Date of Award

12-2025

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Biochemistry and Molecular Biology

Committee Chair/Advisor

Michael Sehorn

Committee Member

Jennifer Mason

Committee Member

Kerry Smith

Committee Member

Meredith Morris

Abstract

Genome stability depends on precise DNA repair pathways such as homologous recombination (HR), where human single-stranded DNA-binding protein 1 (hSSB1) stabilizes single-stranded DNA (ssDNA) intermediates and recruits repair complexes. Post-translational modifications, including acetylation, are known to regulate hSSB1 activity, but their molecular effects remain poorly defined. This study examined the role of lysine 94 (K94) acetylation using a glutamine substitution mutant (K94Q) that mimics the acetylated state. Recombinant hSSB1 wild type (WT) and hSSB1K94Q were expressed in Escherichia coli and purified using Ni–NTA affinity chromatography. Biochemical analyses, including electrophoretic mobility shift assays (EMSA), single-strand annealing assays, and displacement loop (D-loop) formation assays, were used to determine the effect of the K94Q mutation on hSSB1. hSSB1WT displayed strong ssDNA-binding affinity and promoted single-strand annealing and D-loop formation in a concentration-dependent manner, whereas hSSB1K94Q showed significantly reduced DNA-binding and failed to catalyze single-strand annealing and D-loop formation efficiently. These findings indicate that lysine 94 is essential for the DNA-binding and recombination-promoting activities of hSSB1, likely due to its electrostatic potential and proximity to the oligonucleotide/oligosaccharide-binding (OB) fold. The functional loss observed in the hSSB1K94Q mutant highlights the regulatory significance of acetylation at this residue in modulating the contribution of hSSB1 to HR and maintaining genomic stability. Overall, this study provides new insight into the post-translational control of hSSB1 within the DNA damage response network.

Download

Included in

Biochemistry Commons

Share

COinS
 
 

To view the content in your browser, please download Adobe Reader or, alternately,
you may Download the file to your hard drive.

NOTE: The latest versions of Adobe Reader do not support viewing PDF files within Firefox on Mac OS and if you are using a modern (Intel) Mac, there is no official plugin for viewing PDF files within the browser window.