Date of Award

2016

Document Type

Open Access Thesis

Department

Pharmacology, Physiology and Neuroscience

Sub-Department

College of Pharmacy

First Advisor

Douglas Pittman

Abstract

Genomic instability is one of the major components represented in the “Hallmarks of Cancer.” DNA interstrand crosslinks and double-stranded breaks are two of the most severe causes of genomic instability. Homologous recombination (HR) plays a major role in resolving both types of DNA lesions, requiring a homologous template and the RAD51 paralog complex of proteins. One member of this complex is the RAD51D ovarian cancer susceptibility gene product. To better elucidate RAD51D modifications and functions, several protein interaction screens were performed, and one of the novel proteins identified was NONO (a.k.a. p54nrb). Decreased expression of either RAD51D or NONO conferred increased chromosomal instability and cellular sensitivity to DNA interstrand crosslinking agents. To further characterize RAD51D-NONO interaction, I used the yeast two hybrid approach. Interestingly, the yeast two hybrid data not only identified regions of RAD51D necessary for the interaction but indicated that the RAD51D Walker Box A motif and a specific lysine residue within RAD51D decreased the strength of the interaction with NONO. Taken together, the results from this study suggest specific regions or post-translational modifications regulate binding between these two proteins. Another important feature of the NONO protein is that it is a component of a sub-nuclear structure known as the paraspeckle. The second part of this thesis work investigated whether there was a correlation between paraspeckle numbers and levels of DNA damage resulting from etoposide, mitomycin C, or cisplatin. The results suggest that there is a very modest, yet significant, decrease in the numbers of

paraspeckles after double-stranded DNA break induction following etoposide treatment. Additional data are presented suggesting that the absence of RAD51D confers a decrease in paraspeckle number. In conclusion, these studies revealed that the interaction between RAD51D and NONO may be very tightly regulated, perhaps during the DNA damage response, and that there may be a correlation between DNA damage, the paraspeckle structure and/or numbers, and individual paraspeckle components.

Available for download on Wednesday, December 12, 2018

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