UC Santa Cruz

Telomeres and telomerase form a dynamic interplay in order to protect the ends of eukaryotic chromosomes. Telomeres are DNA capping structures that protect the chromosome ends from aberrant recognition as sites of double stranded DNA breaks. Telomerase is a conserved ribonucleoprotein that reverse transcribes G-rich repeats onto the 3’ end of these structures in order to facilitate homeostatic telomere length. The reverse transcription reaction of the telomerase protein component is facilitated by an integral RNA template that codes for telomeric sequence. Catalytically, telomerase differs from canonical polymerases by the ability to add multiple template repeats during a single substrate binding event, an action known as translocation. Herein we attempt to dissect this translocation sub-step through single molecule FRET, a technique used for measuring distances and dynamics within macromolecular complexes. We find that the DNA substrate forms a dynamic interplay between multiple conformations during the catalytic cycle and is stabilized by a telomerase specific N-terminal domain. Similarly, we attempt to dissect the intricacies of the telomerase catalytic cycle by observing the integral RNA component during telomerase activity. We find that the telomerase RNA pseudoknot fold undergoes nanometer scale dynamics during telomerase processivity and likely plays an integral role in priming the telomerase complex for successful translocation of the DNA substrate.

Telomeres are DNA structures with highly repetitive sequence elements (TTAGGG in humans) with a 3’ G-rich tail, which is the product of telomerase synthesis. Many studies have shown that these repetitive G-rich sequences are capable of forming complex, polymorphic tertiary structures known as quadraplexes. In this thesis we investigate the dynamics and structure of human quadruplexes in order to facilitate understanding of how protein binding partners and enzymes act on these unique DNA structures. Our study involved a recently developed methodology that integrates single molecule FRET with Magnetic Tweezers in order to actively manipulate and monitor conformational changes within the DNA. We find that these DNA structures are relatively brittle and can disrupted by destabilizing only a few base pairs of the structure.