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, rif1â 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 rif1â mutant, implicating this domain in Rif1âs role in regulating the fidelity of DSB repair.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-07222016-154428 |
| Date | 01 August 2016 |
| Creators | Obodo, Udochukwu Chinyere |
| Contributors | Kathleen L. Gould, Ph. D., P. Anthony Weil, Ph. D., Katherine L Friedman, Ph. D., Todd R. Graham, Ph. D., Donna J. Webb, Ph. D. |
| Publisher | VANDERBILT |
| Source Sets | Vanderbilt University Theses |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.vanderbilt.edu/available/etd-07222016-154428/ |
| Rights | restricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Vanderbilt University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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