The yeast mitotic spindle will likely be the first such organelle described in complete molecular detail, providing an opportunity to develop an understanding of its function and regulation at a level not achievable for any other organism. The studies proposed here are critical for attaining this goal. The Dam1 complex that we discovered, consists of 7 subunits that associate with the spindle and kinetochores. - The complex has essential roles in spindle integrity and kinetochore function. The goal of the proposed research is to determine the precise mechanisms by which the Dam1 complex contributes to spindle integrity and kinetochore function, and how its activities are regulated in vivo. Because the Daml complex interacts both with inner kinetochore components and with microtubules, and appears essential for establishment of new chromosome attachments, it may bring together chromosomes and spindle microtubules. To develop a deeper understanding of this important function, detailed phenotypic studies of mutants of the Dam1i complex, and of interdependencies in assembly of kinetochore components, will be performed. Interacting partners will be identified genetically to develop a better understanding of the biochemical context within which it functions. Biochemical activities of the complex on microtubules will be identified. To elucidate the roles of individual Dam1i subunits and to provide a foundation for understanding effects of post-translational modifications, interaction domains of each protein in the complex will be mapped and tested for in vivo importance. Three components of the Dam1 complex are phosphorylated in vivo, and their phosphorylation sites have been mapped. Some or all of these phosphates appear to be added by the conserved mitotic kinases Iplip and Mps1p; kinases that provide essential mitotic and mitotic checkpoint functions. Mutagenesis of each identified phosphorylation site will provide a unique opportunity to develop novel insights into mechanisms for orchestrating the complex events of mitosis. Because meiosis I has properties distinct from meiosis II and mitosis, mutants of Dam1 subunits will be studied in a strain that efficiently and synchronously undergoes meiosis. Comparison of how these mutations affect mitosis and meiosis II vs. meiosis I promises to provide a deeper understanding of the unique properties of meiosis I.