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 single-molecule Förster resonance energy transfer (smFRET) and fluorescence detected linear dichroism (smFLD) measurements performed on cyanine labeled DNA fork constructs. These constructs contain a pair of Cy3 chromophores that are rigidly positioned within the opposing sugar-phosphate backbones near the single-stranded (ss) – double-stranded (ds) junction of the DNA fork. The Cy3 dimer supports polarized collective excitations that depend sensitively on the fluctuating local conformations of the sugar-phosphate backbone near the fork junction, and which can be probed by smFLD. The Cy3 dimer can act as a FRET donor to a Cy5 acceptor, which is attached to the end of a short (15 nucleotide) segment of ssDNA attached to the DNA fork construct. These measurements simultaneously probe short-range (5 – 7 Å) distances between Cy3 monomers, and long-range (tens-of-angstroms) distances between donor-acceptor pairs, and thus permit us to track sub-millisecond breathing processes near the fork junction, and the stochastic inter-conversion between various protein-bound and unbound states.

Sub-millisecond single-molecule fluorescence studies of electronically-coupled cyanine dimers in model DNA replication fork constructs

2019 Spring National Meeting

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