Characterization of the human telomerase complex.
Published: 2004
Language: eng
Number of pages: 185
Description: Telomeres are the nucleoprotein structures located at eukaryotic chromosomal termini. Their presence is required at chromosome ends to ensure genomic stability. Telomeric DNA is synthesized de novo by the ribonucleoprotein (RNP) enzyme known as telomerase.This study focuses on characterization of the human telomerase complex. Endogenous human telomerase was partially purified from cells using an anti-sense affinity selection (AAS) technique. Electrophoresis of the AAS purified material on a nondenaturing gel revealed an RNP particle containing the human telomerase RNA subunit. The mobility of the RNP particle was coincident with that of telomerase activity and varied according to purification conditions. UV cross-linking analysis was also performed on the partially purified material using primers containing photoreactive nucleotides. Three proteins were observed to cross-link specifically to telomeric DNA.The biochemical activity of partially purified human telomerase was examined in the second part of this study. Both ciliate and yeast telomerases have an associated nucleolytic activity that is capable of removing telomeric or nontelomeric DNA from the 3' end of an oligonucleotide substrate. In these organisms, an endonuclease is responsible for the cleavage function. The activity is thought to reside within the catalytic core of the enzyme since it copurifies with ciliate and yeast telomerase over several steps and is associated with ciliate telomerase synthesized in rabbit reticulocyte lysate (RRL). The nuclease is thought to assist in proofreading and/or re-initiation of a stalled polymerization complex. In this study, partially purified human telomerase was found to associate with a similar nucleolytic activity. Various chimeric oligonucleotides, containing telomeric and nontelomeric DNA, acted as cleavage substrates. These data are consistent with nucleolytic cleavage occurring at or near the boundary between telomeric and nontelomeric DNA, creating a substrate for subsequent elongation by telomerase. A nuclease activity is also associated with human telomerase synthesized in RRL. These findings suggest that the nuclease activity serves an evolutionarily conserved function in substrate utilization by telomerase.
Notes: Adviser: Lea Harrington.

Thesis (Ph.D.)--University of Toronto, 2004.

Electronic version licensed for access by U. of T. users.

Source: Dissertation Abstracts International, Volume: 65-05, Section: B, page: 2255.