Sister chromatid cohesion role for CDC28-CDK in Saccharomyces cerevisiae

Abstract

High-fidelity chromosome segregation requires that the sister chromatids produced during S phase also become paired during S phase. Ctf7p (Eco1p) is required to establish sister chromatid pairing specifically during DNA replication. However, Ctf7p also becomes active during G2/M in response to DNA damage. Ctf7p is a phosphoprotein and an in vitro target of Cdc28p cyclin-dependent kinase (CDK), suggesting one possible mechanism for regulating the essential function of Ctf7p. Here, we report a novel synthetic lethal interaction between ctf7 and cdc28. However, neither elevated CDC28 levels nor CDC28 Cak1p-bypass alleles rescue ctf7 cell phenotypes. Moreover, cells expressing Ctf7p mutated at all full- and partial-consensus CDK-phosphorylation sites exhibit robust cell growth. These and other results reveal that Ctf7p regulation is more complicated than previously envisioned and suggest that CDK acts in sister chromatid cohesion parallel to Ctf7p reactions.

Figure 1.—

ctf7-203-based synthetic lethal screen identifies a novel cak1 allele. (A) Schematic of synthetic lethal screen: ade2 ade3 ctf7-203 cells (top) produce white colonies (bottom). When transformed with CTF7 URA3 ADE3, cells give rise to red/white sectored colonies on nonselective 23° medium. Random EMS mutation of genes (asterisk) that are synthetically lethal (sl) with ctf7-203 oblige cells to retain the CTF7 URA3 ADE3 plasmid, producing nonsectored red colonies. Transformation of a genomic library (TRP1 SL rescuing plasmid) identified CAK1 as allowing for loss of the CTF7 URA3 ADE3 plasmid. (B) Micrograph shows transformation of synthetic lethal strain with CAK1 genomic clone results in a sectored red and white colony.

Figure 2.—

(A) CDC28-bypass alleles suppress cak1-277 temperature sensitivity. Tenfold serial dilutions of cak1-277 mutant cells containing empty vector (pRS314), a CAK1 genomic clone, or CDC28-bypass alleles (4324 and 43244) were spotted on selective medium plates and incubated at 23°, 30°, or 37°. (B) CDC28-bypass alleles partly suppress double-mutant ctf7-203 cak1-277 synthetic lethality. Tenfold serial dilutions of ctf7-203 cak1-277 double mutants harboring CEN CTF7 URA3 and transformed with either CEN TRP1 vector alone or vector containing CTF7-, CAK1-, CDC28-, or CDC28-bypass alleles (4324 or 43244) grown on medium containing 5-FOA at 23°.

Figure 2.—

(A) CDC28-bypass alleles suppress cak1-277 temperature sensitivity. Tenfold serial dilutions of cak1-277 mutant cells containing empty vector (pRS314), a CAK1 genomic clone, or CDC28-bypass alleles (4324 and 43244) were spotted on selective medium plates and incubated at 23°, 30°, or 37°. (B) CDC28-bypass alleles partly suppress double-mutant ctf7-203 cak1-277 synthetic lethality. Tenfold serial dilutions of ctf7-203 cak1-277 double mutants harboring CEN CTF7 URA3 and transformed with either CEN TRP1 vector alone or vector containing CTF7-, CAK1-, CDC28-, or CDC28-bypass alleles (4324 or 43244) grown on medium containing 5-FOA at 23°.

Figure 3.—

ctf7 cdc28 double-mutant strains are inviable. Tenfold dilutions of ctf7 parental cells and eight spores obtained from ctf7 cdc28 heterozygous diploids. All spores harbor CEN CTF7 URA3 plasmid (growth on SC −Ura medium) but are inviable on rich medium placed at 37°, indicating that each spore contains the temperature-sensitive cdc28 allele. Replica plating onto medium supplemented with 5-FOA counterselects for cells containing the URA3-based plasmid. See text for details.

Figure 4.—

CTF7 mutated at candidate CDK phosphorylation sites maintain cell viability. ctf7 null ade2 ade3 strains harboring CEN ADE3 URA3 CTF7 and CEN TRP1 plasmids: the latter of which contains wild-type CTF7, CTF7 mutated at either the full-consensus CDK phosphorylation site (ctf7^S99A), all candidate CDK phosphorylation sites (ctf7^{S99A S67A T94A S105A} or quad mutant), the ctf7-203 allele in which the quad mutations were also introduced (ctf7-203^quad), or vector alone (Vector). Ability of various ctf7 constructs to support cell growth is observed as white and red sectored colonies on nonselective rich medium held at 23°. Two independent isolates for each strain are shown. Colony size differences reflect only cell crowding and local nutrient conditions, not growth defects associated with ctf7 alleles. Each image in this and subsequent colony sectoring figures corresponds to ∼0.55 cm.

Figure 5.—

Cells harboring mutations in candidate Ctf7p phosphorylation residues as the sole source of Ctf7p function exhibit robust growth not only at 23° but also despite exposure to increased temperatures (30° and 37°) and to genotoxic agents (zeocin and 75 μJ of ultraviolet irradiation). Tenfold serial dilutions of each strain were plated onto rich medium and treated as indicated. See text for details.

Figure 6.—

CTF7 mutated at all candidate CDK phosphorylation residues are competent to support cell viability in the presence of cak1 mutations. See text for details.

Figure 7.—

cdc28 mutant cells exhibit modest cohesion defects. (Top) Flow cytometer profiles reveal DNA contents of wild-type and cdc28 cells during log growth (log), synchronized in early S phase (HU), and then released to the restrictive temperature in the presence of zeocin (Zeo) to obtain preanaphase cells. (Middle) Micrograph pairs of cdc28 mutant cells show cell morphology above and sister chromatid loci immediately below. (Bottom) Quantification of cohesion defects in zeocin-treated wild-type cells and cdc28 mutant cells. Analyses for wild type (open bars) and both cdc28-B119 (shaded bar) and cdc28-B28 (solid bar) are shown.