Structural Studies of the Yeast Dam1 Complex
- Author(s): Ramey, Vincent Harper
- Advisor(s): Nogales, Eva
- et al.
All eukaryotic cells must equally segregate their chromosomes between two daughter cells each time they divide. This process must be extremely robust, as errors cause catastrophic losses or gains of genetic material. Chromosmes are attached to microtubules through an organelle called the kinetochore. Greek for movement place, the kinetochore serves as a platform both for generating the forces required to separate chromatids to opposite poles and a major site of regulation to ensure that all chromosomes are segregated properly. In the budding yeast S. cerevisiae a single microtubule attaches to each kinetochore, making this connection extremely precious. In the last decade a complete parts list of the yeast kinetochore, containing approximately 70 proteins arranged into roughly a dozen complexes, has been established. However, very little structural information exists for most of these components, and the mechanism by which the kinetochore remained attached to a dynamic microtubule plus-end. In the past five years the Dam1 complex emerged as an exciting component of the outer kinetochore that forms rings around microtubules and in vitro recapitulates much of the functionality of kinetochore-microtubule attachment, including remaining attached to ends and using the energy of the depolymerizing microtubule to do work on a load.
To understand Dam1 at the kinetochore we must know how it binds to microtubules, assembles into rings, and how its assembly is regulated by kinases at the kinetochore. In order to answer these questions I used electron microscopy to image Dam1 in various mutant forms both in the presence and absense of microtubules. Computational techniques allowed me to solve structures of the complex alone and bound to microtubules in three different assemblies. I also mapped 5 of the 10 proteins in the complex onto the structure. Integrating all this information I generated a structural model for Dam1 at the kinetochore that substantially advances our understanding of its function and will direct future experiments in the field.