We identified a truncated allele of dam1 as a multicopy suppressor of the sensitivity of cdc13-117 (cyclin B) and mal3-1 (EB-1) cells to thiabendazole, a microtubule poison. We find that Dam1 binds to the plus end of spindle microtubules and kinetochores as cells enter mitosis and this is dependent on other components of the fission yeast DASH complex, including Ask1, Duo1, Spc34 and Dad1. By contrast, Dad1 remains bound to kinetochores throughout the cell cycle and its association is dependent on the Mis6 and Mal2, but not Mis12, Nuf2 or Cnp1, kinetochore proteins. In cells lacking Dam1, or other components of the DASH complex, anaphase is delayed due to activation of the spindle assembly checkpoint and lagging sister chromatids are frequently observed and occasionally sister chromatid pairs segregate to the same spindle pole. We find that the mitotic centromere-associated Klp5/Klp6 kinesin complex is essential in cells lacking components of the DASH complex. Cells lacking both Dam1 and Klp5 undergo a first cell cycle arrest in mitosis due to a failure to establish bipolar chromosome attachment.

After completion of the Schizosaccharomyces pombe genome sequence, we have carried out a pilot gene deletion project to assess the feasibility of a genome-wide deletion project and to estimate the percentage of essential genes. Using a PCR-based gene deletion procedure, we investigated 100 genes within a 253-kb region of chromosome II. Eight of nine genes located within a region of 18 kb could not be deleted, suggesting that systematic deletion of all fission yeast genes may be difficult to achieve using this PCR approach. The percentage of essential genes was found to be 17.5%. Further deletion of selected S. pombe genes revealed that whether a gene is essential or not is correlated with the timing of its appearance on the tree of life and its conservation within all branches of the tree. None of the investigated ancient genes in fission yeast that have been lost in the Saccharomyces cerevisiae lineage are essential. In agreement with S. cerevisiae and Caenorhabditis elegans genome analyses, our data suggest that natural selection has preferentially kept the genes required for vital functions. We propose that many of the essential eukaryotic genes appeared with the first eukaryotic cell and have remained conserved in all species

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Radiation has a limited but relevant role in the adjuvant therapy of gastric cancer (GC) patients. Since Chk1 plays a critical function in cellular response to genotoxic agents, we aimed to analyze the role of Chk1 in GC as a biomarker for radiotherapy resistance. We analyzed Chk1 expression in AGS and MKN45 human GC cell lines by RT-QPCR and WB and in a small cohort of human patient’s samples. We demonstrated that Chk1 overexpression specifically increases resistance to radiation in GC cells. Accordingly, abrogation of Chk1 activity with UCN-01 and its expression with shChk1 increased sensitivity to bleomycin and radiation. Furthermore, when we assessed Chk1 expression in human samples, we found a correlation between nuclear Chk1 accumulation and a decrease in progression free survival. Moreover, using a luciferase assay we found that Chk1’s expression is controlled by p53 and RB/E2F1 at the transcriptional level. Additionally, we present preliminary data suggesting a posttranscriptional regulation mechanism, involving miR-195 and miR-503, which are inversely correlated with expression of Chk1 in radioresistant cells. In conclusion, Chk1/microRNA axis is involved in resistance to radiation in GC, and suggests Chk1 as a potential tool for optimal stratification of patients susceptible to receive adjuvant radiotherapy after surgery.