Date of Award

Spring 1-1-2014

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry & Biochemistry

First Advisor

Thomas R. Cech

Second Advisor

Deborah S. Wuttke

Third Advisor

Arthur Pardi

Fourth Advisor

Roy Parker

Fifth Advisor

Norman R. Pace

Abstract

Telomeres are the nucleoprotein endcaps of linear chromosomes. Telomeres shorten with each cell division, limiting the capacity of telomeres to protect chromosomal DNA. The enzyme telomerase counteracts telomere attrition by synthesizing new telomeric DNA. Telomerase is a ribonucleoprotein complex comprised of TElomerase Reverse Transcriptase (TERT) and Telomerase RNA (TR). To maintain a homeostatic telomere length, telomerase must assemble, traffic to the telomere, and interact with a host of protein cofactors. Since the discovery of telomerase, many key components of the telomerase holoenzyme and telomeric protein cap have been identified. This thesis presents detailed investigations of the molecular interactions of telomerase and associated proteins.

In Saccharomyces cerevisiae the Ku heterodimer binds directly to a hairpin of TR to promote the nuclear localization of telomerase. Footprinting experiments examined the Ku binding site and chemical probing investigated the hairpin secondary structure. Heterologous mixing experiments and RNA mutagenesis tested which stem-loop elements mediate Ku binding. These experiments suggest that Ku binds to the terminal loop and proximal stems of the hairpin, recognizing a critical bulge motif in a sequence-independent but structure-specific manner.

The TEL-patch consists of amino acids on the surface of the human telomere protein TPP1 that are necessary for telomerase recruitment and processivity stimulation, but the TEL-patch contributions are not fully understood. Single-turnover translocation, substrate-competition, and substrate-telomerase dissociation rate assays tested the impact of the TEL-patch on telomerase catalysis. The competition experiments constitute a step in developing an in vitro telomerase recruitment assay. Furthermore, the TEL-patch increases the apparent rate and efficiency of telomerase translocation, slows the rate of substrate dissociation, and contributes to the preferential binding and extension of TPP1-bound substrates by telomerase.

The exact component of telomerase that interacts with the TEL-patch of TPP1 has been unknown. Direct telomerase extension assays identified human TERT separation-of-function alleles that disrupt the telomerase-TPP1 interaction. Perturbation of the interaction resulted in compromised telomere maintenance in cells. A deleterious mutation in the TEN-domain of hTERT was rescued by introducing a compensatory charge-swap mutation in the TEL-patch of TPP1 that restored telomerase stimulation in vitro, telomere maintenance in vivo, and suggests that these proteins interact directly.

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