Single-molecule FRET and linear dichroism studies of DNA breathing and helicase binding at replication fork junctions

• DOI: 10.1073/pnas.1314862110
• Publication Date: 2013-09-24T05:25:39Z
• Authors: Carey Phelps; Wonbae Lee; Davis Jose; Peter H. von Hippel; Andrew H. Marcus
• Keywords: Single-molecule FRET; Replisome

DNA "breathing" is a thermally driven process in which base-paired sequences transiently adopt local conformations that depart from their most stable structures. Polymerases and other proteins of genome expression require access to single-stranded coding templates located the double-stranded "interior," it likely fluctuations sugar-phosphate backbones dsDNA result mechanistically useful base pair opening reactions can be exploited by such regulatory proteins. Such motions are difficult observe bulk measurements, both because they infrequent often occur on microsecond time scales not easy experimentally. We report single-molecule fluorescence experiments with polarized light, tens-of-microseconds rotational internally labeled iCy3/iCy5 donor-acceptor Förster resonance energy transfer fluorophore pairs have been rigidly inserted into replication fork constructs simultaneously detected using fluorescence-detected linear dichroism signals. Our results reveal significant ∼100-μs range, reasonable scale for breathing potential relevance DNA-protein interactions. Moreover, we show magnitudes relaxation times these backbone significantly perturbed interactions construct nonprocessive, weakly binding bacteriophage T4-coded helicase hexamer initiation complex, suggesting may play fundamental role initial binding, assembly, function processive helicase-primase (primosome) component complex.