Thermal fluctuations of DNA play an important role in the regulation of DNA replication, recombination and repair, which depends on the ability of protein machinery to recognize and to bind to selected conformations of DNA lattices. Obtaining information about the nature of these functionally relevant DNA conformations and their time scales of interconversion is critical for understanding the detailed mechanisms of protein-DNA interactions. In this talk, I will discuss experiments to measure the local conformations, conformational disorder, and dynamics of probe-labeled DNA.

The first of these studies examines the local backbone fluctuations of single-stranded (ss) oligo-deoxythymidine o(dT)15 templates using single-molecule Fӧrster Resonance Energy Transfer (smFRET). The smFRET signal depends on the distance between Cy3 and Cy5 probes, which label the distal ends of the o(dT)15 ‘tail’ region of a primer-template (p/t) DNA construct. From microsecond-resolved measurements of the photon data stream, we determine two-point and four-point time-correlation functions (TCFs) and the equilibrium probability distribution of FRET efficiencies. Our results indicate that the o(dT)15 tail of a p/t DNA construct interconverts on sub-millisecond time scales between three macrostates with distinctly different end-to-end distances: a ‘compact’ macrostate that represents the dominant species at equilibrium, a ‘partially extended’ macrostate that exists as a minority species, and a ‘highly extended’ macrostate that is present in trace amounts. We modeled our data using a kinetic master equation to obtain information about the salient features of the free energy surface.

I will also discuss ensemble and single-molecule fluorescence experiments on model DNA fork constructs, which contain a pair of Cy3 chromophores incorporated into the sugar-phosphate backbones at various positions relative to the ss – double-stranded (ds) DNA fork junction. The Cy3 dimer supports polarized excitons that depend sensitively on the local conformations of the labeled backbones near the DNA fork junction. Analysis of absorption and circular dichroism (CD) spectra determine the local conformations, while two-dimensional fluorescence spectroscopy (2DFS) determines the conformational disorder at the probe labeling sites. Single-molecule experiments, in which the polarization of the source laser is swept and the signal stream is phase-tagged, monitor backbone fluctuations at the DNA fork junction.