Date of Award

Spring 2024

Project Type

Dissertation

Program or Major

Genetics

Degree Name

Doctor of Philosophy

First Advisor

Kevin M Culligan

Second Advisor

Anna O’Brien

Third Advisor

Estelle Hrabak

Abstract

DNA damage is a constant threat for all organisms. The most severe form of DNA damage is a double-strand break (DSB) that can have disastrous consequences to the cell including genome truncation and programmed cell death. As such, organisms have multiple complex pathways dedicated to repairing DSBs. These repair pathways begin with a signaling cascade that results in the resecting of the ends of the break to create 3’ OH overhangs which are immediately bound by a single-stranded binding protein called Replication Protein A (RPA). This is followed by the cell either undergoing programmed cell death or utilizing non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), or a homologous recombination repair (HRR) mechanism. HRR uses either single-strand annealing (SSA) or synthesis-dependent strand annealing (SDSA) to repair a DSB, but in either case, RPA must bind to the single-stranded DNA (ssDNA) prior to initiation of repair. RPA is a heterotrimeric ssDNA binding protein that is highly conserved across all eukaryotes. While animal and yeast genomes typically only have a single copy of each subunit, plants have multiple paralogs of each. In plants the number of paralogs of each subunit varies considerably depending on the species. The model organism Arabidopsis (Arabidopsis thaliana) has five RPA1 paralogs and two each of RPA2 and RPA3. The RPA1 paralogs are divided into three groups by function. The group C paralogs (RPA1C and E in Arabidopsis) are involved in DNA damage repair and have a C-terminal extension that is only found in group C paralogs. This C-terminal extension contains a zinc finger motif (ZFM) that is highly conserved and is therefore hypothesized to be critical to the functionality of the paralogs during DNA damage repair. This dissertation investigated the role of RPA in determining how the DSB is repaired and how RPA functions during DSB repair. The role of the C-terminal ZFM in DSB repair was examined via the CRISPR-Cas9 mediated alteration of RPA1 genes in wild-type Arabidopsis plants. CRISPR mutant plants lacking the ZFM of the group C RPA1 paralogs were exposed to DNA damaging agents and their phenotypes were compared to wild-type Arabidopsis as well as to previously characterized T-DNA null mutants for group C paralogs (rpa1c and rpa1e). These plant lines were also used for the characterization of HRR usage with GUS gene reporter lines. This dissertation details how the ZFM of the group C paralogs is involved in the functionality of the paralogs as well as the overall usage of the group C paralogs in HRR.

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