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.

Since the discovery by Blackburn in 1978, telomere has been the subject of intense research focus due to its close relationship with aging and cancer. Despite extensive research efforts more than three decades, the structural properties of telomere remain elusive. In this dissertation, I used single molecule techniques to examine the physical properties of telomere. These studies have identified the kinetically favorable telomere G-quadruplex (GQ) structure, revealed the mechanical unfolding pathway of telomere GQ, and characterized the mechanical properties of duplex telomere DNA and the formation of the telomere D-loop.

Since the discovery by Blackburn in 1978, the telomere has been the subject of intense research focus due to its close relationship with aging and cancer. Despite extensive research efforts for more than three decades, the structural properties of telomere remain elusive. In this dissertation, I used single molecule techniques to examine the physical properties of telomere. These studies have identified the kinetically favorable telomere G- quadruplex (GQ) structure, revealed the mechanical unfolding pathway of telomere GQ, and characterized the mechanical properties of duplex telomere DNA and the formation of the telomere D-loop. Telomeres are specialize DNA sequence that prevent degradation and aberrant fusion of chromosome ends. The foundation of human telomeres consists of 5-10 kb of duplex TTAGGG repeats follow by a 50 to 200 nucleotides of 3' single stranded overhang. The G-rich single stranded telomere has a propensity to fold into a secondary structure known as GQ. In Chapter 2, single-molecule Förster resonance energy transfer (smFRET) was used for constructing the distribution of telomere DNA GQ conformations under physiological salt conditions. With circular dichroism, the kinetic trapped GQ conformation was discovered. In Chapter 3, smFRET and magnetic tweezers (MT) was used to dissect the folding property telomere GQ. Under stretching force, the unfolding pathway of telomere GQ was characterized by a distance less than 1nm. In Chapter 4, the magnetic tweezers assay revealed that telomere is more resistant to torque-induced denaturation. The direct observation of telomere strand invasion suggested that the stretching force can influence the rate of the invasion and the formation of the telomere displacement loop. The subsequent chapters detail the setup and experimental protocol for the MT-FRET, and describe how to use MT to manipulate DNA. These single molecule assays setup a foundation for investigating the interaction between the telomere and its associated proteins and serve as an experimental platform for telomere drug assessment. Ultimately, with these single molecule assays we will enhance our knowledge on how telomere regulates telomerase activity.

Telomerase is an enzyme that maintains telomeres, the protective structures at the ends of chromosomes. Telomerase dysfunction is associated with premature aging disorders and inappropriate telomerase activation is associated with ~90% of all cancers, which has motivated studies to better understand the function of this crucial enzyme. Telomerase has two essential components, the protein component telomerase reverse transcriptase (TERT) and the telomerase RNA (TER). TERT functions by reverse transcribing a small template region of TER into telomere DNA. TER also has several conserved secondary and tertiary structures known to be important for telomerase function.

In this thesis research, I describe our attempts to better understand how conserved elements of TERT and TER assemble to form a functional enzyme. In Chapter II, using the well-characterized model system Tetrahymena thermophila, I describe our characterization of the assembly protein p65. p65 functions in Tetrahymena by binding TER and reorganizing important RNA motifs, placing them in the correct orientation to facilitate TERT binding. We identified a C-terminal domain of p65 which is necessary and sufficient for this activity. We then further characterized this domain, demonstrating that it binds a conserved region of the RNA and affects basepairing interactions in an important stem-loop region of the RNA. This work was originally published in the journal RNA.

In Chapter III, I further explain our efforts to characterize TERT-TER interactions. In this work we used a biochemical technique known as site-directed hydroxyl radical probing to identify sites of interaction between protein and RNA within the enzyme. Our work uncovered a specific interaction between a conserved RNA element known as the template boundary element (TBE) and a conserved protein element known as the CP2 motif. Mutagenesis experiments identified the most crucial amino acid determinants of the CP2 motif and demonstrated that CP2 motif mutations severely affect telomerase activity. This work originally appeared in the Journal of Biological Chemistry.

Another focus of my thesis work has been telomerase dynamics. Telomerase activity involves coordinated motions between protein, RNA, and DNA subunits within the holoenzyme. Understanding these dynamics is crucial to our understanding of telomerase function. In order to study dynamics within the telomerase holoenzyme, we used a technique known as single-molecule Förster resonance energy transfer (smFRET). FRET describes the distance-dependent energy transfer between a donor and acceptor fluorophore at sub-nanometer distances. When these fluorophores are site-specifically incorporated into a molecule of interest, real-time changes in FRET can be used to measure conformational changes within the molecule.

In Chapter IV, I describe a smFRET telomerase binding assay we developed to measure conformational motions during telomerase catalytic activity. We identified reciprocal motions in telomerase RNA on either side of the template that occur during telomerase extension activity. Using smFRET, we demonstrated that RNA on either side of the template undergoes a cycle of expansion and compactions as template RNA is pulled through the active site. This work was originally published in Nature Structural and Molecular Biology.

In Chapter V, I describe smFRET experiments which demonstrate that DNA bound to telomerase is an equilibrium between two conformations: an active state and an alternative state. Furthermore, we discovered that a conserved domain of TERT, the telomerase essential N-terminal (TEN) domain, is responsible for stabilizing the active state and TEN domain mutations which favor the alternative state disrupt the rate of telomerase processivity. Our work conclusively identifies a new DNA binding mode for telomerase and demonstrates an unappreciated function for a conserved telomerase protein domain.

In Chapter VI of this thesis, I describe preliminary work we have performed to further study transitions between the active and the alternative state described in Chapter V. I describe efforts to develop new methodologies to study FRET dynamics across the DNA primer, as well as our efforts to study protein-DNA and protein-RNA motions. These experiments are too preliminary to yield conclusive results, however they demonstrate great promise for future smFRET studies of telomerase.

In the final chapter of this thesis, I describe potential future experiments on telomerase. Having extensively discussed the TEN domain in Chapters V and VI, I focus on potential studies on real-time measurements of telomerase extension activity. I also discuss experiments on further structural characterization of protein-RNA interactions in the telomerase holoenzyme. I feel both of these topics merit further study in order to better understand the mechanism of this important enzyme.

Telomeres are nucleoprotein structures that protect the ends of linear chromosomes. Telomeres serve two functions within the cell: to protect against genetic loss due to chromosomal shortening as a result of the end replication problem, and to distinguish chromosomal termini from sites of DNA damage that would otherwise elicit an unwanted DNA damage response. The foundation of telomere structures begins with a hexameric double-stranded repeat DNA sequence (TTAGGG in vertebrates) ranging from 2-20kb in length and terminate in a 3’ G-rich single-stranded DNA overhang ranging from 50-300bp in length. Telomeric chromatin is known to contain tightly spaced nucleosomes, as well as a telomere-specific protein complex known as the shelterin complex. It is known that the shelterin complex is responsible for providing telomere end protection, however the mechanistic details of how this complex establishes end protection is lacking. Furthermore, how this complex transitions the telomere from a protected state required for end protection into an open deprotected state required for telomere replication remains largely unknown. This thesis outlines single-molecule studies of both telomeric DNA and telomere-interacting proteins to further our understanding of how these structures protect the genome.

Telomerase is a unique eukaryotic enzyme responsible for maintaining the genomic integrity of chromosome ends, through the addition of protective DNA sequences called telomeres. The telomerase holoenzyme is composed of a protein component called TERT, and an RNA component called TR. TERT is a well conserved protein that contains many domains, one of which is the reverse transcriptase responsible for processive addition of telomere repeats to the chromosomal DNA. TER carries several necessary functional regions, which play critical roles in the activity of telomerase. These include the RNA template that is used as the basis for telomere replication, as well as a pseudoknot fold and a three-way junction called CR4/5. Overall TER displays much less sequence conservation than its protein counterpart, but its functional domains bear significant structural similarity throughout Eukarya. The CR4/5 region plays a significant, yet enigmatic role in the mechanism of telomerase. In this work I aim to further define the functional roles of the CR4/5 region through study of its structural dynamics, using single-molecule FRET. I investigate the conformational interconversions displayed by the yeast Kluyveromyces lactis CR4/5, and the impact that magnesium and junction sequence have on these dynamics. Further, I examine how models of the K. lactis CR4/5 correspond to the solved structures of CR4/5 from other organisms, how the dynamics found in K. lactis can be related to these organisms, and what insights are gained by real-time observations of this region. Lastly, I discuss the potential roles played by the CR4/5 region in the overall telomerase mechanism

Telomeres are protective DNA caps that terminate each chromosome in eukaryotic cells. In humans, telomeres are comprised of hexameric GGTTAG repeats and form a single-stranded 3’ DNA overhang. To distinguish chromosome ends from DNA repair machinery, telomeres are bound by protein complexes called shelterin. Shortening of telomeres with each cell cycle can compromise the protective properties of telomeres. Critically short telomeres result in senescence and eventually apoptosis of these cells. In highly proliferative cells, such as bone marrow cells, telomere length is maintained by the enzyme telomerase. While mutations that compromise telomerase activity leads to fatal diseases such as dyskeratosis congenita, aberrant reactivation of telomerase activity in somatic tissues confers immortality in about 90% of cancer cells. This makes telomerase an attractive target for therapeutic intervention for these diseases.The catalytically active core of telomerase is minimally comprised of protein (telomerase reverse transcriptase or hTERT in humans) and RNA (telomerase RNA or hTR in humans) components. During S-phase of the cell cycle, telomerase is recruited to telomeres by shelterin components. Once at the 3’ end telomerase uses its internal RNA template to add GGTTAG repeats to the 3’ end of telomeres. After each round of repeat addition, the enzyme must rearrange its RNA template in a process called repeat addition processivity. In this thesis, I interrogate several aspects of telomerase activity. In chapter two, I focus on a novel single-molecule Förster Resonance Energy Transfer (smFRET) methodology that leverages existing sequencing machinery to observe the incorporation of nucleotides by telomerase in real-time, allowing us to study the kinetic properties of the telomerase catalytic cycle. Chapter three describes my approach to investigate how DNA structure formation in telomeres and presence of shelterin protein components regulate telomerase recruitment efficiency in vitro. I show that interaction of the shelterin subcomplex TPP1-POT1-TIN2 (TPT) with telomerase positively regulates telomerase recruitment efficiency while potassium-induced ssDNA structure negatively regulates telomerase recruitment, even in the presence of TPT. In chapter 4, I study how use of the nucleotide analog 6-thio-dGTP by telomerase leads to inhibition of telomerase activity and show that incorporation of the analog leads to telomerase staling on the ssDNA, possibly due to defects in the template rearrangement mechanism. Finally, in the last chapter I discuss single molecule approaches that can advance ongoing studies of telomerase recruitment and catalysis and the challenges that these studies must overcome to be completed.

Eukaryotic chromosomes are linear DNA strands that terminate with telomeres, protective regions of repetitive sequences that are coated in DNA-binding proteins. Telomeres gradually shorten with each cell division until they reach a critical length that induces a ‘telomere crisis’, resulting in programmed cell death or senescence. The telomerase enzyme prevents telomere crisis by using a reverse transcriptase (TERT) and integral telomerase RNA (TR) to synthesize new telomeric DNA and counteract DNA loss. While telomerase is beneficial for preserving self-renewing tissues, it is also a hallmark of most human cancers.

Despite decades of work since telomerase’s discovery in 1984, zero telomerase-targeting therapeutics are approved for clinical use today. A key obstacle in telomerase drug development was the lack of structural information available, but recent high-resolution cryogenic electron microscopy (cryo EM) models in combination with other structural studies have now eliminated this barrier. While a growing body of research is illuminating telomerase in higher molecular detail, much remains to be learned about the process of telomerase assembly; how the various protein and RNA components of this ribonucleoprotein (RNP) assemble to produce the active complex. The knowledge gained on this topic will accelerate therapeutic development and better enable us to understand diseases caused by telomerase deficiencies.

This thesis focuses on human telomerase biogenesis from an RNA structural perspective, highlighting the interplay between human TR (hTR) structure and protein-binding events that must be properly orchestrated to achieve the assembly of the active enzyme. I will focus first on in vitro studies that reveal hTR adopts heterogenous folds, and describe a discrete hTR conformational change that occurs upon TERT binding. Next, I will focus on how alternatively folded conformations of hTR persist in the cellular environment and their impact on the biogenesis of telomerase. In addition, I provide a detailed guide to designing, executing, and analyzing in-cell RNA chemical probing experiments from start to finish. Finally, I describe the future directions of my unfinished projects on the topics of hTR folding chaperones and disease-associated hTR mutations.

Eukaryotic organisms contain linear chromosomes that are protected by telomeres; long repeated sequences that act as a protecting buffer from the shortening of chromosomes during each replication cycle, and as a signal that identify the ends from double stranded breaks. Telomeres are maintained in most organisms by a ribonucleoprotein called telomerase. While telomeres and telomerase have been implicated in many human diseases, including cancer, very little is known about the unique enzymatic mechanism of telomerase, making it difficult to develop medicinal therapeutics.

The protein p65 is required for telomerase assembly in Tetrahymena thermophila. Using RNase footprinting and single molecule FRET, we show that the C-domain of p65 is not only required, but is sufficient for telomerase assembly, paving the way for understanding possible p65 homologues in other organisms. Preliminary crystals of complexes consisting of the RNA binding domain of the catalytic subunit and the RNA component of T. thermophila were also obtained. Because the catalytic subunit is quite conserved between species, any structure obtained from these crystals will prove valuable in understanding the RNA and protein organization of telomerase.

For a more ambitious goal, a novel method was pursued to solve the structure of telomerase: inserting telomerase RNA into ribosomal RNA, assembling the telomerase complex on the ribosome, using the well known ribosome purification methods and crystallization conditions, and thus using the ribosome as a crystallization chaperone and scaffold. While crystals failed to diffract to a desired resolution, considerable strides were made toward this method that could also be extended to other ribonucleoproteins.

Technology guides the practice of scientific inquiry. In the biological sciences, DNA sequencing has encouraged the commingling of traditional experimental biology and computer science. In this thesis, I describe computational and biochemical methods to advance nanopore DNA sequencing technology. Nanopore DNA sequencing is an single-molecule technology that shows promise in the area of read lengths, instrument portability, and, as shown in this work, chemical modification detection. A nanopore sequencing device contains a nanometer-sized pore that separates two electrolyte buffer reservoirs. A voltage potential is applied across the nanopore and the device records the ionic current through pore. As DNA polymers translocate through the pore they modulate the ionic current by partially obstructing the pore in a sequence-dependent manner. Commercial sequencing devices contain arrays of pores that are individually controlled.

The first chapter of this work provides a technical background and practical application of the technology. In the following chapters I describe new algorithms that improve the utility of the technology. In Chapter 2 I describe a hidden Markov model for the nanopore ionic current. I describe how multiple model topologies were implemented, one of which included modeling the time domain. I describe how a hierarchical Dirichlet process can be used to model new non-canonical (modified) bases affording a HMM-HDP model. In chapter \ref{chap:detecting_modifications} I show how the model can be trained to detect multiple chemical modifications on synthetic DNA and genomic DNA samples. In the final section of this chapter I show how the model was incorporated into a cloud-based pipeline that allows for horizontal scaling of the datasets.

The last section of the paper describes a biochemical method to re-read DNA sequences using a nanopore and a helicase enzyme. Multiple biochemical techniques have been used to increase the accuracy of nanopore sequencing; re-reading the same strand allows for stochastic errors to be differentially placed in multiple passes.