Title: Structure of the yeast DASH complex, a kinetochore-microtubule interface
Description: Kinetochores mediate the process of chromosome segregation by attaching centromeric DNA to the mitotic spindle. Maintaining this attachment during anaphase, however, is complicated by the dynamic nature of the microtubule end. The budding yeast S. cerevisiae is a good model system for studying this problem because only one microtubule attaches to each kinetochore on a small centromere. The DASH complex is an essential microtubule-binding component of the kinetochore. In order to study DASH, we optimized a system for the polycistronic coexpression of multiple proteins in E. coli. Using this system, we purified a single complex, an approximately 210 kD heterodecamer with an apparent stoichiometry of one copy of each subunit. Hydrodynamic properties of the recombinant assembly are indistinguishable from those of the native complex in yeast extracts. The structure of DASH alone and bound to microtubules was visualized by electron microscopy. The free heterodecamer is relatively globular. In the presence of microtubules, DASH oligomerizes to form rings and paired helices that encircle the microtubules. A reconstruction of decorated microtubules was obtained with cryoelectron tomography. We characterized the microtubule binding properties of truncations and subcomplexes of DASH, thus identifying candidate polypeptide extensions involved in establishing the DASH-microtubule interface. The acidic C-terminal extensions of tubulin subunits are not essential for DASH binding. We measured the molecular mass of DASH rings on microtubules with scanning transmission electron microscopy. Approximately twenty-five DASH heterodecamers assemble to form each ring.

The nature of the interface between DASH and the microtubule suggests that DASH translates through the dynamic association and relocation of multiple flexible appendages along the surface of the microtubule. We discuss potential roles for DASH rings in maintaining microtubule attachment during chromosome segregation.