SUMOylation of Cdc13 at a single lysine peaks in early to mid S phase. (a) Cdc13 SUMOylation peaks in early to mid S phase. Alpha-factor–arrested G1 cells (0 min) containing Cdc13-TAP were released and samples at indicated time points were examined for protein SUMOylation (left) and by FACS analysis (right). The position of SUMOylated Cdc13 is labeled as Cdc13-S. (b) Schematic map of Cdc13. Stn1- and Pol1-interacting domains as well as telomerase recruitment (RD) and DNA binding (DBD) domains are labeled^12,32–36. The dot represents the Cdk1 phosphorylation site. The SUMOylation consensus site and the position of the SUMOylated lysine are depicted. (c) K909R abolishes Cdc13 SUMOylation. Wild-type Cdc13 (WT) or Cdc13-K909R proteins tagged with TAP were examined as in Figure 1d. (d) cdc13-snm does not affect protein levels. Protein extracts before (−) and after (+) MMS treatment were examined by immunoblots using anti-ProA antibody. Cdc13 and Cdc13-snm were tagged with TAP. Equal loading is shown by amido black staining.

cdc13-snm weakens Stn1 interaction and increases telomere length in yku70Δ, tel1Δ and est1 cells. (a,b) Cdc13-snm affects Stn1 interaction in yeast two-hybrid assays. Two-hybrid strain containing pairs of indicated Gal4 DNA-binding domain (GBD) and Gal4 activation domain (GAD) plasmids were grown on medium lacking leucine and tryptophan (–LEU–TRP). The activation of reporters was scored by replica plating onto –LEU–TRP–ADE and –LEU–TRP–HIS media (a) and by assaying the activity of β-galactosidase (b). Cdc13-snm specifically weakens interaction with Stn1 (P = 0.0018) but not with Pol1 (b). Error bars represent s.d. of six trials. (c–e) cdc13-snm leads to longer telomeres in yku70Δ, tel1Δ and hypomorphic est1 cells. Hypomorphic est1 (est1-myc) was generated by tagging Est1 at its N terminus with 13MYC.

CDC13-SUMO leads to shorter telomeres and enhanced Stn1 interaction, and the two modifications of Cdc13 act antagonistically. (a) CDC13-SUMO leads to shorter telomeres and cdc13-T308A-SUMO results in a greater degree of telomere shortening than either cdc13-T308A or CDC13-SUMO. (b) CDC13-SUMO fusion enhances Stn1 interaction. Untagged and Myc-tagged Stn1 cells both contain Cdc13-HA. Protein extracts from these cells were co-immunoprecipitated with anti-Myc antibody, and Cdc13 and Cdc13-SUMO were examined in the immunoprecipitated fraction with anti-HA antibody. The numbers indicate the relative amounts of co-purified Cdc13 in wild-type and CDC13-SUMO strains based on the average of four trials (mean ± s.d.). Cdc13 and Cdc13-SUMO input levels are similar (data not shown). (c,d) Abolition of Cdk1-mediated phosphorylation of Cdc13 does not affect its SUMOylation, and vice versa. HA-tagged wild-type and mutant Cdc13 were examined for SUMOylation (c) and phosphorylation (d) after immunoprecipitation. (e) Lack of Cdk1-mediated Cdc13 phosphorylation and lack of Cdc13 SUMOylation affect telomere length in an antagonistic manner. Telomere length was examined in wild-type (WT) and cdc13 mutant cells, and telomere length of cdc13-T308A snm cells was found to be inbetween those of cdc13-T308A and cdc13-snm cells. (f Models for the roles of the two protein modifications on Cdc13. See text for details.

Defects in SUMOylation enzymes lead to longer telomeres, and multiple telomere proteins are SUMOylated. (a–c) ubc9 mutants (a) and two SUMO E3 double mutants (b), but not SUMO E3 single mutants (c), show longer telomeres. ubc9-MYC and ubc9-HA are the Myc- and hemagglutinin (HA)-tagged UBC9 genes, respectively. mms21-CH (mms21) contains two point mutations in the SUMO E3 domain²⁵. (d) Rap1, Yku70, Cdc13, Pif1 and Yku80 are SUMOylated. Proteins were tagged with TAP (not labeled) or Myc and were immunopurified before (−) and after (+) MMS treatment. Unmodified proteins were detected on immunoblots by anti-ProA or anti-Myc antibodies (below) and SUMOylated forms of the proteins by anti-SUMO antibody (above). Asterisks label the positions of SUMOylated proteins before MMS treatment. The occasional difference in signals on the anti-tag blots reflects variation in loading rather than protein levels. (e) SUMOylation of Rap1, Yku70, Cdc13 and Pif1, but not Rad52, increases at 37 °C.

cdc13-snm leads to longer telomeres in a telomerase-dependent manner. (a) cdc13-snm cells show increased telomere length. (b) Deleting the telomerase component TLC1 abolishes telomere lengthening caused by cdc13-snm. (c) cdc13-snm and tlc1Δ show no synthetic defects in senescence. Senescence experiments were conducted by repeatedly streaking strains (number of times streaked is indicated for each).

stn1 alleles are epistatic to cdc13-snm and specifically enhance Cdc13 SUMOylation. (a,b) stn1 alleles are epistatic to cdc13-snm. The double mutants of cdc13-snm with stn1-myc (a) or stn1-ΔC199 (b) have telomere lengths similar to those of the stn1 single mutants. (c,d) stn1 alleles enhance the SUMOylation of Cdc13 but not that of Rap1 and Yku70. The effects of stn1-myc (-myc) and stn1ΔC199 (ΔC) on SUMOylation of Cdc13-TAP (c) and of Rap1 and Yku70 (d) were examined. (e,f) Cdc13-TAP SUMOylation is not altered in rif1Δ (e) or in yku70Δ, tel1Δ and est1-myc cells (f).