Functional analysis of the interaction between Cdc13 and Stn1, two factors critical for telomere capping and length regulation in yeast

Telomeres prevent chromosome ends from being recognized as double strand breaks by protecting them against degradation, end-to-end fusions and illegitimate recombination. An interplay between double stranded and single stranded telomere sequence binding proteins facilitate chromosome end capping by regulating telomere elongation by telomerase and protecting against chromosome degradation, fusions and illegitimate recombination. Mutations in these telomere binding proteins have been associated with DNA damage response, chromosomal instability, cancer or cellular death. We investigated the role of telomere binding proteins in length regulation and capping in the yeast model system Saccharomyces cerevisiae. In this system, Cdc13, Stn1 and Ten1 are three essential proteins that protect the chromosome end from degradation and regulate telomere length. Cdc13 binds telomere single stranded DNA in vitro and associates with telomeres in vivo. Pair wise interactions have been identified between Cdc13, Stn1 and Ten1. Loss of function alleles of stn1 or ten1 share some phenotypic similarities to loss of function alleles of cdc13, including misregulation of telomere length, and capping defects. Cdc13, Stn1 and Ten1 have been proposed to function as one complex to cap chromosome ends and regulate telomere length. The research presented in this dissertation tests this hypothesis by focusing primarily on defining domains of Cdc13 and Stn1 that are critical for their interaction, and examining the functional significance of this interaction. We find that the N-terminus of Stn1 interacts with Ten1 and is essential for viability. The C-terminus of Stn1 interacts with Cdc13 and is required for normal telomere length regulation as constructs expressing only the N-terminus of Stn1 have long telomeres. We further show that over-expression of the Stn1 N-terminus with Ten1 can completely circumvent the essential Cdc13 function in protecting chromosome ends from degradation but not the Cdc13 role in promoting telomere repeat addition. In the absence of Cdc13, cells rely on a telomere capping mechanism that is critically tied to the DNA replication machinery. In summary, we have demonstrated for the first time that Cdc13, Stn1 and Ten1 may form multiple complexes that make independent contributions to telomere length regulation and capping.