Telomerase is a ribonucleoprotein (RNP) enzyme responsible for the replenishment of repetitive DNA sequences present at the ends of most eukaryotic chromosomes. Telomerase is minimally composed of a protein catalytic subunit, the telomerase reverse transcriptase (TERT), and an RNA subunit. Using a small single-stranded segment (7--11 nucleotides) of the telomerase RNA as a template, the active site of the TERT catalytic subunit adds complementary nucleotides onto telomeric DNA. The primary goal of the work presented in this thesis was to biochemically and functionally characterize the human telomerase reverse transcriptase (hTERT) and the human telomerase RNA (hTR), as well as to identify novel telomerase-associated proteins. First, we demonstrated that the budding yeast Saccharomyces cerevisiae possesses the cellular machinery to fully assemble a catalytically active human telomerase RNP in vivo. We also analyzed telomerase activity and binding of hTR to hTERT in rabbit reticulocyte lysates by expressing different hTERT and hTR variants. Our results identified two distinct regions of hTR that can independently bind hTERT in vitro. Furthermore, sequences or structures that include the conserved CR4--CR5 domain of hTR were found to be important for hTERT-hTR interactions and telomerase activity reconstitution. Human TERT carboxy- and amino-terminal amino acid deletions indicated that the polymerase and RNA binding functions of hTERT are separable. We also found that the product of the survival of motor neuron ( SMN) gene, a protein involved in the biogenesis of certain RNPs, is a telomerase-associated protein. Our results demonstrate that the human TR and the human TERT are not associated with Sm proteins, in contrast to Saccharomyces cerevisiae telomerase. Taken together, the work presented in this thesis indicate that the reconstitution of human telomerase activity in vitro requires regions of hTERT that (i) are distinct from the conserved reverse transcriptase
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.38460 |
| Date | January 2002 |
| Creators | Bachand, François |
| Contributors | Autexier, Chantal (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Anatomy and Cell Biology.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001941026, proquestno: NQ85681, Theses scanned by UMI/ProQuest. |
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