@PhdThesis{duepublico_mods_00075233,
  author = 	{Dudziak, Alexander},
  title = 	{Biochemical and genetic analysis of Dam1c ring assembly at the budding yeast kinetochore},
  year = 	{2021},
  month = 	{Dec},
  day = 	{22},
  abstract = 	{During mitotic cell division, chromosomes containing the genetic information of an organism must be equally and accurately distributed to the two new cells. Chromosomes attach to dynamic microtubules of the mitotic spindle and are pulled apart harnessing forces generated by microtubule depolymerization and spindle elongation. Microtubules bind to a well-defined proteinaceous structure, the kinetochore, which is specifically assembled on centromeric chromatin of each chromosome. Besides forming microtubule attachments, kinetochores perform additional functions such as sensing the attachment status of each chromosome and initiating spindle assembly checkpoint signaling. An essential component of the Saccharomyces cerevisiae kinetochore is the Dam1 complex (Dam1c). The ten-subunit complex has the propensity to oligomerize into rings with a 16-fold symmetry that encircle microtubules and track their dynamic plus ends. Together with the Ndc80 complex (Ndc80c), which serves as a kinetochore receptor for Dam1c, Dam1c provides the physical link between the kinetochore and spindle microtubules and eventually translates microtubule depolymerization into chromosome movement. Even though many features of the Dam1 complex have been investigated during the past two decades, employing genetic, cell biological, biochemical, biophysical and structural biological approaches, it is still poorly understood how the complex is specifically recruited to the plus ends of kinetochore microtubules. Furthermore, relatively little is known about interactions of the complex with other proteins such as microtubule-associated proteins (MAPs). In the present study, the interaction between Dam1c with the autonomous plus end-tracking protein Bim1/EB1 is characterized using a combination of genetics, cell biology and biochemical reconstitution. Furthermore, insights into the structure of the Dam1c-Bim1 complex have been obtained by negative stain electron microscopy and chemical crosslinking. It is demonstrated that Bim1 closely associates with the protrusion domains of the Dam1 complex by binding a conserved SxIP motif located in the C-terminus of the Duo1 subunit. Binding of Bim1 to the complex is required for maximum loading of Dam1c onto kinetochores in metaphase and ensures timely mitotic progression. Phosphorylation by the conserved kinase Mps1 promotes the interaction between Dam1c and Bim1 and overexpression of Mps1 affects the localization of Dam1c during metaphase. In contrast, binding of Bim1 to Dam1c is refractory to phosphorylation by Ipl1/Aurora B. Biochemical and structural analyses reveal that Bim1 induces oligomerization of Dam1c into partially assembled rings with well-defined curvature. Furthermore, Bim1 recruits Bik1/CLIP-170 to Dam1c and by this induces the assembly of Dam1c into complete rings even in the absence of microtubules. Hence, binding of Bim1 and subsequent recruitment of Bik1/CLIP-170 is a novel regulatory mechanism for Dam1c ring assembly. Simultaneous disruption of Bim1-binding to Dam1c and interfering with Cdk1- and Ipl1/Aurora B-regulated Dam1c oligomerization mechanisms is detrimental to viability of yeast cells and negatively affects growth especially at low temperatures, suggesting that oligomerization of Dam1c is essential for formation of mature kinetochore-microtubule attachments. Dam1c finally engages with the Ndc80 complex to form load-bearing kinetochore-microtubule attachments. Biochemical reconstitution assays suggest that Dam1c engages either with Bim1-Bik1 or with Ndc80c. This implies that the Dam1c-Bim1-Bik1 and Dam1c-Ndc80c complexes presumably represent two distinct biochemical entities of Dam1c that might exist at different timepoints during the formation of kinetochore-microtubule attachments. These results demonstrate that binding of Bim1 to Dam1c is an important step in the regulation of outer kinetochore assembly and serves to control oligomerization and kinetochore recruitment of the complex. Kinetochore-localized Mps1 activity promotes binding of Bim1 and ensures specific accumulation of Dam1c in proximity of unattached kinetochores, but not at any other part of the mitotic spindle such as the midzone. Furthermore, Bim1 might serve as temporary placeholder that initially brings Dam1c to the microtubule plus end and is subsequently replaced by the Ndc80 complex to form mature end-on attachments. Previous reports about physical interactions between the metazoan Ska complex, the functional homolog of Dam1c, and the end-binding protein EB1 suggest that the mechanistic principles described in this thesis may be more widely conserved among eukaryotes.},
  doi = 	{10.17185/duepublico/75233},
  url = 	{https://duepublico2.uni-due.de/receive/duepublico_mods_00075233},
  url = 	{https://doi.org/10.17185/duepublico/75233},
  file = 	{:https://duepublico2.uni-due.de/servlets/MCRFileNodeServlet/duepublico_derivate_00074958/Diss_Dudziak.pdf:PDF},
  language = 	{en}
}