Site-specific free energy surface parameters from single-molecule fluorescence measurements of exciton-coupled (iCy3)2 dimer probes positioned at DNA replication fork junctions

Abstract

Single-stranded (ss) - double-stranded (ds) DNA replication forks and primer-template junctions are important recognition sites for the assembly and function of proteins involved in DNA replication, recombination and repair. DNA breathing - i.e., thermally-induced local fluctuations of the sugar-phosphate backbones and bases - can populate metastable conformational macrostates at positions near such junctions and likely play key roles in the functional interactions of the regulatory proteins that bind at these sites. Recently, Maurer et al. (1) performed polarization-sweep single-molecule fluorescence (PS-SMF) studies on exciton-coupled (iCy3)2 dimer-labeled ss-dsDNA fork constructs, which revealed that the nucleobases and backbones immediately adjacent to the dimer probes undergo conformational fluctuations on time scales ranging from hundreds-of-microseconds to hundreds-of-milliseconds. The local conformations sensed by the dimer probes consist of four quasi-stable macrostates whose populations and dynamics depend on dimer probe position relative to these junctions. Here we present theoretical analyses of these PS-SMF data that quantify the relative stabilities and activation barriers of the free energy surfaces (FESs) of site-specific DNA breathing events at key positions within these junctions. Our results suggest a detailed molecular picture for DNA breathing at these positions, thus providing insights into understanding the molecular mechanisms of the proteins that operate at these sites.