UC San Diego

In the budding yeast S. cerevisiae, the telomerase enzyme is composed of a 1.3 kb TLC1 RNA, which forms a complex with Est2 (the catalytic subunit) and two regulatory proteins, Est1 and Est3. Telomerase acts at a particular period of the cell cycle, late S phase, to sufficiently counteract chromosome erosion and cellular senescence. However, the telomerase complex is very limiting in expression, which argues for a highly regulated process for getting telomerase to where it needs to act. Although yeast telomerase has been the subject of intense investigation by numerous laboratories, a high-resolution view of the precise mechanistic contributions by each subunit to telomere homeostasis has not yet been achieved. To address this, I have employed a biochemical method that provides a detailed view of the stoichiometry of the telomerase holoenzyme combined with a genetic strategy intended to characterize the functional surface of telomerase. First, I developed a genetic strategy that targets functionally important residues on the surface of a protein. This identified dominant negative mutations in EST3 that confer a telomere replication defect when over- expressed in a wild-type yeast strain. This approach was continued by other members of the lab and has resulted in a large collection of separation-of-function alleles in each of the three EST genes. The biochemical method relies on a yeast strain designed to allow quantitative immunoprecipitation of the limiting subunit of the holoenzyme (Est2), followed by equivalent detection of each protein subunit by western blotting. I used this stoichiometry assay to evaluate how Est1 associates with a long helical arm on the TLC1 RNA. I also show that an enzyme complex containing Est1 assembles early in the cell cycle, well before telomere elongation occurs, in accordance with prior genetic observations, whereas the Est3 subunit associates much later in the cell cycle. Integrating the mutations from the genetic strategy with the biochemical method has identified four functionally distinct interfaces on Est1, as well as additional interfaces on Est2 and Est3. Finally, I have uncovered evidence that telomerase exists as two separate complexes with different stoichiometries, arguing for yet an additional level of telomerase regulation