To ensure accurate segregation, the major cell cycle control mechanism in mitosis, the mitotic checkpoint, delays anaphase onset until all chromosomes have properly attached to spindle microtubules. This is achieved through the production of a "wait anaphase" inhibitor(s) that blocks recognition of cyclin B and securin by Cdc20- activated APC/C, an E3 ubiquitin ligase which targets them for destruction. Using physiologically relevant levels of Mad2, Bub3, BubR1, and Cdc20, unattached kinetochores on purified chromosomes are demonstrated to catalyze generation of a soluble Cdc20 inhibitor or inhibition of Cdc20 already bound to APC/C. Antibody inhibition of Mad1 and dimerization deficient Mad2 are used to demonstrate that the chromosome-produced inhibitor requires both recruitment of Mad2 by Mad1 stably bound at unattached kinetochores and dimerization competent Mad2. By acting directly on Mad2, but not BubR1, purified chromosomes promote BubR1 binding to Cdc20 and APC/C, supporting a model in which immobilized Mad1/Mad2 at kinetochores provides a template for initial assembly of Mad2 bound to Cdc20 that is then converted to BubR1-Cdc20 as sequentially produced mitotic checkpoint inhibitor
