The carboxyl-terminus of Cdc28 is essential for chromosome stability
and regulation by its activating kinase Cak1.
Ana A. Kitazono, Stephen J. Kron
Mol. Gen. and Cell Biology, University of Chicago, 924 E. 57th St. R322,
Chicago, IL 60637, U.S.
Several mutations induce genomic instability in yeast. The altered genes
are diverse and include some involved in DNA repair, recombination and
replication; checkpoint response, chromosome cohesion and separation,
chromatin structure, etc. Many of these pathways are regulated by
the cyclin-dependent kinase Cdc28. In turn, Cdc28 is regulated through
the association with cyclins, inhibitors and accessory factors; and
activating and inhibitory phosphorylations. We identified a conserved
domain at the C-terminus of Cdc28 that is essential for genomic
stability. We randomly mutagenized codons 282 and 287 through 298, and
screened for cdc28 mutants that enhance the temperature
sensitivity of the kinetochore mutant ctf13-30, a phenotype that
is linked to chromosome instability. We isolated six
cdc28CST mutants that progress through mitosis despite
the defective kinetochores, which normally induces cell-cycle arrest.
Sequencing of the CDC28 ORF in each of the mutants revealed
multiple substitutions, ranging from four to seven of the thirteen
randomized residues. Single cdc28CST mutants are
temperature sensitive and cultures treated at semi-restrictive
temperature accumulate aneuploids and exhibit chromosome loss.
Interestingly, the activating kinase Cak1 was found to suppress the
temperature sensitivity completely, and the genomic instability
partially. Our data implicate the C-terminal domain of Cdc28 in
surveillance function and regulation by Cak1.
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