Abstract #34

The telomerase accessory protein, Est1p, is a cell cycle regulated activator of telomere-bound telomerase in vivo. Creighton T. Tuzon, Virginia A. Zakian. Princeton University, Molecular Biology Department, Princeton, NJ USA.
   Telomeres, the nucleoprotein complexes that define the ends of most linear eukaryotic chromosomes, make essential contributions to chromosome stability and cellular proliferation. In most eukaryotes, including yeast, chromosome ends are replicated by the enzyme telomerase to counteract telomere attrition due to incomplete chromosomal replication. In Saccharomyces cerevisiae, telomerase (Est2p) and its templating RNA subunit (TLC1) requires additional proteins, either which confer additional in vivo properties or that augments telomerase activity, for telomere maintenance. Deletion or mutations in any or all five genes, EST1, EST2, EST3, TLC1, or CDC13, result in progressive telomere shortening that consequently leads to cell death, the so-called est phenotype. Despite the identification of these proteins and other factors that influence telomere functions, the mechanism of telomere length homeostasis remains unclear.
   Our lab previously demonstrated that in S. cerevisiae, Est2p is telomere-associated throughout most of the cell cycle with peak association occurring in G1 and late S/G2 phases. The peak telomeric association of the telomerase accessory factor, Est1p, coincides with the second peak of Est2p telomere association and with telomere replication late in S/G2 phase, suggesting that Est1p may be a molecular switch controlling telomere length homeostasis. We developed a novel assay, nucleotide resolution analysis of immunoprecipitated chromatin (NRAChIP), to directly evaluate whether Est1p is indeed a cell cycle regulated activator of telomere-bound telomerase in vivo. We hypothesize that Est1p demarcates the subset of telomeres that will be extended by telomerase and predict that Est1p would be associated with the majority of telomeres that are elongated by telomerase. The utility of and the results from this assay will be discussed along with evaluating the genetic requirements for Est1p telomere association to this subset of extendible telomeres.

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