Telomeres , the ends of linear eukaryotic chromosomes, are replicated by a specialized RNA-protein enzyme called telomerase. The long-term goal of this project is to contribute to the understanding of the assembly, structure, activity and regulation of telomerase, with special emphasis on its RNA component. Because telomerase is inactive in most human somatic cells but reactivated in most tumors, analysis of this enzyme may contribute to the development of cancer chemotherapeutics. Key features of telomerase are phylogenetically conserved, so experimental systems are chosen to allow the most incisive analysis. In the first set of specific aims, the structure of the telomerase RNA will be determined in the yeast Saccharomyces cerevisiae, which facilitates genetic as well as molecular analysis. Specific functions, such as binding a protein subunit or positioning the active site relative to the template, will be assigned to RNA structure elements. The second set of specific aims involves the telornerase of the ciliate Euplotes aediculcitus, chosen because of its simplicity and biochemical tractability. A protein subunit called p43 is a putative chaperone for assembly of the telomerase holoenzyme, and its RNA-binding and functional roles will be analyzed in detail. Because telomerase action is regulated in large part by proteins that bind to the telomeric DNA, the third specific aim concerns the recently discovered Pot1 (Protection Of Telomeres) protein, which is widely conserved among eukaryotes including humans and fission yeast. The goal here is to determine which telomeric DNA nucleotides are recognized by which amino acids, providing an understanding of how the Pot1 proteins from different organisms recognize different telomeric DNA sequences.
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