Graduate Studies, UNL

 

Dissertations and Doctoral Documents, University of Nebraska-Lincoln, 2023–

First Advisor

Alan Christensen

Degree Name

Doctor of Philosophy (Ph.D.)

Committee Members

Etsuko Moriyama, Jeffrey Mower, Kristi Montooth, Rebecca Roston

Department

Biological Sciences

Date of this Version

12-2025

Document Type

Dissertation

Citation

A dissertation presented to the faculty of the Graduate College of the University of Nebraska in partial fulfillment of requirements for the degree Doctor of Philosophy (Ph.D.)

Major: Biological Sciences

Under the supervision of Professor Alan Christensen

Lincoln, Nebraska, December 2025

Comments

Copyright 2025, Moira Rodriguez. Used by permission

Abstract

Plant mitochondrial genomes are complex when compared to most other eukaryotic mitochondrial genomes. They are large and variable in size, expand and rearrange frequently, abundant in non-tandem repeats, but maintain a very low synonymous substitution rate in the remaining coding regions. Many of these complexities can be explained by the increased use of double-strand break repair for DNA damage. To advance the understanding of mitochondrial genome dynamics as it relates to double-strand break repair, we performed a forward genetics screen to identify proteins involved in DNA repair in plant mitochondrial genomes. Through this screen, we identified an RNA/DNA helicase, Suv3, that when mutated in Arabidopsis thaliana, causes no change in phenotype unless plants are stressed. Further characterization of suv3- mutants in A. thaliana revealed its role in repeat recombination and maintaining the structure of the mitochondrial genome. Many of the known plant mitochondrial DNA repair genes are homologous to those of E. coli, however characterization of the active sites has not been done in these proteins. We attempted to make site-directed mutants of these proteins in A. thaliana to characterize these active sites, in order to compare them to their homologs in E. coli. One hinderance to understanding the mechanism of double-strand break repair in plant mitochondrial genome is the difficulty in making targeted breaks in plant mitochondrial DNA. The most commonly used system to create breaks, CRISPR-Cas9, cannot be employed to mitochondria due to the inability of a gRNA to enter the organelle. Some methods, like mitoTALENs, allow targeted changes but are labor intensive and only allow for a few targets at a time. To get around these issues, we designed a nuclear-encoded, mitochondrial-targeted, inducible restriction enzyme line that creates 35 simultaneous double-strand breaks throughout the mitochondrial of genome of A. thaliana and analyzed the repair choices and consequences on the genome structure. This dissertation contributes to the field of plant mitochondrial genome dynamics by further characterizing a mitochondrial helicase, providing tools to study active sites of mitochondrial DNA repair proteins, and by showing proof-of-concept of a novel method to make targeted changes in A. thaliana mitochondrial DNA.

Advisor: Alan Christensen

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