Telomerase and Telomeres: Structure, Function and Regulation

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DESCRIPTION (provided by applicant): Telomerase, required for the replication of chromosome ends in most eukaryotes, is unusual in that it is comprised of RNA as well as protein subunits. The goal of this project is to better understand structure- function relationships in yeast and human telomerases, with special attention to the regulation of telomerase action via the accessibility of its telomeric substrate. Understanding the fundamentals of telomerase action is important for human health, as 85 - 90% of cancers involve reactivation of telomerase; thus, there is substantial interest in anti-telomerase chemotherapeutic agents and in TERT (telomerase reverse transcriptase) as an antigen for cancer vaccines. In studies of yeast telomerase, we have proposed that the RNA subunit provides a flexible scaffold to tether essential proteins into the complex. In specific aim #1, we will critically test this model in vitro and in vivo, and determine the structure and function of the TERT (Est2p)-binding region of the RNA. In specific aim #2, we will analyze structure-function relationships of the yeast regulatory subunit Est1p and determine its functional interactions. We will test the specific hypothesis that the N-terminal region of Est1p is a 14-3-3 phosphoaminoacid-binding adapter involved in recruiting and activating telomerase at the telomere. Specific aim #3 extends this work to the human system, taking advantage of our recent crystal structure of the chromosome-capping protein hPOT1 bound to telomeric single-stranded DNA. We will test the hypothesis that hPOT1 is switched from telomerase-inhibitory to faciliatory binding modes in a sort of "chromatin remodeling" reaction, driven by post-translational modification and/or interaction with human EST1 proteins.