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Developmentally regulated telomerase activity from Euplotes crassus
xii, 176 leaves : illustrations ; 28 cm.
Telomerase synthesizes telomeric DNA repeats via reverse transcription of a templating domain in its RNA subunit. It serves a dual role at telomeres, maintaining tracts of telomere repeats and forming telomeres de novo on broken chromosomes in a process called chromosome healing. In a ciliate organism E. crassus both reactions are readily observed. Vegetatively growing cells maintain pre-existing telomeres, while cells undergoing macronuclear development fragment their chromosomes and form telomeres de novo. This study provides the first evidence for developmentally regulated initiation of DNA synthesis by telomerase. In vitro, telomerase from developing E. crasser cells can add telomeric [G4T4] repeats directly onto non-telomeric primer 3' ends in a reaction mediated by a dissociable chromosome healing factor (CHF). Telomerase from vegetatively growing cells does not initiate synthesis directly onto non- telomeric DNA. Instead, it requires primers with at least four to five nucleotides of 3' terminal complementarity to the telomerase RNA template for efficient elongation. Initiation of non-telomeric 3' end extension occurs in the absence of Watson-crick base pairing between the primer 3' end and the telomerase RNA template. The instability of the primer-template interaction during extension of non- telomeric primer 3' end results in reiterative copying of C residues in the telomerase RNA templating domain if dTTP is omitted from telomerase reactions. Indiscriminate do addition observed under these conditions most likely occurs by a slippage mechanism described for other polymerizes and is confined to telomerase from developing cells. 80th developmental and vegetative forms of telomerase are associated with a DNA endonuclease that removes nucleotides from a primer 3' terminus prior to telomere repeat addition. Flexible positioning of the endonuclease site relative to the telomerase RNA template results in elimination of single nucleotide mismatches in do tracts of telomeric DNA. Thus, the telomerase-associated nucleate could provide a proofreading function in telomere synthesis by eliminating mismatches between the DNA primer and the 5, region of the telomerase RNA template.
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Includes bibliographical references: pages 155-171.