Errors in chromosome segregation result in the loss of damage of DNA which leads to genomic instability, a hallmark of many cancer cells. To ensure proper chromosome separation, a functional mitotic spindle must be assembled before the cell divides. The regulatory pathway which ensures this coordination is the spindle assembly, or mitotic checkpoint. It appears that this pathway acts through a kinetochore-based signal which inhibits cell-cycle progression when the kinetochore is not properly attached to the mitotic spindle. The proteins involved in 'sensing' proper kinetochore attachment to spindle and generating a cell-cycle 'stop' signal are unknown. In S. cerevisiae, however, many of the proteins associated with the kinetochore have been identified as have many components required for the spindle-assembly checkpoint. The experiments I propose in this application are designed to determine the proteins that are required to generate the checkpoint signal. I will approach this problem from two directions: I will determine whether yeast kinetochore components are required for generating the spindle-assembly checkpoint signal, and will also investigate the regulation of the putative checkpoint signaling protein, Bub1p. Bub1p is a kinase that functions upstream in the spindle-assembly checkpoint pathway and localizes to the kinetochores in human cells making it a good candidate for the protein involved in generating the checkpoint signal. These experiments will contribute to our understanding of the mechanism which limits genomic instability and which controls cell division.
