Biochemical, Computational, and Functional Characterization of Zf-GRF-Containing Proteins: From Nucleic Acid Binding to Inhibitor Discovery

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Candidate Name: Joshua Matos Program: Biology Committee Chair: Dr. Shan Yan Committee Members: Dr. Ian Marriot, Dr. Kausik Chakrabarti, Dr. Junya Tomida, and Dr. Jun-Tao Guo

Abstract:Nucleic acid recognition by accessory domains contributes to the regulation of genome maintenance proteins. This dissertation examines the zinc finger–glycine-arginine-phenylalanine (Zf-GRF) domain through comparative analysis, NEIL3 binding experiments, predicted interface analysis, and screening for compounds that perturb APE2–DNA interactions. Sequence alignments and phylogenetic analysis of 70 Zf-GRF domain sequences from eight vertebrate species identify a conserved framework alongside variation associated with protein identity and zinc finger position. Electrophoretic mobility shift assays with wild-type and mutant Xenopus laevis NEIL3 tandem Zf-GRF motifs reveal substrate-dependent contributions of selected aromatic and basic residues. Substitutions reduce apparent ssDNA binding, while R-loop binding is substantially retained at higher protein concentrations; the basic-residue mutants display distinct responses with ssRNA, and the double aromatic-residue mutant shows lower ssRNA binding than either single mutant. Computational analysis of 120 predicted NEIL3-Nucleic Acids complexes extends these observations by identifying changes in contact organization and the relative contributions of the two Zf-GRF motifs across substrates. High-throughput Screening 2,459 compounds identifies anthracyclines and anthracenediones for APE2 follow-up experiments. Doxorubicin, idarubicin, daunorubicin, and mitoxantrone reduce APE2–ssDNA binding and exonuclease processing in biochemical assays. Zf-GRF deletion reduces APE2's DNA binding and exonuclease processing. Exploratory clinical analyses identify survival associations involving recorded Topoisomerase II inhibitor treatment and BRCA1/2 mutation status but an APE2-dependent therapeutic mechanism of action warrants further investigations. Together, these findings support substrate-dependent Zf-GRF contributions to nucleic acid engagement and identify biochemical perturbations for mechanistic investigation.