Visualizing protein-DNA interactions at the single-molecule level

Abstract

Recent advancements in single-molecule methods have allowed researchers to directly observe proteins acting on their DNA targets in real-time. Single-molecule imaging of protein-DNA interactions permits detection of the dynamic behavior of individual complexes that otherwise would be obscured in ensemble experiments. The kinetics of these processes can be monitored directly, permitting identification of unique subpopulations or novel reaction intermediates. Innovative techniques have been developed to isolate and manipulate individual DNA or protein molecules, and to visualize their interactions. The actions of proteins that have been visualized include: duplex DNA unwinding, DNA degradation, DNA packaging, translocation on DNA, sliding, superhelical twisting, and DNA bending, extension, and condensation. These single-molecule studies have provided new insights into nearly all aspects of DNA metabolism. Here we focus primarily on recent advances in fluorescence imaging and mechanical detection of individual protein-DNA complexes, with emphasis on selected proteins involved in DNA recombination: DNA helicases, DNA translocases, and DNA strand exchange proteins.

Figure 1. Schematic representations of methods for manipulating DNA at the single-molecule level

(A) Single optical tweezer. Biotinylated DNA is attached to a μm-sized, streptavidin-coated, polystyrene bead. The DNA-bead complex is captured by the optical trap. DNA can be visualized by bound fluorescent dye. (B) Dual optical tweezers. DNA is attached at both ends to beads. Tension on the DNA can be created by either moving the traps apart, or protein-dependent changes in DNA conformation. (C) Magnetic tweezer. DNA is tethered at one end to a glass slide and the other end is bound to a paramagnetic bead. Magnets can be moved in the vertical direction to control DNA tension or rotated in the horizontal plane to change superhelicity. (D) Surface attachment and TIRFM. DNA can be tethered to a surface and extended by buffer flow. An array of hundreds of DNA molecules can be created to form “DNA curtains”. The fluorescent DNA or bound proteins are visualized by TIRFM.

Figure 2. Visualization of DNA unwinding and χ-recognition by a single molecule of the RecBCD helicase/nuclease

Translocation on ssDNA and resultant unwinding of dsDNA by RecBCD can be monitored by either the displacement of YOYO-1 from the DNA or by labeling the enzyme with a fluorescent nanoparticle (not shown). The RecBCD-DNA-bead complex is captured by an optical trap, and translocation is initiated by movement to an ATP-containing buffer channel in a multi-channel flow cell. Single molecule visualization can detect a brief pause by the enzyme at the χ sequence followed by a resumption of translocation at approximately one-half of the rate. Single-stranded DNA loops are created before and after χ recognition due to the different translocation velocities of the RecB and RecD subunits. Adapted from [16].

Figure 3. Visualization of translocation on duplex DNA by a single Rad54/Rdh54

(A) Still frames and graphs of upstream and downstream translocation by Rad54 on single dsDNA molecules. Rad54 is fluorescently tagged with a fluorescein-labeled antibody. The Rad54-DNA-bead complex is captured by a single optical trap and translocation is initiated by movement to an ATP-containing buffer channel in a multichannel flow cell. Reprinted from [23] with permission from Elsevier. (B) Diagram of DNA surface attachment used to visualize Rdh54 translocation by TIRFM. Translocation of Rdh54 multimers on DNA can extrude DNA loops. Adapted from [25] with permission from Elsevier.

Figure 4. Direct observation of individual RecA/Rad51 filament dynamics by single-molecule fluorescence

(A) Assembly of fluorescent RecA (RecA-FAM) protein onto a single dsDNA molecule. DNA length increases by ∼60%. Data points and still frames from time-dependent “dipping” in a flow channel containing fluorescent RecA show nucleation followed by extensive growth. Reprinted by permission from Macmillan Publishers Ltd: Nature, [34], 2006. (B) Single-molecule FRET assay of RecA binding to ssDNA. The high FRET state exists in the absence of RecA, when the FRET-pair fluorophores are in close proximity. The low FRET state occurs upon RecA binding and DNA extension when the FRET pair is beyond their Förster distance. Reprinted from [35] with permission from Elsevier. (C) Rad51 binding to a single dsDNA molecule which is fluorescently labeled at one end. Filament formation extends the DNA by 65% as seen by the movement of the fluorescent end-label, Cy3. Adapted from [44]. (D) Dissociation of fluorescent Rad51 filaments (Rad51-AF555) from DNA held in a dual optical trap. ATP hydrolysis by Rad51 creates tension which stalls the disassembly process. Disassembly is resumed upon tension release. Reprinted by permission from Macmillan Publishers Ltd: Nature, 2009 [45].