Development of biological structural tools for the study of protein-DNA complexes

PROJECT SUMMARY/ABSTRACT Interactions between proteins and DNA control several fundamental biological processes, including gene regulation, DNA repair, replication, transcription, translation, and recombination. The PI’s previous research in instrument design, biological mass spectrometry, ion mobility spectrometry, and structural biology have enabled the study of the intra- and inter- molecular interactions and conformational landscape that drive protein, DNA, protein-protein and protein-DNA interactions in several model systems. The goal of this project is to advance mass spectrometry (MS) based techniques with complementary structural tools to better describe biomolecular complexes in their native solution environment. Coupling solution- and gas- phase separations (e.g., accessible solvent area labeling, ion-neutral reactions, and trapped ion mobility spectrometry), new dissociation methods (e.g., electron- and UV- based methods) with new developments in ultra-high, resolution MS will provide new structural biology tools capable of dissecting the molecular complexity and energy landscape of protein isoforms and protein-DNA complexes. These advanced MS tools will be used for the characterization of model systems of the non-histone chromosomal HMG protein family (e.g., high mobility group AT-hook 2 protein-HMGA2), type IA topoisomerase (TOP1) family and nucleosome dynamics (e.g., interplay of histone composition and histone post-translational modifications, PTMs). We will study HMGA2 transitions from unstructured to structured that allow HMGA2 to be involved in multiple biological processes, including DNA replication, translation, recombination, and gene regulation. We will capture the structural intermediates of the TOP1-DNA complexes associated with the catalytic cycle to search for new antibiotic candidates. We will study the influence of histone variants and PTMs in the nucleosome dynamics (e.g., partial, and full assemble); nucleosome dissociation and alteration of nucleosomal structure are crucial features by which chromatin regulates gene expression and DNA replication. The development of MS-based technologies capable of studying intrinsically disordered and dynamic biomolecular systems will significantly advance our understanding of the biological machinery associated with disease mechanisms, identification of therapeutic targets and disease prevention.