To prevent genetic instability, the kinetochores of replicated chromosomes must form proper bipolar attachments to the mitotic spindle. Cells with improperly attached kinetochores are prevented from entering anaphase by the actions of the spindle checkpoint. This mechanism is conserved and defects have been linked to human cancer. The components of the spindle checkpoint were originally identified in the budding yeast S. cerevisiae. We propose to utilize the experimental tractability of this yeast to define how the anaphase inhibitory signal is generated by unattached kinetochores and how this signal is transmitted to downstream targets. We have demonstrated that the Bub1p protein kinase acts in concert with Bub3p at an early step of the spindle checkpoint pathway. As has been demonstrated for other species, our data indicate that Bub1p is acting at S. cerevisiae kinetochores. The Bub1p-kinetochore interaction will be examined by both physical and genetic techniques and its characterization will be critical to defining how the checkpoint signal is initiated. We have recently found that, in addition to its checkpoint-signaling role, Bub1p is influencing kinetochore activity. The roles of Bub1p in signaling and kinetochore function will be defined. The Bub1p kinase itself undergoes changes in phosphorylation in a cell cycle specific manner. It is likely that these changes are correlated with Bub1p activity changes. We will assess the role of phosphorylation of and by the Bub1p kinase. A target of the spindle checkpoint is the APC/C, a protein complex required for entry into anaphase. Studies by others have demonstrated a role for the Mad protein components of this checkpoint in inhibiting the APC/C. However, we have found an association between Bub3p and the APC/C, suggesting another mechanism of signal transduction. This interaction will be defined, and we will determine if this represents either a parallel or interdependent mechanism of signal transduction. Finally, a major effort will be undertaken to identify genes whose transcription is activated by the actions of the spindle checkpoint. The identified genes will report checkpoint activation and will allow a powerful dissection of this signal transduction pathway.
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