ORCID ID
Graduation Date
Fall 12-19-2025
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Programs
Immunology, Pathology & Infectious Disease
First Advisor
Gloria Borgstahl
Abstract
Replication protein A (RPA) is the major human single-stranded DNA-binding protein essential for DNA replication, recombination, and repair. As a flexible heterotrimer composed of RPA70, RPA32, and RPA14 subunits, RPA engages a broad range of DNA and protein partners and is extensively regulated by post-translational modifications, particularly phosphorylation. Although RPA has been studied for decades, a complete structural understanding of how phosphorylation modulates its dynamic architecture and functions remains incomplete. In this work, we investigated how G2-phase cell-specific phosphorylation and the DNA damage response (DDR) associated with double-strand breaks (DSBs) influence RPA structure, single-stranded DNA (ssDNA) binding, and protein-protein interactions, all of which are central to the DDR. Eleven candidate serine and threonine phosphorylation sites across RPA70 and RPA32 were mutated to glutamic acid to mimic phosphorylation, and seventeen engineered phosphomimetics were evaluated for expression and stability. Five phosphomimetic proteins were successfully purified as stable heterotrimers and used to assess how phosphorylation affects RPA’s affinity for ssDNA and RAD52, as well as its capacity to transfer ssDNA to RAD52 during homologous recombination.
To determine the structural consequences of phosphorylation, we integrated multiple biophysical approaches – including thermal denaturation, circular dichroism, small-angle X-ray scattering (SAXS), and X-ray footprinting mass spectrometry (XFMS) – to characterize each phosphomimetic in the absence and presence of ssDNA. All phosphomimetics exhibited decreased disorder and increased compaction upon ssDNA binding, as revealed by low-resolution SAXS electron density, which showed a denser, more compact molecular envelope in the presence of ssDNA. XFMS further identified residue-specific protection patterns throughout the protein, including previously unrecognized ssDNA-induced protection within RPA14 and the F domain on RPA70, suggesting a direct role for this subunit in DNA engagement and structural stabilization. Together, these findings demonstrate that phosphorylation measurably alters RPA’s structural integrity and regulatory behavior while preserving its global compaction response to ssDNA. This work advances our understanding of how RPA phosphorylation modulates DNA binding and repair functions and provides new structural insights – including an unexpected contribution from RPA14 and RPA70F – that may inform future mechanistic studies and therapeutic targeting of RPA-mediated DNA repair pathways.
Recommended Citation
Fousek-Schuller, Valerie J., "Phosphorylation Modulates the Structure and Function of Replication Protein A" (2025). Theses & Dissertations. 1015.
https://digitalcommons.unmc.edu/etd/1015
Included in
Amino Acids, Peptides, and Proteins Commons, Biophysics Commons, Molecular Biology Commons, Nucleic Acids, Nucleotides, and Nucleosides Commons, Structural Biology Commons
Comments
2025 Copyright, the authors