(A) G1-arrested wild type S. cerevisiae cells were released in the cell cycle in the presence of nocodazole. FACS analysis at the indicated time points shows that cells first undergo DNA replication and subsequently arrest with 2C DNA content, indicative of mitotic checkpoint arrest. (B) bub3Δ cells are checkpoint deficient, fail to arrest, and re-replicate they DNA. (C) A functional checkpoint is re-established upon expression of wild type Bub3 in bub3Δ cells. (D) Bub3R217A–R239A is unable to restore a functional checkpoint when expressed in bub3Δ cells. Panels A–D report experiments that were carried out at the same time and at least twice. (E) Re-budding in the presence of nocodazole was taken as an independent indication of checkpoint deficiency. Wild type cells, and bub3Δ cells reconstituted with wild type Bub3 were able to maintain the checkpoint arrest and did not re-bud during the time of observation. Conversely, bub3Δ cells and cells reconstituted with Bub3R217A–R239A re-budded, indicative of checkpoint failure. (F) The binding affinity of Bub1–Bub3 for individual MELTP is high. This predicts that multiple Bub1–Bub3 complexes may become bound to a single Spc105/Knl1 molecule. Mad3/BubR1 requires Bub1 for kinetochore recruitment, indicating that it is not able to target autonomously to kinetochores. Because Mad3/BubR1 is, like Bub1, constitutively bound to Bub3, it is plausible that Mad3/BubR1 suppresses the MELTP-binding activity of Bub3. Whether this occurs, and how, are purely speculative at this time.