An Essential Function of Yeast Cyclin-dependent Kinase Cdc28 Maintains Chromosome Stability

Abstract

Multiple surveillance pathways maintain genomic integrity in yeast during mitosis. Although the cyclin-dependent kinase Cdc28 is a well established regulator of mitotic progression, evidence for a direct role in mitotic surveillance has been lacking. We have now implicated a conserved sequence in the Cdc28 carboxyl terminus in maintaining chromosome stability through mitosis. Six temperature-sensitive mutants were isolated via random mutagenesis of 13 carboxyl-terminal residues. These mutants identify a Cdc28 domain necessary for proper mitotic arrest in the face of kinetochore defects or microtubule inhibitors. These chromosome stabilitydefective cdc28CST mutants inappropriately continue mitosis when the mitotic spindle is disrupted at 23°C, display high rates of spontaneous chromosome loss at 30°C, and suffer catastrophic aneuploidy at 35°C. A dosage suppression screen identified Cak1, a kinase known to phosphorylate and activate Cdc28, as a specific high copy suppressor of cdc28 CST temperature sensitivity and chromosome instability. Suppression is independent of the kinase activity of Cak1, suggesting that Cak1 may bind to the carboxyl terminus to serve a non-catalytic role in assembly and/or stabilization of active Cdc28 complexes. Significantly, these studies implicate Cdc28 and Cak1 in an essential surveillance function required to maintain genetic stability through mitosis.From the onset of S phase until return to G 1 , surveillance pathways collaborate to maintain chromosome stability, monitoring completion of DNA replication and repair and determining attachment and tension in the mitotic spindle while regulating sister chromatid cohesion and mediating chromosome condensation (1). The budding yeast Saccharomyces cerevisiae offers a powerful model system with which to dissect these multiple determinants and pathways. Several genetic screens have identified genes that are implicated in chromosome stability (2, 3) and/or kinetochore function (4 -7). The yeast kinetochore is a centromere binding complex and attachment point for the plus-end of a single spindle microtubule, linking each sister chromatid to one spindle-pole body (SPB).1 Furthermore, the kinetochore is an assembly point for components of the spindle checkpoint, which mediates the dependence of mitotic progression on attachment and tension (8).The cyclin-dependent kinase Cdc28 (Cdk1 and Cdc2) is the master regulator of the yeast cell cycle (9, 10). It coordinates bud emergence, SPB duplication, and DNA replication at Start and directs spindle assembly and function in mitosis. Whereas the abundance of Cdc28 is constant, its activity is regulated by associations with cyclins, stoichiometric inhibitors, and accessory factors as well as by activating and inhibitory phosphorylations (9, 10). Even subtle changes in Cdc28 function affecting mitotic progression may impinge on chromosome stability. Indeed, mutations of several Cdc28 partners and substrates have been associated with genomic instability, whereas mutations of Cdc28 itself, such as cdc28-1...

Sister Chromatid Cohesion Role for CDC28-CDK in Saccharomyces cerevisiae

“…These cdc28 alleles were of particular interest because they exhibit defects in genetic stability, consistent with a model that Cdc28p functions in chromosome segregation (Kitazono and Kron 2002). The resulting diploids were sporulated and eight His1 Leu1 Ura1 strains that also exhibited temperature sensitivity at 37°(attributable to cdc28) selected for further analyses (Figure 3).…”

Role of Dot1-Dependent Histone H3 Methylation in G1 and S Phase DNA Damage Checkpoint Functions of Rad9

“…To generate the rad9-Tyr798Gln mutation, an EcoRI-BamHI fragment encoding the 5Ј region of RAD9 obtained by digesting pDL847, a pRS416-based vector carrying a full-length RAD9 clone (6), kindly provided by D. Lydall, was cloned into pUC18 and PCR mutagenized (30) to encode a Tyr798Gln substitution by using the primers YQMUT1 (5Ј-CGTGG AATTACAAATTTCAGCCGGGTATTTTATTGG-3Ј) and YQMUT2 (5Ј-CC AATAAAATACCCGGCTGAAATTTGTAATTCCACG-3Ј) (mutations underlined). The mutation was confirmed by sequencing and the fragment was recloned into pDL847 (6) to form pRS416-rad9-Tyr798Gln.…”

“…Cell cycle synchronization, culture manipulation, and flow cytometry analysis of DNA content were as previously described (16,30). To induce DNA damage, cells were irradiated at 30 Gy/min in a Gammacell 220 60 Co source (Atomic Energy of Canada, Ltd.), treated with 0.03% MMS, exposed to 50 J/cm 2 at 254 nm in a Stratalinker 1600 (Stratagene), or incubated in 25 g of phleomycin (Sigma)/ml.…”

Yeast G1 DNA damage checkpoint regulation by H2A phosphorylation is independent of chromatin remodeling

“…epl1⌬ [EPL1-LEU2] and epl1⌬ [epl1-380-LEU2] are in the S288C background (45). Cell cycle synchronization, DNA damage, flow-cytometry analysis, and microscopy were as described (23,51).…”