Telomeres are specialized elements found at the termini of linear chromosomes. They are required to protect chromosome ends from degradation and fusion with other ends; in addition, telomeres also promote accurate chromosome segregation. In humans, missegregation of chromosomes can lead to a number of disorders, such as cancer and birth defects. Defects in telomere replication in yeast and Tetrahymena lead to senescence, suggesting that this could be a contributing factor in mammalian cellular senescence as well. A primary determinant for maintaining a constant chromosomal telomere length is telomerase, identified biochemically in ciliates and humans. Telomerase has been shown to be a novel reverse transcriptase-like enzyme which carries an internal RNA responsible for templating the newly synthesized telomeric DNA. Telomerase activity has not yet been biochemically identified from a genetically tractable organism such as yeast, which has limited the analysis of factors that interact with this enzyme. In addition, nothing is yet known about the protein composition of telomerase. The long term objective of the proposed research is to dissect the apparatus responsible for replicating telomeres, using the yeast S. cerevisiae as a model system. A starting point for these studies is the EST1 gene of yeast. Yeast cells lacking a functional EST1 gene show a continuous decline in telomere length and a senescence phenotype. Although a number of mutants of yeast have been identified which alter telomere length, this is the only mutant of yeast which coordinately affects both telomere length and senescence. No exhaustive search has previously been done in any organism for genes which, when mutated, coordinately affect telomere replication and senescence. S. cerevisiae lends itself to such a search, both because it is a genetically tractable system and because isolation of the est1 mutation provides a precedent for this particular class of mutations. The first section of this proposal describes a genetic approach to identify all other genes besides EST1 which, when mutated, coordinately affect telomere replication and senescence; this should include the predicted yeast telomerase RNA Recovery of such genes is an important step towards understanding how different components involved in telomere replication contribute to cellular senescence in a lower eukaryote.
The second aim of this research is to identify additional genes that encode components that interact with the EST1 gene product, using three different genetic approaches; characterization of these genes should provide additional information about Est1 function. A third goal is to biochemically characterize activities associated with the Est1 protein. Two hypotheses will guide these biochemical experiments: first, that Est1 is a component of telomerase, and second, that Est1 is a telomere binding protein.
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