SUMOylated PCNA regulates telomere length.

A. Southern blot (Teloblot) showing that lack of SUMOylation of PCNA prevents telomere elongation. Independently-created colonies were passaged ten times, its DNA extracted, digested with XhoI and run in an agarose gel. The DNA was then transferred to a nitrocellulose membrane, which was incubated with a radioactive probe that detects telomeres, and a size marker. B. Teloblot showing that overexpression of wild type PCNA or Pol30-RR-SUMO fusion, but not Pol30-ubiquitin fusion or Pol30-RR causes telomere elongation.

Genetic and physical interaction between ELG1 and STN1.

A. Teloblot showing epistasis between elg1Δ and stn1 mutants. B. Co-Immunoprecipitation experiment showing physical interaction between Elg1 and Stn1 and reduced physical interaction between Elg1-sim and Stn1. No interaction could be detected between Elg1 and Cdc13 or Ten1.

Determinants of the Elg1-Stn1 interaction.

A. Schematic representation of the Elg1 protein. The 3 SIM motifs, the WalkerA motif and the 2 threonines at the interface with PCNA are shown. B. Yeast-Two-Hybrid (YTH) interaction of Cdc13 and Elg1 in a wild type strain. AD: protein fused to the activating domain of Gal4; BD: protein fused to the DNA binding domain of Gal4. e.v.: empty vector.C. YTH interaction of Stn1 and Elg1 in a wild type strain. D. YTH experiments in the siz1Δ siz2Δ and pol30-RR background. E. Co-Immunoprecipitation experiment showing physical interaction between Cdc13 and PCNA.

Summary of all YTH interactions presented.

The unloading activity of Elg1 and Cdc13 SUMOylation are necessary for the Elg1-Cdc13 interaction.

A. YTH experiment in a elg1Δ strain. B. YTH experiment in a elg1-sim strain. C. YTH experiment in a elg1-TT386/7DD strain. D. YTH experiment in a elg1-sim+DD strain. E. YTH experiment in a elg1-Walker AB strain. F. Lack of interaction between cdc13-snm and Elg1 (in a wild type strain).

Timing of Elg1-Stn1 interaction.

A. Co-IP experiment with synchronized cells. Aliquots were taken at time intervals, Elg1 was immunoprecipitated, and the level of Stn1 and Pol3 (the large subunit of DNA polymerase Delta) were monitored by Western blot. Strains with single tags are shown as controls. Whole Cell Extract results are shown in Figure S5. B. Quantitation of the Western shown in A. C. DNA content of the cells used in A, by cell cytrometry. D. Chromatin immunoprecipitation at telomeres (Telo-ChIP) in synchronized cells showing PCNA occupancy. E. Interaction between Stn1 and Cdc13 in a wild type and a elg1Δ strain. F. Quantitation of three independent biological repeats of the experiment shown in E. **:p<0.001.

Model for the role of SUMOylated PCNA and Elg1 in telomere length regulation.

A. Cdc13 binds ssDNA at the telomeres, Elg1 moves with the replisome at the lagging strand, unloading PCNA in each Okazaki fragment. B. Arrival of the SUMOylated PCNA at the fork to Cdc13 promotes telomerase activity. C. Elg1 interacts with Stn1, which could be SUMOylated. Cdc13 becomes SUMOylated. Elg1 unloads PCNA and leaves telomeres. D. Stn1 is now able to interact with Cdc13, evicting Est1 and terminating telomerase activity.

Yeast strains used in this study.

Plasmids used in this study