3602–3611 Nucleic Acids Research, 2009, Vol. 37, No. 11 doi:10.1093/nar/gkp235 Published online 9 April 2009 Rapid Cdc13 turnover and telomere length homeostasis are controlled by Cdk1-mediated phosphorylation of Cdc13 Shun-Fu Tseng, Zih-Jie Shen, Hung-Ji Tsai, Yi-Hsuan Lin and Shu-Chun Teng* Department of Microbiology, College of Medicine, National Taiwan University, Taipei, Taiwan Received January 15, 2009; Revised March 13, 2009; Accepted March 30, 2009 ABSTRACT Budding yeast telomerase is mainly activated by Tel1/Mec1 (yeast ATM/ATR) on Cdc13 from late S to G2 phase of the cell cycle. Here, we demon- strated that the telomerase-recruitment domain of Cdc13 is also phosphorylated by Cdk1 at the same cell cycle stage as the Tel1/Mec1-dependent regulation. Phosphor-specific gel analysis demon- strated that Cdk1 phosphorylates residues 308 and 336 of Cdc13. The residue T308 of Cdc13 is critical for efficient Mec1-mediated S306 phosphorylation in vitro. Phenotypic analysis in vivo revealed that the mutations in the Cdc13 S/TP motifs phosphory- lated by Cdk1 caused cell cycle delay and telomere shortening and these phenotypes could be partially restored by the replacement with a negative charge residue. In the absence of Ku or Tel1, Cdk1- mediated phosphorylation of Cdc13 showed no effect on telomere length maintenance. Moreover, this Cdk1-mediated phosphorylation was required to promote the regular turnover of Cdc13. Together these results demonstrate that Cdk1 phosphorylates the telomerase recruitment domain of Cdc13, thereby preserves optimal function and expression level of Cdc13 for precise telomere replication and cell cycle progression. INTRODUCTION Telomeres are dynamic DNA–protein complexes that pro- tect the ends of linear chromosomes, prevent detrimental chromosome rearrangements and defend against genomic instability and the associated risk of cancer (1–3). Telomeres, consisting of tandem repeats of short G-rich sequences, are synthesized by the enzyme telomerase (4,5). The catalytic core of telomerase is composed of a reverse transcriptase and an RNA subunit. The reverse transcriptase utilizes the RNA component as a template to add the G-rich repeats onto the 30-ends of the chromo- some (4–6). In most human somatic cells, telomerase activity is absent, and telomeres are gradually shortened with successive cell divisions due to incomplete replica- tion, which eventually causes replicative senescence. Once telomeres become suﬃciently short, they are thought to lose the ability to protect the ends of the chromosomes from being recognized as broken ends, and being sub- jected to nuclease digestion and active recombinational repair. Continuous telomere shortening in human ﬁbro- blasts leads to chromosome fusions, crisis and apoptosis (7). Very few human cells can bypass this crisis either through telomerase reactivation or through an alternative recombination pathway for telomere lengthening (8–10). In budding yeast Saccharomyces cerevisiae, genes encoding components of telomerase have been identiﬁed and mutations in these genes cause a gradual loss of telo- mere length (11,12). EST2 and TLC1 encode the reverse transcriptase catalytic protein subunit and the templating RNA, respectively (12–14). In addition, the protein encoded by EST1 is associated with the RNA component of telomerase (15–18). Other accessory factors, such as Cdc13, are required for the in vivo action of telomerase. Cdc13 is a single strand telomere-binding protein (19,20). It forms a complex with Stn1 and Ten1. Cdc13, Stn1 and Ten1 show homology to Rfa1, Rfa2 and Rfa3, respec- tively. This replication protein A-like heterotrimeric com- plex speciﬁcally binds single strand telomeric sequences (21) and is required for both telomere protection and telomerase recruitment (19,20,22). It recruits telomerase to its site of action through an electrostatic interaction between Cdc13 and Est1 (22,23). DNA replication must take place only once per cell cycle in eukaryotes. Cyclin-dependent kinases (CDKs), particularly the budding yeast Cdk1 (Cdc28), have been proven to phosphorylate and regulate a number of DNA replication factors, including components of the origin recognition complex, Orc1, Orc2 and Orc6, and MCM proteins (24,25). Previous studies also demonstrated that *To whom correspondence should be addressed. Tel: +886 2 23123456 (ext. 88294); Fax: +886 2 23915293; E-mail: shuchunteng@ntu.edu.tw Present address: Hung-Ji Tsai, Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455, USA 2009 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.