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Abstract:
To maintain genome integrity, the genetic material has to be equally distributed during cell division. The spindle assembly checkpoint is a conserved surveillance mechanism that delays anaphase as long as any of the chromosomes is not properly attached to the mitotic spindle. Malfunction of this checkpoint causes erroneous chromosome segregation and has been implicated in tumorigenesis. Despite a wealth of information on the localization and interaction of checkpoint proteins, the in vivo signaling mechanism is still only partially understood. A complex between the proteins Mad1 and Mad2 is crucial for the spindle assembly checkpoint. Kinetochore-bound Mad1:Mad2 binds a second molecule of Mad2 through Mad2:Mad2 dimerization, which is necessary for binding of this second Mad2 to the anaphase activator Cdc20. This ultimately prevents anaphase. Work of several labs, using different organisms, consistently showed that Bub1 is required for kinetochore recruitment of Mad1 and that kinetochore-bound Mad1 serves as binding platform for Mad2. Employing a combination of biochemical methods, yeast molecular genetics and live cell imaging approaches, we recently showed that the roles of Mad1 and Bub1 go beyond this role in bringing Mad2 to the kinetochore. We introduced mutations in the Mad1 C-terminus that preserved Mad1 kinetochore localization, its interaction with Mad2, and the capability for Mad2 dimerization, but nevertheless impaired checkpoint functionality. Similarly, artificial tethering of Mad1 to the kinetochore in cells depleted of Bub1 also did not rescue checkpoint functionality, although Mad2 was co-recruited with Mad1 and was able to dimerize. These results indicate that Mad1 and Bub1 are more than mere recruitment factors for Mad2. We are in the process of determining, which step, downstream of Mad2 dimerization, is defective and use the separation-of-function mutants of Mad1 to investigate the molecular role of the Mad1 C-terminus in this process.
