Budding yeast Cdc13-Stn1-Ten1 (CST) complex plays an essential role in telomere protection and maintenance, and has been proposed to be a telomere-specific replication protein A (RPA)-like complex. Previous genetic and structural studies revealed a close resemblance between Stn1-Ten1 and RPA32-RPA14. However, the relationship between Cdc13 and RPA70, the largest subunit of RPA, has remained unclear. Here, we report the crystal structure of the N-terminal OB (oligonucleotide/oligosaccharide binding) fold of Cdc13. Although Cdc13 has an RPA70-like domain organization, the structures of Cdc13 OB folds are significantly different from their counterparts in RPA70, suggesting that they have distinct evolutionary origins. Furthermore, our structural and biochemical analyses revealed unexpected dimerization by the N-terminal OB fold and showed that homodimerization is probably a conserved feature of all Cdc13 proteins. We also uncovered the structural basis of the interaction between the Cdc13 N-terminal OB fold and the catalytic subunit of DNA polymerase α (Pol1), and demonstrated a role for Cdc13 dimerization in Pol1 binding. Analysis of the phenotypes of mutants defective in Cdc13 dimerization and Cdc13-Pol1 interaction revealed multiple mechanisms by which dimerization regulates telomere lengths in vivo. Collectively, our findings provide novel insights into the mechanisms and evolution of Cdc13.

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The Hsp90 proteomic network is expansive and includes a variety of cell processes operating within the cytoplasm and nucleoplasm. Though the functional significance of the extensive interactions has not been defined, we suggest that the Hsp90 molecular chaperone machinery promotes dynamic behaviors for client proteins that is critical to achieve homeostasis. A general rapid action by cell factors would permit both proper assembly of biological complexes and efficient transitions between distinct structures. Here, we describe why the properties that are inherent to molecular chaperones place these proteins in a unique position to drive the dynamic cellular environment.

The Hsp90 molecular chaperone is a highly abundant eukaryotic molecular chaperone. While it is understood that Hsp90 modulates a significant number of proteins, the mechanistic contributions made by Hsp90 to a client protein typically are not well understood. Here we investigate the yeast Hsp90 regulatory roles with telomerase. Telomerase lengthens chromosome termini by specifically associating with single-stranded telomeric DNA and appending nucleotides by reverse transcription. We have found that the yeast Hsp90 homolog Hsp82p promotes both telomerase DNA binding and nucleotide addition properties. By isolating telomerase from different allelic backgrounds we observed distinct defects. For example, in an hsp82 T101I strain telomerase displayed decreased nucleotide processivity, whereas both DNA binding and extension activities were lowered in a G170D background. The decline in telomerase DNA binding correlated with a loss of Hsp82p association. No matter the defect, telomerase activity was recovered upon Hsp82p addition. Importantly, telomere length and telomerase telomere occupancy was yeast Hsp90 dependent. Taken together, our results indicate that Hsp82p promotes telomerase DNA association and facilitates DNA extension once telomerase is engaged with the DNA.The Hsp90 molecular chaperone is a highly conserved and abundant protein that has evolved into an essential eukaryotic protein (4, 13, 41). Given recent proteomic and genetic screens, Hsp90 has a role in a multitude of normal cellular functions and is involved in a number of diseases ranging from conformational protein folding problems to cancer (31). Accelerating the interest in Hsp90 is its developing use as a therapeutic target for diverse diseases (27,42). Presumably, the broad therapeutic spectrum results from Hsp90's role in maintaining the conformation, stability, and activity of many key cellular proteins that includes intracellular hormone receptors, cRaf, Her2, Akt, Cdk4, p53, and telomerase. Despite its apparent central role in the eukaryotic molecular chaperone system and disease relevance, its mechanistic role in client protein regulation is not well understood. Here we investigate the yeast Hsp90 contributions to telomerase activities.Telomerase maintains genomic integrity, in part, by preserving chromosome length after DNA replication (5, 34). Since conventional DNA polymerases require priming events to initiate synthesis, the extreme terminus of each lagging strand cannot be completed-commonly referred to as the end replication problem (39). In the absence of a compensatory process, this limitation would lead to chromosome erosion with each round of replication. Almost all eukaryotes circumvent this problem by adding a tandem array of simple sequence repeats to each terminus that buffers against the loss. Depending upon the organism, telomerase increases chromosome ends between a few hundred to a few thousand nucleotides to create the telomeric DNA end (34). Perhaps unexpectedly, it was realized that the length of each telomere is not ad...