Analysis of the S. cerevisiae Telomere Capping Protein Stn1's Function in Global DNA Replication and S phase Checkpoint Control

Telomeres, the nucleoprotein structures that comprise the natural ends of eukaryotic chromosomes, play a critical role in maintaining genomic stability. Their essential function is to cap chromosome ends, thus preventing these natural ends from being inappropriately fused to one-another or repaired by resection/recombination. Telomeres are distinct from internal DNA double-stranded breaks because they do not elicit a DNA damage checkpoint response. In addition to their capping function, telomeres also help overcome the DNA end-replication problem. Because all DNA polymerases synthesize only in the 5' to 3' direction and require a pre-existing 3' hydroxyl group as a primer, the most terminal portion of a chromosome cannot be completely duplicated. Through the concerted actions of two enzymes, telomerase and DNA polymerase alpha (Pol-α), telomeres are able to facilitate complete and faithful duplication of the entire genome. Both the capping of ends and their complete replication are integral in maintaining genomic stability. In the budding yeast S. cerevisiae, three proteins, Cdc13, Stn1 and Ten1, participate in both the capping and replication of telomeres. The three proteins are hypothesized to form a heterotrimeric complex (CST), which binds to chromosome termini, forming a physical cap. Furthermore, Cdc13 has been shown to physically interact with telomerase, and all three CST components interact with Pol-α. Genetic analysis suggests that these interactions are critical for efficient replication of telomeres. Current models within the field propose that Cdc13 first recruits telomerase to the chromosome end, which uses an intrinsic RNA template to elongate one of the DNA strands. Then, Pol-α is recruited to the end, which "fills-in" the complementary strand to achieve full duplication of the telomere. There are several outstanding questions within this model. 1) How is the switch between telomerase dependent elongation and Pol-α dependent fill-in synthesis regulated? 2) Are these two events temporally separated, or are they coordinated? 3) Does the CST complex simply recruit Pol-α to the end, or does it facilitate its catalytic activity? 4) Can the CST complex regulate Pol-α dependent synthesis at non-telomeric sites throughout the genome? The majority of research presented in this dissertation examines the hypothesis that Stn1 can facilitate global DNA replication in budding yeast. Using a variety of genetic, biochemical, cellular and molecular biology approaches, we demonstrate that Stn1 has newly discovered roles in DNA metabolism in addition to its previously characterized function at the telomere. In chapter 3, we analyze the functional significance of the interactions between the CST complex and Pol-α, in both telomere capping and length regulation. The results demonstrate that multiple redundant interactions between the capping and replication complexes reinforce each other, thereby generating functional telomeres