Cdk1 regulates telomere replication (26–28). cdk1 cells display defective telomere elongation and long single G strand tails (26,28). Furthermore, very recently, Cdk1 has been reported to activate Cdc13 (29).In eukaryotes, the maintenance of genome integrity relies on checkpoint to properly detect and repair DNA damage caused by environmental stresses or irregularities during DNA metabolisms. Damage and replication defects are recognized by the putative protein complex containing protein kinases such as Tel1 and Mec1. We previously reported that Cdc13 is a target of Tel1/Mec1-kinases (30). This regulation occurs from late S to G2 phase of the cell cycle and is required for telomerase recruitment (31–33). In addition, we also observed a TEL1/MEC1- independent phosphorylation of Cdc13 (30). We wondered how these phosphorylations mediate Cdc13 functions at telomere replication and which kinase contributes to the TEL1/MEC1-independent phosphor- ylation. In this work, we show that Cdc13 is phosphor- ylated in a cell cycle-dependent manner. Similar to the Tel1/Mec1-dependent regulation, Cdk1-mediated phos- phorylation of Cdc13 occurs at the same cell cycle stage. We identiﬁed two phosphorylation sites. Inactivation of both Cdk1 phosphorylation sites in Cdc13 results in telo- mere shortening and delays the regular proteolysis of Cdc13 and cell cycle progression. Thus, we suggest that phosphorylation of Cdc13 by Cdk1 facilitates telomere replication. MATERIALS AND METHODS Strains, plasmids, yeast and telomere experiments All the yeast operations were performed by standard methods (34). Yeast strains used in the study were deriva- tives of YPH501 (MATa/MAT ura3-52/ura3-52 lys2-801 amber/lys2-801 amber ade2-101 ochre/ade2-101 ochre trp1 D63/trp D63 his3 D200/his3 D200 leu2- D1/leu2- D1). The yeast strains carrying mec1 (all combined with sml1), tel1, cdc13, hdf1 and CDC13-Myc9 were described previously (15,35,36). cdc28-4 (37) and cdc28-as1 (38) strains were kindly provided by Steven Reed and David Morgan, respectively. pRS304cdc13-Myc9 was obtained from Virginia Zakian (36,39) and pRS306cdc13 was constructed by PCR containing the CDC13 open read frame and the downstream 200 nt. Point mutations were introduced into CDC13 using QuikChange site-directed mutagenesis (Stratagene). To generate chromosomal cdc13 mutants, pRS306cdc13 mutants were XhoI-digested and trans- formed into CDC13 strains, and the URA3 pop-out mutants were selected from the 5-FOA-resistant colonies using PCR analysis. pLD1(YIP, pGAL1-Sic1 N) (40) plasmid was kindly provided by John Diﬄey. pKB174(CEN CDC28-HA TRP1) and pKB173(CEN cdc28K40L-HA TRP1) (41) plasmids were generous gifts from Orna Cohen-Fix. All primer sequences for PCR and mutagenesis are available upon request. Telomere blot analysis was performed as previously described (42,43). Data shown are representatives of three or more experi- ments from independent colonies. Nucleic Acids Research, 2009, Vol. 37, No. 11 3603 Immunoprecipitation, gel electrophoresis, western blot analysis and cell cycle analysis To detect Cdc13, cells were grown in YPD broth. Whole cell proteins were extracted by TCA precipitation (44) and resolved by sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS–PAGE) or SDS–PAGE contain- ing 10 mM of acrylamide-pendant Phos-tagTM (Phos-tag Consortium) (45) as previously described (46). Cdc13 was detected with a Myc antibody (Santa Cruz Biotech- nology). A Rad53 antibody (gift of J. Diﬄey) was used to detect Rad53. Images were captured and quantiﬁed by a bioluminescence imaging system (UVP BioSpectrum AC Imagine System, UVP). Cells were stained with propidium iodide and the cell cycle analysis was performed by a ﬂow cytometer (FACSCalibur 200, Becton Dickinson). Inhibition of Cdk1 in cdc28-4 and cdc28-as1 strains CDC13 in cdc28-4, W303 and cdc28-as1 strains was chromosomally tagged with Myc9. cdc28-4 arrests cells in G1, the stage that Cdc13 is usually under-phosphory- lated, preventing us from distinguishing the lack of phos- phorylation being directly due to the absence of Cdk1 from an indirect consequence of cell cycle arrest. To cir- cumvent this problem, cdc28-4 cells were ﬁrst arrested with nocodazole at 238C and shifted to 378C for 3 h. The cdc28-as1 cultures were split into halves for the 0.5 mM 4-amino-1-tert-butyl-3-(10-naphthylmethyl) pyra- zolo [3,4-d] pyrimidine (1-NMPP1) (47) and the mock treatments. Phosphatase treatment of immune complexes of Cdc13 Agarose-bound Cdc13 immunocomplexes were washed twice with basic phosphatase (PPase) buﬀer (50 mM Tris–HCl, pH 7.9, 100 mM NaCl, 1 mM dithiothreitol, 10 mM MgCl2). The beads were then incubated with 100 U of calf intestinal PPase (New England Biolabs) for 30 min at 308C. The reaction was stopped by addition of ice-cold basic kinase buﬀer containing 10 mM Na3VO4 and 10 mM NaF. Recombinant protein purification pGEX-4TCDC13(1–252) was constructed by ligating the PCR product containing amino acids 1–252 of Cdc13 from pRS314CDC13 into the NdeI- and EcoRI-treated pGEX-4T (GE). pGEX-4TCDC13(252–491) was con- structed by ligating the EcoRI- and ClaI/Klenow-treated fragment, which contains amino acids 252–491 of Cdc13 from pRS314CDC13 into the EcoRI- and XhoI/Klenow- treated pGEX-4T. pGEX-4TCDC13(601–781) was con- structed by ligating the BglII- and SacI/Klenow-treated fragment, which contains amino acids 601–781 of Cdc13 from pRS314CDC13 into the BamHI- and XhoI/Klenow- treated pGEX-4T. pGEX-4TCDC13(276–332) was con- structed as previously described (30). Recombinant proteins were overexpressed in E. coli and puriﬁed as pre- viously described (30). The bound GST-fusion proteins were detected by Coomassie blue staining.