Figure 6. Cdk1-mediated phosphorylation regulates the stability of Cdc13. Western blot analysis of the stability of Cdc13 was performed. (A) Wild-type, cdc13-T308A and cdc13-T308D strains were treated with cycloheximide. After cycloheximide treatment, cells were collected at 30-min intervals for 90 min. Lysates were TCA-precipitated, subjected to a 10% SDS–PAGE and transferred for western blot analysis using a Myc antibody. Pgk1 was served as a loading control. (B) Strains were arrested by nocodazole. Western blot analysis was performed as described in (A). The percentages of the expression levels of Cdc13 compared to that of the wild-type are shown below. Nucleic Acids Research, 2009, Vol. 37, No. 11 3609 and the experimental strains proceeded with indistinguish- able kinetics of telomere addition through the subsequent cell cycle (Figure 7B). These results indicated that phos- phorylation of Cdc13 at amino acids 308 and 336 are not essential for de novo telomere addition. DISCUSSION Cdc13 recruits telomerase to its site of action through an electrostatic interaction with a telomerase component Est1 (22,23,55). We previously showed that Tel1 and Mec1 phosphorylate Cdc13 and govern the telomerase recruit- ment function of Cdc13 (30). Consistent with the recent ﬁnding that Cdk1 regulates Cdc13 (29), in this article we additionally demonstrated that Cdk1 phosphorylates Cdc13 at the telomerase recruitment domain. While it currently lacks structural information between Cdc13 and Est1 in the recruitment interface, we speculate that the hypernegatively charged domain of Cdc13 contributed by Tel1, Mec1 and Cdk1 may provide an optimal interface to recruit the potential positively charged domain near the amino acid 444 lysine residue of Est1 (56). This hyperne- gatively charged domain of Cdc13 forms at the late S and lasts to G2 phase, right at the timing for telomerase to function on telomeres. Figure 7. De novo telomere addition in cdc13 mutants. (A) cdc13 mutants were introduced into UCC5706. Strains were arrested by nocodazole. Galactose was added to arrested culture to induce the expression of HO endonuclease. DNA was isolated after 0, 1, 2, 3 and 4 h and digested with SpeI. Southern blot analysis was performed with a probe that recognizes sequence distal to the HO recognition site. De novo telomere addition is marked with an asterisk. (B) Strains were arrested in G1 by a factor, galactose was added to induce HO expression for 2 h, and cells were resuspended in prewarmed YEPD to release. Samples were taken at diﬀerent time points for 24 h for analysis as described in (A).