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Zusammenfassung:
The spindle assembly checkpoint (SAC) is a highly conserved eukaryotic surveillance mechanism that maintains genomic integrity by delaying mitotic progression until all chromosomes have become properly attached to the mitotic spindle via their kinetochores. Malfunction of this checkpoint leads to chromosome segregation errors and has been implicated in tumorigenesis. SAC protein localization to unattached kinetochores is considered to be required for checkpoint signaling. This study employs the model organism Schizosaccharomyces pombe to investigate the role of different checkpoint components and their interactions with each other during the SAC signaling cascade. We examined the link between Mad1 and Bub1 to explore the connection between upstream and downstream events during checkpoint signaling. We found that conserved motifs in Bub1 and Mad1 are essential for Mad1 localization to the kinetochore and checkpoint activity. Furthermore, we revealed a hitherto unknown additional function of Mad1 in creating the checkpoint signal. Bub1 seems to act upstream of Mad1, and certain motifs in one of the kinetochore proteins are required for kinetochore recruitment of the Bub3-Bub1 complex. Here we provide evidence that a subset of these motifs is sufficient for this recruitment and checkpoint activity. The ultimate effector of checkpoint signaling is the mitotic checkpoint complex. It was recently found that the composition of this complex is different from previously assumed. While early work suggested the presence of one Cdc20 molecule in the complex, latest results revealed that the mitotic checkpoint complex actually contains two Cdc20 molecules when bound to the APC/C. We observed the same situation in fission yeast and describe the role of conserved motifs within the checkpoint protein Mad3 in binding to those Cdc20 molecules. We furthermore indicate a function of the APC/C subunit Apc15 in the checkpoint that was unexpected based on work in other model organisms. Taken together, we added new facets to the picture of spindle assembly checkpoint signaling and highlight similarities and differences between organisms, which illustrate how conserved, yet versatile this signaling pathway is.
