An investigation into factors affecting condensin association with mitotic centromeres

The SMC protein family (Structural Maintenance of Chromosomes) consists of a group of highly conserved protein complexes, central to chromosome dynamics and key cell cycle events. Condensin is a member of the SMC protein family, best known for its role in chromosome condensation and segregation in mitosis. The condensin complex becomes enriched at specific chromosome loci in a cell-cycle specific manner. However, the details of how it becomes associated with chromatin remain unclear. A particular area of interest regarding condensin association and activity is at the centromeres and pericentromeres, where condensin has been consistently shown to be enriched specifically during mitosis. This work is comprised of four results chapters, investigating factors affecting condensin association with mitotic centromeres in Saccharomyces cerevisiae, using chromatin immunoprecipitation (ChIP). We started by establishing a robust ChIP assay suitable for probing condensin enrichment at the centromeric regions. We conducted genetic control experiments to ensure the functionality of the experimental technique. In the next chapter we explored the importance of the kinetochore with regards to condensin enrichment, and found that perturbing the budding yeast kinetochore results in a loss of centromeric condensin association during mitosis. We then used condensin phosphorylation site and mitotic kinase mutants to examine the role of condensin subunit phosphorylation in its association with chromatin. Our results showed that Ipl1 (Aurora B kinase) and condensin phosphorylation is important for its enrichment at the centromere, but rather surprisingly that Cdc5 (polo-like kinase) a known activator of condensin does not appear to be. The final chapter investigates the function of condensin’s intrinsic ATPase activity, and we found that ATP-binding activity but not ATP-hydrolysis is important for condensin association with chromatin.