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Title page for ETD etd-07222016-154428


Type of Document Dissertation
Author Obodo, Udochukwu Chinyere
URN etd-07222016-154428
Title Not Restricted to the Ends: Yeast Telomere Proteins Rif1 and Cdc13 Function in Double-Strand Break Repair Pathways with Implications for Genome Stability
Degree PhD
Department Biological Sciences
Advisory Committee
Advisor Name Title
Todd R. Graham, Ph. D. Committee Chair
Donna J. Webb, Ph. D. Committee Member
Katherine L Friedman, Ph. D. Committee Member
Kathleen L. Gould, Ph. D. Committee Member
P. Anthony Weil, Ph. D. Committee Member
Keywords
  • Double-strand breaks
  • telomeres
  • de novo telomere addition
  • Cdc13
  • non-homologous end joining
  • Rif1
Date of Defense 2016-06-21
Availability restricted
Abstract
DNA double-strand breaks (DSBs) are the most lethal form of DNA damage; a single DSB, if left unrepaired, can cause cell death. Inaccurately repaired DSBs are sources of mutations, chromosomal rearrangements and genome instability. Telomeres, TG-rich nucleoprotein structures at the termini of eukaryotic chromosomes, protect chromosome ends from nucleolytic degradation and distinguish chromosome ends from those generated by DSBs. In most eukaryotes, proper telomere maintenance requires a specialized reverse transcriptase, telomerase, that utilizes an enzyme-associated RNA as template for new telomere synthesis. Increasingly, telomere-associated proteins are being recognized as playing roles in the repair of internal DSBs.

At endonuclease-induced DSBs immediately preceded by ectopic, short telomere seeds, the telomeric single-stranded DNA binding protein Cdc13 recruits telomerase, promoting the addition of new (de novo) telomere sequences to the telomere seed. However, spontaneous DSBs occur very infrequently and as such, are rarely expected to occur immediately adjacent to telomere-like seed sequences. An experimental system in which a DSB is induced at a distance (~3 kb) from endogenous Sites of de novo Telomere Addition (SiRTAs) in budding yeast was utilized as a more representative model of de novo telomere addition following spontaneous DSBs. These telomere-like SiRTAs incurred high frequencies of de novo telomere addition relative to flanking sequences. Additionally, de novo telomere addition at these SiRTAs required a bipartite structure in which Cdc13 binding to a stimulatory (Stim) sequence strongly stimulates telomere addition at a nearby target (Core) sequence.

The role of yet another budding yeast telomere-associated protein, Rif1, in DSB repair was also explored using an experimental system of an endonuclease-induced DSB that is primarily repaired by imprecise non-homologous end joining (NHEJ). Wild-type repair junctions contained only deletions. However, rif1∆ repair junctions contained relatively smaller deletions, as well as insertions, indicative of ends undergoing less resection in the absence of Rif1. A rif1 mutant lacking the protein phosphatase-1-interacting domain phenocopies the rif1∆ mutant, implicating this domain in Rif1’s role in regulating the fidelity of DSB repair.

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