Structure and Dynamics of Telomerase

Telomeres are repetitive, G-rich DNA sequences, along with DNA-associated proteins, that cap the ends of linear chromosomes. Telomeres serve a two-fold function; they protect the ends of chromosomes from being recognized as sites of DNA damage and they prevent fraying of chromosomes into coding regions during successive rounds of DNA replication. In proliferative cells where unregulated chromosome end shortening would be a critical problem, telomere lengths are maintained by the specialized reverse transcriptase enzyme, telomerase. Because of its requirement in proliferative cells, telomerase is also upregulated in about 90% of cancer, making it an attractive target for cancer therapeutics. Telomerase is a ribonucleoprotein, composed of a protein component, TERT, and an RNA component, TR. Telomerase reverse transcribes telomere repeats onto the 3’ end of chromosomes through its integral template within TR. Telomerase is unique among reverse transcriptases in that it is able to add multiple telomere repeats during a single binding event. The precise conformational rearrangements required for this processive telomerase action are not well understood. In this thesis, I focus on the interaction between telomerase and the nascent telomere and how reverse transcription of multiple repeats affects the actively extending telomerase complex. First, I focus on how template boundary definition ensures the faithful synthesis of the required hexameric telomere sequence and demonstrate that critical regions within both TERT and TR are responsible for establishing strict template boundary definition. Second, I focus on the interplay between DNA structure formation within the nascent telomere and the actively extending telomerase complex and show that formation of G-quadruplexes within the telomere sequence affects telomerase activity. Lastly, I discuss ongoing work using single molecule techniques to investigate dynamics of human telomerase during the telomerase catalytic cycle and the problems that must be overcome to complete this work