The spindle
assembly checkpoint prevents anaphase in nocodazole treated cells.
Mutations affecting essential kinetochore proteins are lethal in
combination with checkpoint mutants suggesting that defective
kinetochores activate the checkpoint. ts ndc10-1 mutants don't
arrest in nocodazole and behave as checkpoint mutants. Therefore the
checkpoint is dependent on intact kinetochore function. Furthermore,
nocodazole's effect is mediated through Ndc10p suggesting that the only
lesion activating the checkpoint in nocodazole is impaired kinetochore
function. We have begun a genetic dissection of the checkpoint within
the kinetochore. All kinetochore mutants are benomyl sensitive and could
lack checkpoint function. Checkpoint activity is unaffected in:
cep1 (deletion), cse4 (4 alleles), mif2 (4
alleles), cep2 (2 alleles) ctf13-30 , skp1-4 and a
skp1-4 ctf13-30 double mutant. These data suggest that there are
two types of benomyl sensitivity associated with kinetochore mutants.
One is due to impaired checkpoint function and the other is presumably
due to impaired attachments to microtubules. Furthermore, checkpoint
function is limited to a subset of proteins within the kinetochore. We
have constructed degron tagged alleles for each of the genes to generate
null alleles. Degron tagged cep3 mutants have a phenotype similar
to ndc10-1 suggesting that the checkpoint is dependent on both
Ndc10p and Cep3p (CBF3). Data on the other null alleles will be
presented.
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