Quantitative measurement of the number of Cse4, CBF3, and Ndc80 proteins at kinetochores reveals a 2.5–3-fold increased copy number relative to prior estimates.

Regions of highly repetitive DNA, such as those found in the nucleolus, show a self-organization that is marked by spatial segregation and frequent self-interaction. The mechanisms that underlie the sequestration of these sub-domains are largely unknown. Using a stochastic, bead-spring representation of chromatin in budding yeast, we find enrichment of protein-mediated, dynamic chromosomal cross-links recapitulates the segregation, morphology and self-interaction of the nucleolus. Rates and enrichment of dynamic crosslinking have profound consequences on domain morphology. Our model demonstrates the nucleolus is phase separated from other chromatin in the nucleus and predicts that multiple rDNA loci will form a single nucleolus independent of their location within the genome. Fluorescent labeling of budding yeast nucleoli with CDC14-GFP revealed that a split rDNA locus indeed forms a single nucleolus. We propose that nuclear sub-domains, such as the nucleolus, result from phase separations within the nucleus, which are driven by the enrichment of protein-mediated, dynamic chromosomal crosslinks.

Mechanisms that drive DNA damage-induced chromosome mobility include relaxation of external tethers to the nuclear envelope and internal chromatin–chromatin tethers. Together with microtubule dynamics, these can mobilize the genome in response to DNA damage.

A new posttranslational modification is found of centromeric histone H3 variant Cse4. Cse4 is sumoylated by E3 ligases Siz1 and Siz2 and ubiquitinated by Slx5, a Sumo-targeted ubiquitin ligase. Slx5 regulates ubiquitin-mediated proteolysis of Cse4 and prevents it from being mislocalized under normal physiological conditions.

Depletion analyses and nanometer-scale mapping of spindle assembly checkpoint proteins reveal how these proteins are integrated within the substructure of the kinetochore.