mapped subtelomeric heterochromatin. This observation implies that subtelomeric heterochromatin in fission yeast differs from heterochromatin at centromeres, in the mating type region, and in ribosomal DNA, since these regions replicated at least as efficiently in wild-type cells as in checkpoint-mutant cells.</p> <p>Conclusion</p> <p>The fact that ~97% of fission yeast replication origins – both early and late – are not significantly affected by replication checkpoint mutations in HU-treated cells suggests that (i) most late-firing origins are restrained from firing in HU-treated cells by at least one checkpoint-independent mechanism, and (ii) checkpoint-dependent slowing of S phase in fission yeast when DNA is damaged may be accomplished primarily by the slowing of replication forks.</p

Development of a high throughput yeast-based screening assay for human carbonic anhydrase isozyme II inhibitors

Abstract Carbonic anhydrase (CA; EC 4.2.1.1) catalyzes the reversible hydration of carbon dioxide (CO2) to bicarbonate and proton. There are 16 known isozymes of α-CA in humans, which differ widely in their kinetics, subcellular localization and tissue-specific distribution. Several disorders are associated with abnormal levels of CA, and so the inhibition of CA has pharmacological application in the treatment of many diseases. Currently, searching for novel CA inhibitors (CAI) has been performed using in vitro methods, and so their toxicity remains unknown at the time of screening. To obtain potentially safer CAIs, a screening procedure using an in vivo assay seems to have more advantages. Here, we developed a yeast-based in vivo assay for the detection of inhibitors of the human CA isozyme II (hCAII). The yeast Saccharomyces cerevisiae mutant deprived of its own CA (Δnce103 strain) can grow under a high CO2 condition (5% (v/v) CO2) but not at an ambient level. We constructed Δnce103 strains expressing various levels of hCAII from a plasmid harboring the hCAII gene arranged under the control of variously modified GAL1 promoter and relying on the expression of hCAII for growth under low CO2 condition. Using a multidrug-sensitive derivative of the Δnce103 strain expressing a low level of hCAII, we finally established a high throughput in vivo assay for hCAII inhibitors under a low CO2 condition. Cytotoxicity of the candidates obtained could be simultaneously determined under a high CO2 condition. However, their inhibitory activities against other CA isozymes remains to be established by further investigation

A novel yeast-based screening system for potential compounds that can alleviate human α-synuclein toxicity

Aims This study aimed to establish a yeast-based screening system for potential compounds that can alleviate the toxicity of α-synuclein (α-syn), a neuropathological hallmark of Parkinson’s disease, either inhibition of α-syn aggregation or promotion of ubiquitin-mediated degradation of α-syn. Methods and Results A powerful yeast-based screening assay using the rsp5A401E-mutant strain, which is hypersensitive to α-syn aggregation, was established by two-step gene replacement and further overexpressed the GFP-fused α-syn in the drug-sensitive yeast strain with a galactose-inducible multicopy plasmid. The rsp5A401E-mutant strain treated with baicalein, a known α-syn aggregation inhibitor, showed better α-syn toxicity alleviation than the same background wild type strain as accessed by comparison on the reduction kinetics of viable dye resazurin fluorometrically (λex540/λem590 nm). The rsp5A401E-mutant yeast-based assay system showed high sensitivity as it could detect as low as 3.13 µmol l−1 baicalein, the concentration that lower than previously report detected by the in vitro assay. Conclusions Our yeast-based system has been effective for screening potential compounds that can alleviate α-syn toxicity with high sensitivity and specificity. Significance and Impact of the Study Yeast-based assay system can be used to discover novel neuroprotective drug candidates which may be either efficiently suppress-α-syn aggregation or enhance ubiquitin-dependent degradation

Clausmarin A, Potential Immunosuppressant Revealed by Yeast-Based Assay and Interleukin-2 Production Assay in Jurkat T Cells

<div><p>Small-molecule inhibitors of Ca²⁺-signaling pathways are of medicinal importance, as exemplified by the immunosuppressants FK506 and cyclosporin A. Using a yeast-based assay devised for the specific detection of Ca²⁺-signaling inhibitors, clausmarin A, a previously reported terpenoid coumarin, was identified as an active substance. Here, we investigated the likely mechanism of clausmarin A action in yeast and Jurkat T-cells. In the presence of 100 mM CaCl₂ in the growth medium of Ca²⁺-sensitive Δ<i>zds1</i> strain yeast, clausmarin A exhibited a dose-dependent alleviation of various defects due to hyperactivation of Ca²⁺ signaling, such as growth inhibition, polarized bud growth and G2 phase cell-cycle arrest. Furthermore, clausmarin A inhibited the growth of Δ<i>mpk1</i> (lacking the Mpk1 MAP kinase pathway) but not Δ<i>cnb1</i> (lacking the calcineurin pathway) strain, suggesting that clausmarin A inhibited the calcineurin pathway as presumed from the synthetic lethality of these pathways. Furthermore, clausmarin A alleviated the serious defects of a strain expressing a constitutively active form of calcineurin. In the human Jurkat T-cell line, clausmarin A exhibited a dose-dependent inhibition of IL-2 production and IL-2 gene transcription, as well as an inhibition of NFAT dephosphorylation. The effects of clausmarin A observed in both yeast and Jurkat cells are basically similar to those of FK506. Our study revealed that clausmarin A is an inhibitor of the calcineurin pathway, and that this is probably mediated via inhibition of calcineurin phosphatase activity. As such, clausmarin A is a potential immunosuppressant.</p></div

Clausmarin A alleviates the growth inhibition of a constitutively active calcineurin.

<p><b>(A)</b> The effect of clausmarin A on the YRC1 yeast strain expressing an inducible constitutively active form of calcineurin. The experimental procedures were similar to those described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136804#pone.0136804.g001" target="_blank">Fig 1</a>, except the YRC1 strain with a chromosomally integrated construct for the galactose-inducible constitutively active form of the calcineurin catalytic subunit (<i>CMP2</i>ΔC) and the appropriate medium (SC) without CaCl₂ addition for induction of <i>GAL1</i> promoter was used. Legend: SC medium with no addition (□); 250 μM clausmarin A (▲); 250 nM FK506 (△). The <i>GAL1</i> promoter was induced by the addition of 2% (w/v) galactose and incubated at 30°C with shaking for the indicated period of time. *Statistically different with <i>p</i>-value <0.001. <b>(B)</b> The samples obtained after 12 h of incubation were observed under phase-contrast microscopy (left), fluorescence microscopy of Hoechst 33342-stained cells (middle) or flow cytometric analysis of Hoechst 33342-stained cells (right). Data shown are from one trial and are representative of those seen from three independent trials.</p

Clausmarin A alleviates the Ca²⁺-dependent growth inhibition of the YNS17 (Δ<i>zds1</i>) yeast strain.

<p>YNS17 cells (5 x 10⁶ cells/mL) were incubated for 30 min in YPD medium containing the indicated concentration of clausmarin A before the addition of CaCl₂ to 100 mM final concentration and incubating at 30°C with shaking for the indicated time. Legend: 100 mM CaCl₂ (■);100 mM CaCl₂ + 125 μM clausmarin A (□<b>)</b>;100 mM CaCl₂ + 250 μM clausmarin A (▲);100 mM CaCl₂ + 250 nM FK506 (△); none (○) and 250 μM clausmarin A (●). Data shown are from one trial and are representative of those seen from three independent trials. *Statistically different with <i>p</i>-value <0.0001.</p

Yeast strains used in this study.

<p>Yeast strains used in this study.</p