Biochemical and Structural Analyses of Budding Yeast Telomere Associated CST Complex

Abstract

Telomeres are specialized protein-DNA complexes that compose the natural termini of linear chromosomes. Telomeres prevent chromosome ends from deleterious degradation and fusion events and ensure the complete replication of chromosomes. In Saccharomyces cerevisiae, Cdc13, Stn1 and Ten1 are essential for both chromosome capping and telomere length homeostasis. These three proteins have been proposed to fulfill their roles at chromosome termini as a telomere-dedicated RPA (Replication Protein A, including Rpa70, Rpa32 and Rpa14) complex on the basis of several parallels with the conventional RPA. However, no direct evidence has been provided for this hypothesis. Here I provided the first direct evidence based on our crystal structures. Structural and functional analyses of Candida albicans Stn1-Ten1 revealed striking similarities with Rpa32-Rpa14 and critical roles for these proteins in suppressing aberrant telomerase activities at telomeres. All proved that Stn1-Ten1 is an Rpa32-Rpa14-like complex at telomere. However, the relationship between Cdc13 and Rpa70 remained unclear. The crystal structures of multiple OB (oligonucleotide/oligosaccharide binding)-folds at the N- and C-terminal ends of Cdc13 established an Rpa70-like domain organization, although the structures of Cdc13 OB-folds are significantly different from their Rpa70 counterparts. Furthermore, our structural and biochemical analyses revealed unexpected Cdc13 dimerization by either N- or C-terminal OB-fold and showed that homodimerization is probably a conserved feature of all Cdc13s. We also uncovered the versatility of Cdc13 dimerization in mediating interaction with different targets. The structural characterization of the interaction between the Cdc13 N-terminal OB-fold and Pol1, the catalytic subunit of DNA polymerase α, demonstrated a role for N-terminal dimerization in Pol1-binding. The discovery of Candida spp. Cdc13 dimerization through its OB4 domain revealed its important role in high affinity telomere DNA binding. Collectively, our findings provided novel insights into the mechanisms and evolution of Cdc13. Additionally, we have shown Cdc13’s role in regulating the synthesis of telomere by interacting with telomerase subunit Est1. The interaction involves the second OB-fold in addition to the previously recognized recruitment domain of Cdc13. The finding significantly furthered our understandings about the synthesis of leading and lagging strands of chromosome and the essential role of Cdc13 in solving the end-replication problem.