DNA ‘breathing’ is a thermally driven process in which nucleotide residues near single-strand (ss) - double-strand (ds) DNA forks and junctions temporarily adopt local conformations that depart from their most stable structures. It is thought that the transient occurrence of these ‘open’ conformations is centrally involved in the proper function of DNA-protein complexes responsible for replication, transcription, and many other reactions that involve the manipulation of the DNA genome. Such motions are difficult to observe in bulk measurements because they likely occur in the microsecond regime, which is an especially difficult timescale to access experimentally. In this work we report polarized light single-molecule fluorescence experiments in which microsecond rotational motions of the sugar-phosphate backbone are directly observed. These experiments simultaneously detect single molecule FRET and linear dichroism (FLD) signals from dsDNA replication fork constructs containing Cy3 - Cy5 FRET donor-acceptor probes strategically located near the replication fork junction on opposite strands. Our results show that significant local backbone motions are manifested on a ∼100 microsecond timescale, which is a reasonable range for at least some types of DNA ‘breathing’ fluctuations. In addition we have shown that these motions can be perturbed by the presence of the T4 gp41 hexameric helicase.