DNA replication is a core biological process, which occurs in prokaryotic cells at high speeds (~ 1 nucleotide residue added per millisecond) and with high fidelity (fewer than 1 misincorporation event per 10<sup>7</sup> nucleotide additions). The various sub-assemblies of the T4 bacteriophage DNA replication complex may serve as model systems to study the underlying mechanisms of protein-nucleic acid binding, reposition, and dissociation. I will discuss microsecond-resolved single-molecule fluorescence measurements performed on probe-labeled model DNA fork constructs, which contain a pair of Cy3 chromophores incorporated into the sugar-phosphate backbones at various positions relative to the single-stranded (ss) – double-stranded (ds) DNA fork junction. The Cy3 dimer supports polarized excitons that depend sensitively on the local conformations of the sugar-phosphate backbones near the DNA fork junction, and which can be probed by polarization selective (cw excitation) single-molecule fluorescence, and in separate experiments by spectrally selective (pulsed excitation) experiments. We show that this approach can be used to study the local conformations and conformational fluctuations of DNA fork junctions, and how these motions might influence the assembly of replication protein-DNA complexes.

Polarization and spectrally selective single-molecule studies of excitonically-coupled cyanine dimers in model DNA replication fork constructs

ACS Fall 2021

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