http://www.loc.gov/mods/v3 http://www.loc.gov/standards/mods/v3/mods-3-2.xsd
Characterization of the telomerase holoenzyme from the hypotrichous ciliate Euplotes crassus
The holoenzyme telomerase is a specialized reverse transcriptase that uses its RNA subunit as a template to synthesize telomeric repeats and maintain telomeres on chromosome ends. In the hypotrichous ciliate Euplotes crassus, telomerase demonstrates a unique developmentally programmed assembly into three higher order complexes during mating. Here I identify the Euplotes crassus telomerase reverse transcriptase gene (EcTERT), which is the catalytic subunit of telomerase. I show that expression of EcTERT requires programmed ribosomal frameshifting to generate catalytically active protein. Using an antibody directed against the EcTERT amino terminus, I used Western blot analysis to identify full-length 124 kDa TERT and a more abundant 79 kDa protein which corresponds to termination at an internal stop codon. The 79 kDa protein is likely to be catalytically inactive, and is confined to the cytoplasm. Affinity purification of telomerase from the cytoplasm demonstrated that both 124 kDa and 79 kDa TERT assemble with telomerase RNA. Hence, the 79 kD protein could potentially regulate enzyme activity by sequestering telomerase RNA in the cytoplasm. Using an affinity purification approach, I show the co-purification of two different versions of telomeric extension products from E. crassus telomerase particles, indicating that the minimal functional telomerase complex consists of at least two active sites in vivo. In vitro studies indicate that the EcTERT protein can multimerize. By site-directed mutagenesis and subcloning, I identified two independent domains required for this interaction, amino acids 254-384 at the N-terminus and amino acids 755-857 at the C-terminus. My analysis suggests that EcTERT can adopt head-to-head, tail-to-tail and head-to-tail configurations. Finally, I demonstrated that telomerase is physically associated with DNA polymerase α/primase complex in mated E. crassus cells using an affinity purification approach. This association is not found in vegetatively growing cells, indicating that the interaction is developmentally regulated. The physical association of telomerase and the lagging strand DNA replication machinery explains the coordinate regulation of telomeric G- and C-strand synthesis and the efficiency of telomere addition in Euplotes.
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Includes bibliographical references (leaves 163-210).