Analysis of kinetochore assembly in S. cerevisiae

University Of Virginia Charlottesville, Charlottesville VA

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Abstract

DESCRIPTION (provided by applicant) The goal of this project is to understand the dynamics and regulation of kinetochore assembly using S. cerevisiae as a model. Kinetochores are protein complexes bound to centromeres, responsible for mitotic spindle attachment, chromosome segregation, and spindle checkpoint signaling. Dysfunctional kinetochores cause chromosome missegregation, aneuploidy, and cancer, thus understanding the regulation of kinetochore assembly could lead to a better understanding and treatment of cancer. Some current models propose that kinetochore assembly is dependent on DNA replication, thus limiting it to S-Phase of the cell cycle. Other models suggest that it is cell cycle independent. This study will determine whether kinetochores can assemble de novo by destroying kinetochores using temperature-sensitive kinetochore mutants, and assaying for assembly by viability assays and by using chromatin immunoprecipitation and immuno-fluorescence to visualize kinetochore proteins at the centromere, at different stages of the cell cycle. Stoichiometric factors affecting centromere-specific nucleosome formation will be examined by studying the effect on localization of centromere-specific specialized histone, Cse4p, when over-expressed. Finally, kinetochore assembly may be regulated by accessory factors that load proteins onto DNA. Performing a genetic screen for conditional mutants that cannot assemble kinetochores at the restrictive temperature will identify factors required for assembling kinetochore proteins onto centromeres.