Student Theses and Dissertations

Date of Award

2023

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Abstract

Telomeres define and protect the ends of linear chromosomes. Genome stability requires that cells differentiate telomeres from DNA double-strand breaks (DSBs), which humans accomplish with the shelterin complex. The reverse transcriptase telomerase counteracts the shortening of telomeres that occurs with cell division due to incomplete DNA replication and nucleolytic resection. During human development, the expression of the reverse transcriptase component of telomerase (TERT) is restricted to the germ line and select stem cell pools, causing telomeres to shorten in most somatic cells. After a certain number of divisions, one or a few telomeres become too short to adequately suppress DNA damage response signaling, driving cells into senescence or apoptosis. Cells deficient in the p53 and Rb pathways, however, disregard these alarms, continue to divide, and ultimately enter telomere crisis, marked by restricted cell viability due to severe genome instability. During crisis, dysfunctional telomeres undergo repeated breakage-fusion-bridge cycles, forming dicentric chromosomes that snap during mitosis. Ultimately, clinically detectable tumors emerge from this process with shuffled, aneuploid genomes after acquiring a telomere maintenance mechanism, usually by reactivating TERT expression. Canonically, telomerase is believed to function in cancer by rapidly restoring critically short telomeres to a tolerable length and then maintaining replicative immortality thereafter. Nevertheless, this dogma does not explain how cells restore end protection to deeply damaged chromosomes, which likely require a mechanism for reestablishing their telomeres. We suspected that telomerase might aid incipient tumor cells in their escape from telomere crisis by healing broken chromosomes with neotelomeres. In a subset of human patients with terminal chromosome deletions, telomeric repeats directly abut reference genomic sequence at the breakpoint, suggesting that telomerase itself might have directly added to a DSB at that site. Consistent with this interpretation, the breakpoint sequence from one of these patients, known as TS, has been shown to be an excellent primer for telomerase in vitro. Here, we use this TS sequence to develop a sensitive and specific qPCR assay to detect and measure telomeric repeat addition at a programmed DSB in human cells. With this assay, we demonstrate that telomeric repeat addition occurs at a Cas9- induced DSB in a telomerase-dependent fashion, though such events are rare in cells with physiologic levels of telomerase activity. We identify long-range DSB resection and ATR kinase signaling as repressors of telomerase at DSBs in human cells. Finally, we provide preliminary evidence that telomerase can synthesize a fully functional telomere de novo at a DSB. Our findings unveil a new function for telomerase in carcinogenesis and provide a mechanistic explanation for the recent identification of neotelomeres in human tumors.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

Download

Included in

Life Sciences Commons

Share

COinS