It has been well-documented that Mad1, Mad2, and Cdc20 have important roles in the spindle checkpoint in mitosis. In mammalian cells, Mad1 is responsible for Mad2 localization at the kinetochore. The Mad1-Mad2 complex has been shown to have a 'seat belt' binding conformation. This complex can also remain stable in 4m urea or 2M NaCI. Regardless of this extremely stable interaction, there is a Mad2 switch between Mad1 and Cdc20 at the kinetochore. In the presence of Mad2, Cdc20 is not able to activate the anaphase-promoting complex (APC). This inhibition causes cells to remain arrested in metaphase. We will use a collection of four cdc20 mutants to analyze the mechanism of Cdc20-Mad2 inhibition of the cell cycle in mitosis. Three of the cdc20 mutants are deficient in Mad2 binding, and the other has an increased affinity for Mad2. These mutants will also be used to identify other genes involved in the Mad2 switch between Mad1 and Cdc20. Another aim of this proposal is to identify genes involved in the tension-sensing aspect of the spindle checkpoint. We will use temperature sensitive mcd1-1 mutants to screen for genes involved in this process. Mcd1 is a member of the cohesion complex that is essential for sister chromatid association.
