Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Molecular Genetics and Genomics

Language

English (en)

Date of Award

January 2009

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Sheila Stewart

Abstract

Telomeres, protein-DNA structures that distinguish the end of a chromosome from a bona fide DNA double strand break, are integral to genomic stability. High fidelity replication of telomeres is indispensable for their stability. Telomere replication is a challenging task that is completed through the coordinated actions of telomere binding proteins and DNA replication and repair proteins in ways that are not well understood. This work focuses on delineating the function of one DNA replication and repair protein, Flap Endonulcease 1: FEN1), in telomere replication and maintenance. I demonstrate that FEN1 is essential for the efficient replication of telomeres through its ability to re-initiate stalled replication forks. FEN1 depletion leads to telomere dysfunction characterized by the recognition of the telomeres as DNA double strand breaks and the specific loss of telomeres replicated by the lagging strand machinery. Expression of catalytically active telomerase, the reverse transcriptase that adds telomeric repeats to chromosome ends, was sufficient to rescue telomere dysfunction upon FEN1 depletion. Genetic rescue experiments revealed that the nuclease activity of FEN1, its interaction with the RecQ helicases and its ability to process DNA bubble structures are essential to prevent telomere loss, whereas its ability to process Okazaki fragments is dispensable. However, FEN1 depletion did not affect cell cycle progression or in vitro DNA replication through non-telomeric substrates and in the absence of telomere dysfunction, FEN1 depletion did not affect overall genomic stability. Further analysis revealed that FEN1 is important for the efficient re-initiation of stalled replication forks and that this function ensures telomere stability. As with telomere loss, FEN1's ability to process bubble DNA structures and its ability to interact with the RecQ helicases are vital for the re-initiation of stalled replication forks. Finally, FEN1 depletion in transformed telomerase-negative ALT-positive cells leads to telomere end-to-end fusions. I propose that FEN1 maintains stable telomeres through the efficient re-initiation of stalled replication forks that occur in the G-rich lagging strand telomere, ensuring high fidelity telomere replication. This model suggests that mutations that arise in FEN1 are detrimental to telomere stability, leading to genomic instability and driving the transformation process.

Comments

Permanent URL: http://dx.doi.org/10.7936/K79Z92X1

Download

Share

COinS