|dc.description.abstract||The eukaryotic cell cycle is governed by molecular checkpoints that ensure genomic integrity
and the faithful transmission of chromosomes to daughter cells. They inhibit the cycle until
conditions prevail that guarantee accurate DNA duplication and chromosome segregation. Two
major mechanisms are the ‘spindle assembly checkpoint’ and the ‘DNA damage checkpoint’.
During pro-metaphase, the spindle checkpoint monitors the orientation process of chromatid
pairs on the bipolar microtubule array nucleated by spindle pole bodies. In the yeasts
Schizosaccharomyces pombe and Saccharomyces cerevisiae, six proteins are at the heart of
spindle checkpoint function: Mad1, Mad2, Mad3, Bub1, Bub3 and Mph1/Mps1. The formation
of spindle checkpoint complexes signals the presence of incorrect spindle microtubule
attachments to kinetochores. These complexes cooperate to suppress the activity of the
anaphase promoting complex (APC) and inhibit the onset of anaphase. By isolating these
distinct complexes and analysing their composition by mass-spectrometry (MS) this work
revealed several intriguing disparities between the two yeast species, and the way in which the
Bub and Mad proteins cooperate to achieve inhibition. The ‘mitotic checkpoint complex’,
which in S.cerevisiae consists of Mad2, Mad3, Bub3 and the APC activator Cdc20, was found to
lack Bub3 in S.pombe. The S.pombe complex was shown to interact with the APC, but no stable
interaction was found to be required in S.cerevisiae cells. And whereas Bub1 and Bub3 were
found to form a complex with Mad1 in S.cerevisiae, in S.pombe they were shown to associate
with Mad3 to form the ‘BUB+ spindle checkpoint complex’.
In addition, MS analysis uncovered TAPAS: a novel S.pombe complex that was found to interact
with the BUB+ complex and revealed to consist of Tfg3, Abo1 (gene product of SPAC31G5.19),
Pob3 and Spt16. TAPAS mutant cells were shown to lose viability as a result of genotoxic
stress, a phenotype that was surprisingly shared with bub1Δ and bub1kd ‘kinase dead’ mutants.
Sensitivity of cells deficient in TAPAS or Bub1 did not appear to be due to the loss of DNA
damage checkpoint or DNA replication checkpoint functions. Further examination revealed
that Bub1 functions in the repair of DNA double strand breaks.
Taken together, this work demonstrates that even though the molecular components of the
spindle checkpoint pathway are conserved, their regulatory connections have to some extent
diverged through molecular evolution. This process not only rewired, but entwined two
molecular processes that together safeguard the genetic heritage of cells.||en