The telomerase reverse transcriptase adds telomeric DNA simple sequence repeats to chromosome ends by copying a template sequence within its integral RNA component. This de novo addition is required to balance the loss of repeats that occurs with incomplete replication of chromosome ends by conventional DNA-dependent DNA polymerases. Cells that do not produce active telomerase, including most cell types in multicellular organisms, lose telomeric repeats with each round of cell division. When telomeric repeat number reaches a critical minimum, short telomeres signal apoptosis or entry into an irreversible replicative senescence. Cancer cells can escape this limitation of proliferative capacity by activating telomerase. Because telomerase-positive cancer cells appear to require telomerase for continued viability, telomerase inhibitors could prove to be potent, selective and broadly useful anti-cancer therapeutics. Telomerase activators may also be useful in enhancing the proliferative capacity of some human tissues such as blood and skin. The desire to understand telomerase function and regulation is hindered by an incomplete knowledge of proteins associated with the telomerase enzyme and by an even more limited knowledge of what factors govern the telomerase-telomere interaction. By using the ciliate Tetrahymena thermophila as a model system, both of these issues can be addressed. Tetrahymena provides the combination of facile genetic manipulation with a relative abundance of telomeres and telomerase. Affinity purification experiments described in Specific Aim I will identify a complete inventory of telomerase protein components in telomerase RNPs with different biological functions.
Specific Aim II describes structural studies of recombinant telomerase that should illuminate the biochemical basis for novel enzyme properties.
Specific Aim III examines Tetrahymena telomere structure using microscopy, biochemistry, and molecular genetics, then uses these same techniques to study the molecular regulation and cellular dynamics of telomere-telomerase interaction.
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