Telomeres are essential chromosome structures required for both chromosome stability and for complete replication of linear eukaryotic chromosomes. The Tetrahymena telomerase enzyme catalyzes the de novo addition of telomere sequences onto the ends of chromosomes. This de novo addition establishes a length equilibrium at the end of the chromosome which may protect chromosomes from the loss of essential sequences. In addition, telomerase may be involved in the process of chromosome """"""""healing"""""""" that is observed at broken chromosome ends in many organisms. The telomerase enzyme is a remarkable DNA polymerase. It specifically recognizes telomeric sequences and it accurately synthesizes the Tetrahymena telomeric sequence TTGGGG given telomeric primers whose 3' ends lie at any nucleotide within that sequence. Telomerase is a ribonucleoprotein complex; the RNa component contains the sequence CAACCCCAA, which probably serves as the template for the synthesis of TTGGGG repeats. Thus telomerase is anew type of DNA polymerase in which the template is an integral part of the enzyme rather than an external substrate.
The aim of this proposal is to investigate both the biochemical mechanism and the regulation of this unusual DNA polymerase. Telomerase will be purified and the protein components identified. The structure of the RNA and the possibility of RNa catalysis will be studied. Experiments will be undertaken to reconstitute active enzyme from purified RNa and protein. Finally, the mechanism by which telomerase adds hundreds of TTGGGG repeats in vitro will be characterized. Since telomere structure is remarkably conserved from protazoans to man, the studies of the Tetrahymena telomerase may later be applied to all eukaryotes.
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