DNA replication and the related processes of genome expression require binding, assembly, and function of protein complexes at and near single-stranded (ss)–double-stranded (ds) DNA junctions. These central protein–DNA interactions are likely influenced by thermally induced conformational fluctuations of the DNA scaffold across an unknown distribution of functionally relevant states to provide regulatory proteins access to properly conformed DNA binding sites. Thus, characterizing the nature of conformational fluctuations and the associated structural disorder at ss–dsDNA junctions is critical for understanding the molecular mechanisms of these central biological processes. Here, we describe spectroscopic studies of model ss–dsDNA fork constructs that contain dimers of “internally labeled” cyanine (iCy3) chromophore probes that have been rigidly inserted within the sugar–phosphate backbones of the DNA strands. Our combined analyses of absorbance, circular dichroism, and two-dimensional fluorescence spectroscopy permit us to characterize the local conformational parameters and conformational distributions. We find that the DNA sugar–phosphate backbones undergo abrupt successive changes in their local conformations—initially from a right-handed and ordered DNA state to a disordered splayed-open structure and then to a disordered left-handed conformation—as the dimer probes are moved across the ss–dsDNA junction. Our results suggest that the sugar–phosphate backbones at and near ss–dsDNA junctions adopt specific position-dependent local conformations and exhibit varying extents of conformational disorder that deviate widely from the Watson–Crick structure. We suggest that some of these conformations can function as secondary-structure motifs for interaction with protein complexes that bind to and assemble at these sites.

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