Nucleic Acids Research, 2009, Vol. 37, No. 11 3607 Figure 3. Cdc13 is phosphorylated by Cdk1 in vivo. (A) Cdc13 is phosphorylated at the G2/M phase. CDC13 wild-type, T308A, S336A and T308S336A strains were synchronized with a factor or nocodazole. Lysates were extracted and the phosphorylated Cdc13 was analyzed by western blot analysis. Hypo- and hyper-phosphorylated Cdc13 are marked with single and double asterisks, respectively. (B) T308 and S336 were phos- phorylated in vivo. Lysates of wild-type and Cdc13 mutants (T308A, S336A and T308S336A) were extracted from nocodazole synchronized cells. Phosphorylated Cdc13 was separated in a SDS–PAGE containing 10 mM of Phos-tag and analyzed by western blot analysis. (C) Cdc13 phosphor- ylation at T308 and S336 were Tel1-independent. Lysates of wild-type and Cdc13 mutants (T308A, S336A, T308S336A) were extracted from nocodazole synchronized TEL1 and tel1 strains. Phosphorylated Cdc13 was analyzed by western blot analysis. Pgk1 was served as a loading control. (D) Phosphorylation of Cdc13 at T308 is Cdk1-dependent. Wild-type and cdc28-as1 strains were subjected to 1-NMPP1 treatment and GAL1-Sic1 N expressed cells were treated with dextrose (D) or galactose (G). Phosphorylation of Cdc13 was analyzed by western blot analysis. Tel1-dependent phosphorylation on Cdc13 (and perhaps on other proteins), Cdk1-mediated phosphorylation of Cdc13 has no measurable eﬀect on telomere length homeostasis. The stability of Cdc13-T308A is increased Many studies have shown that CDK promotes protein degradation, especially on cell cycle regulatory factors. Rapid turnover of Cln2 at the S phase is achieved by Cdk1-dependent phosphorylation (52). Proteolysis of the anaphase-promoting complex early mitotic inhibitor, Emi1, is induced by Cdk1 (53). In addition, TOP2- inhibition leads to Cdk1-dependent degradation of an ORC1/2 reservoir at mitosis (54). To examine whether Cdk1-mediated phosphorylation of Cdc13 also modulates the proteolysis of Cdc13, the relative Cdc13 stabilities in wild-type and mutant strains were measured. Wild-type, cdc13-T308A and cdc13-T308D strains were grown to log phase, and cycloheximide was added to turn oﬀ the pro- duction of Cdc13. The treatment was maintained for 90 min, and the Cdc13 protein levels were evaluated. As shown in Figure 6A, the half-life of Cdc13-T308A was extensively expanded, comparing to that of wild- type Cdc13. Furthermore, replacement of T308 with D could revert this delay of degradation. Since Cdc13 is phosphorylated by Cdk1 at late S/G2 phase, we also inves- tigated the steady-state levels of Cdc13 after the action of Cdk1. Increased level of Cdc13-T308A was observed in G2/M-arrested cells (Figure 6B). These ﬁndings high- lighted that, while Cdc13 protein is maintained in a cell cycle-dependent manner, Cdk1-mediated phosphorylation facilitates the degradation of Cdc13. Cdk1-mediated phosphorylation of Cdc13 is not essential for the de novo telomere addition Lastly, we wish to address whether Cdk1-mediated Cdc13 phosphorylations are essential for de novo telomere addi- tion. We tested the sudden telomere elongation by the de novo telomere addition assay developed by Diede and Gottschling (49). Strains were arrested at M phase by nocodazole treatment, and then subjected to induction of HO expression. Compared to wild-type cells, which showed telomere elongations at the HO cut site, the TG1–3/HO end was also elongated in T308A, S336A and T308AS336A cells (Figure 7A). We next investigated whether some partial defect in de novo telomere addition could be detected in these mutants. After cells were released into the cell cycle from a factor, both the control