Diploid yeast strains heterozygous for wild type and putative Cdk1 phosphorylation site mutants in CDC13 were sporulated and dissected. The telomere length of each individual yeast colony derived from the dissected spore was measured at ~PD100. Mutation of CDC13 threonine 308 to alanine results in telomere shortening (lanes 5-8).

A. ChIP assays show that Cdc13 threonine 308 mutation does not affect binding of Cdc13 to telomeres during cell cycle progression. B. Cdc13-T308A still interacts with Est1 and Stn1 when Est1 and Stn1 are overexpressed. C. Co-immunoprecipitation of Cdc13 and TLC1 in vivo. Reduced telomerase recruitment efficiency by Cdc13-T308A as indicated by reduced association of Cdc13-T308A and TLC1 (p values < 0.01), quantified by real-time QRT-PCR. D. ChIP assays show that recruitment of Est1 to telomeres is reduced in cdc13-T308A yeast compared to wild-type yeast expressing Est1-13myc (p values < 0.01). E. ChIP assays show that cell cycle-dependent recruitment of Est1 to telomeres is reduced in cdc13-T308A yeast compared to wild-type yeast expressing Est1-13myc (p values < 0.01). F. ChIP assays show that cell cycle-dependent recruitment of Est2 to telomeres is reduced in cdc13-T308A yeast compared to wild-type yeast expressing Est2-13myc (p values < 0.01).

A. ChIP assays show that recruitment of Stn1 to telomeres is cell cycle regulated in yeast strain expressing Stn1-13myc. B. Dramatic telomere lengthening in yeast strain harboring stn1-ΔC199 mutation is not affected by Cdc13 threonine 308 to alanine mutation (compare lanes 5 and 6) or RAD52 deletion (compare lanes 1 and 2 to lanes 5 and 6). C. Co-immunoprecipitation of Cdc13 and TLC1 in stn1-ΔC199 yeast. Dramatic increase of telomerase recruitment by Cdc13 in stn1-ΔC199 yeast is not affected by Cdc13 threonine 308 to alanine mutation (indicated by substantially increased co-immunoprecipitation of both Cdc13 and Cdc13-T308A with TLC1, p values = 0.559). D. Over-expression of Stn1 in yeast with wild-type CDC13 results in telomere shortening. Over-expression of Stn1 in yeast with cdc13-T308A results in further telomere shortening. E. Over-expression of Est1 in yeast with wild-type CDC13 results in slight telomere elongation. Over-expression of Est1 in yeast with cdc13-T308A also results in slight telomere elongation, but not complete rescue of the telomere shortening resulting from the cdc13-T308A mutation.

A. A silver stained gel showing the purification of TAP-tagged Cdk1-as1 and co-purified cyclins from asynchronous yeast cell cultures. B. Coomassie blue stain gel showing the input of recombinant 6xHis tagged telomerase subunits and telomere-binding proteins. Cdc13-7A is used as negative control with all 7 potential Cdk1 phosphorylation sites mutated to alanine. Asterisks mark proteins of expected molecular weight. C. Autoradiograph showing the specific phosphorylation of wild-type Cdc13 by Cdk1-as1/cyclin complexes in vitro in the presence of [γ-³²P] N⁶-Benzyl ATP, but not Cdc13-7A, Est1, Est3 or Ten1. D. In vitro phosphorylation assay of mutant Cdc13 proteins that contain mutations at individual putative Cdk1 phosphorylation site as indicated. Mutant Cdc13 with threonine 308 to alanine mutation results in dramatic reduction of phosphorylation by Cdk1-as1/cyclins in vitro. E. In vitro phosphorylation assay of mutant Cdc13 proteins that contain putative Cdk1 phosphorylation site mutations in addition to threonine 308 to alanine mutation. Mutation of both threonine at position 308 and serine at position 336 results in complete loss of phosphorylation by Cdk1-as1 in vitro.

Endogenously expressed Cdc13 is tagged at its C terminus with a 13-myc tag. A. Cdc13 phospho-threonine 308 specific antibody detected wild-type Cdc13, but not Cdc13-T308A mutant protein from asynchronous yeast cell lysates (top panel). The same blot was stripped, then re-probed with anti-myc antibody to detect the total amount of Cdc13 protein (bottom panel). B. Threonine 308 phosphorylation is sensitive to lambda phosphatase treatment. C. Inactivation of Cdk1-as1 activity by 1-NM-PP1 results in rapid loss of Cdc13 phosphorylation in vivo. An asynchronous yeast culture was treated with 10μM 1-NM-PP1, and harvested 0, 15 or 30 min after treatment. D. FACS analysis shows slight changes of cell cycle progression after 1-NM-PP1 treatment. E. Phosphorylation of Cdc13 threonine 308 is cell cycle dependent, enriched around 45-75 minutes after alpha-factor synchronization release. F. FACS analysis showing cell cycle progression after alpha-factor synchronization. At 45-75 minutes after alpha-factor synchronization release, the cell cycle has progressed into late S to G2 phase.

A. Rapid telomere elongation in yeast strains with rif1Δ or rif2Δ is strongly inhibited by Cdc13 threonine 308 to alanine mutation, as shown by teloblot. B. Co-immunoprecipitation of Cdc13 and TLC1 in rif1Δ or rif2Δ yeast. Deletion of RIF1 or RIF2 results in a dramatic increase of telomerase recruitment by Cdc13 (as indicated by dramatically increased co-immunoprecipitation of Cdc13 and TLC1 RNA) that is hampered by Cdc13 threonine 308 to alanine mutation (p values < 0.01). C. ChIP assays show that recruitment of Est1 to telomeres is compromised in cdc13-T308A/rif2Δ yeast compared to CDC13/ rif2Δ yeast expressing Est1-13myc (p values < 0.01).