Single-stranded DNA (ssDNA) is an essential intermediate in various DNA metabolic processes and interacts with a large number of proteins. Due to its flexibility, the conformations of ssDNA in solution can only be described using statistical approaches, such as flexibly jointed or worm-like chain models. However, there is limited data available to assess such models quantitatively, especially for describing the flexibility of short ssDNA and RNA. To address this issue, we performed FRET studies of a series of oligodeoxythymidylates, (dT)N, over a wide range of salt concentrations and chain lengths (10 < or = N < or = 70 nucleotides), which provide systematic constraints for testing theoretical models. Unlike in mechanical studies where available ssDNA conformations are averaged out during the time it takes to perform measurements, fluorescence lifetimes may act here as an internal clock that influences fluorescence signals depending on how fast the ssDNA conformations fluctuate. A reasonably good agreement could be obtained between our data and the worm-like chain model provided that limited relaxations of the ssDNA conformations occur within the fluorescence lifetime of the donor. The persistence length thus estimated ranges from 1.5 nm in 2 M NaCl to 3 nm in 25 mM NaCl.

Photobleaching and blinking of fluorophores pose fundamental limitations on the information content of single-molecule fluorescence measurements. Photoinduced blinking of Cy5 has hampered many previous investigations using this popular fluorophore. Here we show that Trolox in combination with the enzymatic oxygen-scavenging system eliminates Cy5 blinking, dramatically reduces photobleaching and improves the signal linearity at high excitation rates, significantly extending the applicability of single-molecule fluorescence techniques.

Helicases are motor proteins that couple conformational changes induced by ATP binding and hydrolysis with unwinding of duplex nucleic acid, and are involved in several human diseases. Some function as hexameric rings, but the functional form of non-hexameric helicases has been debated. Here we use a combination of a surface immobilization scheme and single-molecule fluorescence assays--which do not interfere with biological activity--to probe DNA unwinding by the Escherichia coli Rep helicase. Our studies indicate that a Rep monomer uses ATP hydrolysis to move toward the junction between single-stranded and double-stranded DNA but then displays conformational fluctuations that do not lead to DNA unwinding. DNA unwinding initiates only if a functional helicase is formed via additional protein binding. Partial dissociation of the functional complex during unwinding results in interruptions ('stalls') that lead either to duplex rewinding upon complete dissociation of the complex, or to re-initiation of unwinding upon re-formation of the functional helicase. These results suggest that the low unwinding processivity observed in vitro for Rep is due to the relative instability of the functional complex. We expect that these techniques will be useful for dynamic studies of other helicases and protein-DNA interactions.

RecA and its homologs help maintain genomic integrity through recombination. Using single-molecule fluorescence assays and hidden Markov modeling, we show the most direct evidence that a RecA filament grows and shrinks primarily one monomer at a time and only at the extremities. Both ends grow and shrink, contrary to expectation, but a higher binding rate at one end is responsible for directional filament growth. Quantitative rate determination also provides insights into how RecA might control DNA accessibility in vivo. We find that about five monomers are sufficient for filament nucleation. Although ordinarily single-stranded DNA binding protein (SSB) prevents filament nucleation, single RecA monomers can easily be added to an existing filament and displace SSB from DNA at the rate of filament extension. This supports the proposal for a passive role of RecA-loading machineries in SSB removal.