Local Structure and Conformational Disorder at Single-Strand--Double-Strand DNA Junctions
Date
2023-03-24
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University of Oregon
Abstract
DNA functions as a stable repository for heritable information across generations. However, the structure of DNA within the cell must be dynamic, allowing for thermally induced fluctuations to facilitate the recognition and assembly of functional protein-DNA complexes. For example, the local conformations of the sugar-phosphate backbones near the replication fork junction are likely recognized by protein components during DNA replication. The presence of local backbone disorder (i.e., the absence of an ordered conformation) within duplex and ss-ds DNA junctions indicates a distribution of local backbone conformations that could, for example, facilitate kinetic competition between distinct protein regulatory factors. By fitting a theoretical model to experimental absorbance and circular dichroism (CD) spectra, the ensemble average conformations (relative orientation and distances) of Cy3 dimer probes within the DNA constructs was determined as a function of insertion site position and temperature. The results of our analyses were subsequently compared using increasingly complex models of exciton coupling between individual Cy3 labeling sites. To investigate local conformational disorder of the sugar-phosphate backbones as a function of temperature and proximity to protein binding sites, two-dimensional fluorescence spectroscopy (2DFS) was used, which allowed for direct characterization of the local conformational disorder at the Cy3 labeling sites. The presence of local disorder at and near ss-ds DNA junctions suggests that these sites undergo rapid interconversion between different conformations, which were studied under varying DNA composition and buffer conditions, and with a novel technique, single molecule polarization sweep microscopy. The effect of assembling T4 bacteriophage helicase loading (gp59) and single strand binding protein (gp32) on the DNA ds-ss junction was examined and some of these conformations can function as secondary-structure motifs for interaction with protein complexes that bind to and assemble at these sites. This dissertation includes previously published and unpublished co-authored material.