Determining Local Conformations and Conformational Disorder at and Near Single-Stranded (Ss) and Double-Stranded (Ds) DNA Forks and Junctions

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. The local conformations of the sugar-phosphate backbones near the replication fork junction are likely recognized by protein components that form a stable helicase-primase sub-assembly during DNA replication. Moreover, the presence of local backbone disorder 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. The exploration of local DNA conformations can be examined by site-specifically inserting cyanine (Cy3) dimers within the sugar-phosphate backbones of DNA constructs. Spectroscopic characterization of these Cy3 dimer labeled DNA constructs can provide detailed, site-specific information about the local conformations and disorder of the sugar-phosphate backbones at the Cy3 labeling site positions. By fitting a theoretical model to experimental absorbance and circular dichroism (CD) spectra, we determined the ensemble average conformations (relative orientation and distances) of Cy3 dimer probes within the DNA constructs as a function of insertion site position and temperature. We compared the results of our analyses 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, we used two-dimensional fluorescence spectroscopy (2DFS), which allowed us to characterize local conformational disorder at the Cy3 labeling sites. The presence of local disorder at ss-ds DNA junctions suggests that these sites undergo rapid interconversion between different conformations. Single-molecule polarization-sweep experiments were used to obtain detailed molecular information about the mechanisms of these conformational transitions.