SUMMARY Chemical inhibitors can help analyze dynamic cellular processes, particularly when probes are active in genetically tractable model systems. Although fission yeast has served as an important model system, which shares more cellular processes (e.g., RNAi) with humans than budding yeast, its use for chemical biology has been limited by its multidrug resistance (MDR) response. Using genomics and genetics approaches, we identified the key transcription factors and drug-efflux transporters responsible for fission yeast MDR and designed strains sensitive to a wide-range of chemical inhibitors, including commonly used probes. We used this strain, along with acute chemical inhibition and high-resolution imaging, to examine metaphase spindle organization in a “closed” mitosis. Together, our findings suggest that our fission yeast strains will allow the use of several inhibitors as probes, discovery of new inhibitors, and analysis of drug action.

Condensin, one of the most abundant components of mitotic chromosomes, is a conserved protein complex composed of two structural maintenance of chromosomes (SMC) subunits (SMC2-and SMC4-type) and three non-SMC subunits, and it plays an essential role in mitotic chromosome condensation. Purified condensin reconfigures DNA structure using energy provided by ATP hydrolysis. To know the regulation of condensin in somatic cells, the expression level, subcellular localization, and phosphorylation status of human condensin were examined during the cell cycle. The levels of condensin subunits were almost constant throughout the cell cycle, and the three non-SMC subunits were phosphorylated at specific sites in mitosis and dephosphorylated upon the completion of mitosis. Subcellular fractionation studies revealed that a proportion of condensin was tightly bound to mitotic chromosomes and that this form was phosphorylated at specific sites. Condensin purified from mitotic cells had much stronger supercoiling activity than that purified from interphase cells. These results suggest that condensin functions in somatic cells are regulated by phosphorylation in two ways during the cell cycle; the phosphorylation of specific sites correlates with the chromosomal targeting of condensin, and its biochemical activity is stimulated by phosphorylation.

Previous studies of Epstein-Barr virus (EBV) replication focused mainly on the viral and cellular factors involved in replication compartment assembly and controlling the cell cycle. However, little is known about how EBV reorganizes nuclear architecture and the chromatin territories. In EBV-positive nasopharyngeal carcinoma NA cells or Akata cells, we noticed that cellular chromatin becomes highly condensed upon EBV reactivation. In searching for the possible mechanisms involved, we found that transient expression of EBV BGLF4 kinase induces unscheduled chromosome condensation, nuclear lamina disassembly, and stress fiber rearrangements, independently of cellular DNA replication and Cdc2 activity. BGLF4 interacts with condensin complexes, the major components in mitotic chromosome assembly, and induces condensin phosphorylation at Cdc2 consensus motifs. BGLF4 also stimulates the decatenation activity of topoisomerase II, suggesting that it may induce chromosome condensation through condensin and topoisomerase II activation. The ability to induce chromosome condensation is conserved in another gammaherpesvirus kinase, murine herpesvirus 68 ORF36. Together, these findings suggest a novel mechanism by which gammaherpesvirus kinases may induce multiple premature mitotic events to provide more extrachromosomal space for viral DNA replication and successful egress of nucleocapsid from the nucleus.DNA viruses adopt various strategies to facilitate their replication and maturation within host cells, including usurping the cellular DNA replication machinery and taking over the nuclear space for viral DNA replication, transcription, and packaging. Small DNA viruses, such as simian virus 40 and papillomaviruses, modulate the cell cycle control pathway and promote entry into S phase. This enables the host DNA polymerase and the increased nucleotide pool to be used for virus replication. For large DNA viruses, such as herpesviruses, which encode DNA replication and nucleotide metabolism enzymes, the viral DNA replication strategy is controlled through even more sophisticated interactions between host and viral machineries (reviewed in references 36 and 57).Epstein-Barr virus (EBV) belongs to the Gammaherpesvirinae and infects most of the human population worldwide. Infection may cause infectious mononucleosis and is closely associated with human malignant diseases, such as nasopharyngeal carcinoma (NPC) and Burkitt's lymphoma (61). Two different mechanisms have evolved to sustain successful infection of EBV. After primary infection, EBV becomes latent and the virally encoded EBNA-1 ensures that the circular episomal genome replicates during the S phase of the cell cycle and partitions equally into the daughter cells at mitosis (38). Upon immunoglobulin (Ig) cross-linking or chemical stimulation, EBV can be reactivated and express two immediate-early transactivators, Zta and Rta, which then turn on a cascade of viral gene expression to initiate lytic virus replication. Simultaneously, Zta and Rta may also modulate the cell c...