Outer kinetochore protein Dam1 promotes centromere clustering in parallel with Slk19 in budding yeast.

A higher order organization of the centromeres in the form of clustering of these DNA loci has been observed in many organisms. While centromere clustering is biologically significant to achieve faithful chromosome segregation, the underlying molecular mechanism is yet to be fully understood. In budding yeast, a kinetochore-associated protein Slk19 is shown to have a role in clustering in association with the microtubules whereas removal of either Slk19 or microtubules alone does not have any effect on the centromere clustering. Furthermore, Slk19 is non-essential for growth and becomes cleaved during anaphase whereas clustering being an essential event occurs throughout the cell cycle. Hence, we searched for an additional factor involved in the clustering and since the integrity of the kinetochore complex is shown to be crucial for centromere clustering, we restricted our search within the complex. We observed that the outermost kinetochore protein Dam1 promotes centromere clustering through stabilization of the kinetochore integrity. While in the absence of Dam1 we failed to detect Slk19 at the centromere, on the other hand, we found almost no Dam1 at the centromere in the absence of Slk19 and microtubules suggesting interdependency between these two pathways. Strikingly, we observed that overexpression of Dam1 or Slk19 could restore the centromere clustering largely in the cells devoid of Slk19 and microtubules or Dam1, respectively. Thus, we propose that in budding yeast, centromere clustering is achieved at least by two parallel pathways, through Dam1 and another via Slk19, in concert with the microtubules suggesting that having a dual mechanism may be crucial for ensuring microtubule capture by the point centromeres where each attaches to only one microtubule.