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Mol. Cell. Biol. doi:10.1128/MCB.02105-06
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Positive and negative regulation of Tetrahymena telomerase holoenzyme

Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA 94720-3204

^* To whom correspondence should be addressed. Email: kcollins{at}berkeley.edu.

	Abstract

Telomerase replenishes the telomeric repeats that cap eukaryotic chromosome ends. To perform DNA synthesis, the active site of telomerase reverse transcriptase (TERT) copies a template within the integral telomerase RNA (TER). In vivo, TERT and TER and additional subunits form a telomerase holoenzyme capable of telomere elongation. We previously purified epitope-tagged Tetrahymena thermophila TERT and characterized two of the associated proteins. Here we characterize the remaining two proteins that were enriched by TERT purification. The primary sequence of the p75 polypeptide lacks evident homology with other proteins, whereas the p20 polypeptide is the Tetrahymena ortholog of a conserved multi-functional protein Skp1. Genetic depletion of p75 induced telomere shortening without affecting the accumulation of TER or TERT, suggesting that p75 promotes telomerase function at the telomere. Affinity purification of p75 co-enriched telomerase activity and each other known telomerase holoenzyme protein. On the other hand, genetic depletion of Skp1p induced telomere elongation, suggesting that this protein plays a negative regulatory role in the maintenance of telomere length homeostasis. Affinity purification of Skp1p did not detectably enrich active telomerase but did co-purify ubiquitin ligase machinery. These studies reveal additional complexity in the positive and negative regulation of Tetrahymena telomerase function.