2D Fluorescence Spectroscopy is used to Probe Local Conformations and Conformational Disorder of the Sugar-Phosphate Backbones of DNA at and Near DNA Replication Fork Junctions

Abstract

DNA replication requires the binding of genome-regulatory proteins to non-sequence-specific positions near single-stranded (ss) - double-stranded (ds) DNA junctions. The local conformation of DNA near ss-dsDNA junctions must undergo fluctuations within an unknown distribution of functionally relevant states to permit the proper binding of replication proteins. As a consequence, the nature and extent of conformational disorder at DNA junctions may be critical for controlling the molecular mechanisms of this binding. DNA constructs that contain dimers of Cy3 chromophore probes rigidly incorporated into the sugar-phosphate backbones at defined positions near ss-dsDNA fork junctions have been studied using absorption, circular dichroism (CD) and two-dimensional fluorescence spectroscopic (2DFS) techniques to determine local backbone conformations and the degree of disorder experienced by these dimer probes. Analysis of these data provides insight into position- and temperature-dependent conformational heterogeneity at various sites within these DNA constructs, and suggests that quantifiably different energetic barriers to conformational exploration exist at the ss-ds junction when compared to DNA base pairs located deeper within the duplex DNA. We have employed an optimization routine to fit linear and nonlinear spectra in order to characterize structural and disorder parameters associated with conformations at and near ss-dsDNA junctions that are stabilized by the binding of relevant proteins, as well as to examine structural distortions induced by DNA-associated small molecules and ions.