Graduation Date

Spring 5-5-2018

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Biochemistry & Molecular Biology

First Advisor

Dr. Gloria E.O. Borgstahl

Abstract

Human cells are routinely exposed to DNA-damaging conditions, from both external sources like ionizing radiation and internal sources like normal oxidative metabolism. Damage in the form of double strand breaks (DSBs) is especially problematic. DSBs occurring outside of replication forks can be repaired through two forms of homologous recombination. The first of these is genetic conversion involving either RPA, BRCA1, PALB2, BRCA2, and RAD51, or RPA, RAD52, RAD51, and other unknown factors. The second is single strand annealing involving RPA and RAD52. Familial breast cancers, among numerous others, are characterized by homozygous pathological mutations in the BRCA2 pathway and must therefore rely on the RAD52 pathway for remediation of DSBs. Inhibiting the interaction between RPA and RAD52 should therefore selectively terminate such cancer cells without harming healthy cells. Structural information regarding RAD52 and the phosphorylation state of RPA during active DNA repair must be elucidated to achieve this. Initial structural data for RAD52 was acquired using small angle X-ray scattering (SAXS). Using the available RAD52(1- 212) crystal structure we were able to estimate the orientation of the RAD52(1-303) SAXS structure. The application ITASSER allowed for the modeling of one of the RAD52(213-303) sections which is outside of the RAD52(1-212) crystal structure, and which includes the RPA binding domain. Utilizing available information about known DSB-induced phosphorylation sites of RPA, paired with data from Phosida and PhosphositePlus, eleven candidate sites were selected for structural and DNA binding studies. Phosphorylation was mimicked by mutation of candidate sites to glutamic acid, and 6 of the combinations tested retained heterotrimer stability. Phosphomimetic mutations to the RPA70 subunit decreased DNA binding affinity. Identification of these stable phosphomimetics with confirmed DNA binding activity provides tools for experiments delving into the activities of RPA functioning in BIRDSB repair, these tools will also be used for structural experiments involving the binding of RPA to DSB repair proteins, including the SAXS compatible RAD52(1-303).

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