Date of Award

Summer 8-15-2015

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Molecular Genetics & Genomics)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

High fidelity DNA replication is essential for genomic stability and cell survival; this fact is underscored by the redundancy present in DNA replication and repair pathways. The complexity of these pathways is most evident at challenging DNA templates, such as those with repetitive sequence and transcribed loci. Among these challenging templates are telomeres, which are terminal, highly repetitive sequences that maintain genomic stability by preventing aberrant end-to-end chromosome fusions. In the absence of accurate, complete telomere replication, genomic instability results, ultimately leading to cell death or transformation. Here, we describe two unique roles in telomere stability for the DNA replication and repair protein flap endonuclease 1 (FEN1). First, we find that FEN1 maintains telomere stability by facilitating replication fork reinitiation on the lagging strand-replicated telomere. In the absence of FEN1, sister telomere loss (STL) occurs at lagging strand-replicated telomeres. Genetic knockdown-rescue experiments demonstrated that FEN1’s nuclease activity, interactions with DNA repair proteins via its C-terminus, and gap endonuclease activity are essential for preventing STL. Similarly, an analysis of FEN1’s ability to reinitiate stalled replication forks revealed that it is dependent on the same activities as its ability to prevent STL, suggesting that FEN1’s role in reinitiating stalled replication forks is responsible for its ability to suppress STL on the lagging strand. Second, we show that FEN1 maintains telomere stability by limiting telomere fragility on the leading strand-replicated telomere. Strikingly, this activity is biochemically and genetically distinct from FEN’s role in preventing lagging strand- specific STL; FEN1’s ability to suppress telomere fragility depends only on its flap endonuclease activity, while its C-terminal interactions and gap endonuclease activity are dispensable. We show that FEN depletion-induced telomere fragility is increased by RNA polymerase II inhibition and rescued by ectopic ribonuclease H1 expression, suggesting that FEN1 limits leading strand-specific telomere fragility by processing RNA:DNA hybrid/flap structures that arise following co-directional replisome–RNAP collisions at the telomere. Notably, this is the first known role for FEN1 in leading strand DNA replication, and the first molecular mechanism for telomere fragility at the leading strand. Lastly, we demonstrate that while FEN1 interacts directly with the shelterin protein TRF1, which is required to prevent telomere fragility, this interaction does not contribute to FEN1’s ability to suppress telomere fragility. Together, these data indicate that FEN1 has two functionally separate roles in maintaining telomere replication and stability: preventing STL on the lagging strand by facilitating replication fork reinitiation, and suppressing telomere fragility on the leading strand by processing intermediates that result from replisome–RNAP collisions.

Language

English (en)

Chair and Committee

Sheila A Stewart

Committee Members

Peter M Burgers, Susana Gonzalo, Barry P Sleckman, Zhongsheng You,

Comments

Permanent URL: https://doi.org/10.7936/K7DB800F

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