Connecting Structure with Kinetics of Single-Stranded DNA Fluctuations that Provide Potential Binding Sites for Replication Proteins

Abstract

The local conformations of DNA at and near replication forks and primer-template (p/t) junctions likely regulate the non-sequence-specific binding of replication proteins. Thermal fluctuations can drive the exploration of local DNA conformations, thereby providing opportunities for proteins to stably bind to transiently formed secondary structure motifs. In this work, we apply microsecond-resolved single-molecule Förster resonance energy transfer (sm FRET) experiments to study the equilibrium distribution and conformational transitions of the end-to-end distances of an oligo-deoxythymidine chain (dT)15 that serves as the single-stranded (ss) region of a Cy3-Cy5 labeled p/t DNA construct. By comparing our experimental data to the results of a kinetic network model, we determined that the system exists as three distinct macrostates with different end-to-end extensions, and that transitions between these macrostates occur over time scales ranging from ∼ 10 microseconds to ∼ 100 milliseconds. In addition, we measured the relative stabilities and transition barrier heights that characterize the free energy landscape of the (dT)15 p/t DNA system. The magnitudes of the activation barriers between macrostates of varying chain extension likely depend on the rates of rearrangement of the various populations of base stacked segments within the single-stranded portion of the p/t DNA construct. In ongoing work, we are investigating the physical nature of the transition barriers between macrostates in terms of local base-stacking interactions, using a combination of Monte Carlo simulation and spectroscopy of fluorescent base analogue substituted DNA constructs. Certain conformations within these macrostates are likely to represent targets for the binding of proteins, such as the single-stranded DNA binding protein (gp32) of bacteriophage T4, to ssDNA templates in the assembly and function of the replication complex.