Telomerase is an unusual reverse transcriptase (RT) that catalyzes the de novo addition of telomeric DNA repeats to telomeres. Telomerase activity counteracts the progressive loss of telomeric DNA over successive rounds of DNA replication, and is important for the immortality of most eukaryotic cells. Telomerase is distinct from other RTs in that its catalytic subunit (TERT: telomerase reverse transcriptase) stably associates with a telomerase RNA (TR) component that contains a short template used to direct synthesis of telomeric repeats. Telomerase also exhibits a unique, repeat addition form of processivity that permits the addition of multiple telomeric repeats to a single substrate by repetitive reverse transcription of the template. The TERT proteins consist of a central region of RT-conserved motifs, which is flanked by extensive telomerase-specific N- and C-terminal sequences. The N terminus constitutes nearly half of the TERT protein, and is an excellent candidate site for telomerase-specific catalytic functions. Using a mutagenic approach, we investigated the contributions of human TERT N-terminal sequences to telomerase catalytic function and nucleic acid interactions. We found that the hTERT N terminus contains two RNA interaction domains, RID1 and RID2. RID1 was functionally and physically separable from the remainder of hTERT, and may constitute an hTERT polymerase accessory domain. We investigated the catalytic function of two RID1- and RID2-interacting regions in the human TR, the pseudoknot/template domain and the P6.1 helix. RID1-pseudoknot/template domain interactions were essential for repeat addition processivity, and RID2-P6.1 interactions mediated telomerase assembly and were required for basic polymerase function. Repeat addition processivity is thought to be partly dependent on an anchor site(s) in TERT that stabilizes telomerase-DNA interactions. We found that RID1 mutations also reduced the affinity of human telo
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.85628 |
| Date | January 2005 |
| Creators | Moriarty, Tara J. |
| 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: 002207917, proquestno: AAINR12913, Theses scanned by UMI/ProQuest. |
Page generated in 0.0015 seconds