Force extension and manipulation of DNA-protein interactions
Here, using optical tweezers, a single dsDNA molecule is caught and tethered between two optically trapped beads. Next, the molecule is coated with RecA – a class of repair proteins that form helical filaments around DNA. We can calculate the mechanical properties of the DNA and investigate how RecA affects its properties, by stretching the molecule while measuring the force and extension.
Figure 1 shows the force-distance curve of a dsDNA molecule, shown before and after being coated with RecA. The observed shift of the curve is caused by an increased stiffness of the DNA molecule due to the formation of RecA filaments. Less force is necessary to unravel the DNA-RecA complex as the filaments prevent it to coil.
Combining the experiment with simultaneous fluorescence measurements allows correlating the mechanical properties of the DNA with the binding location and quantity of DNA repair proteins.
1 Force-distance curves of DNA in the absence (left) and presence (right) of RecA.
Visualization of DNA-protein interactions
In this experiment, a DNA molecule is tethered between two beads while multiple fluorescently labeled proteins are interacting with it. We can visualize these interactions and track them over time using multicolor confocal or STED fluorescence microscopy. The resulting kymograph unveils the number, position, diffusion and (un)binding events of the proteins along the DNA.
The kymograph in Figure 2 shows the position of bound XRCC4 and XLF on DNA over time at protein concentrations of 5 nM. These are two repair proteins involved in non-homologous end joining which can associate with each other to form complexes capable of bridging DNA. From the figure we can observe and quantify the dynamics (N=94 events) of XRCC4 (green, 9%), XLF (red, 62%) and XRCC4-XLF complexes (yellow, 29%).
The kymograph gives real-time insights in the DNA-protein interactions and protein-protein interactions involved in DNA repair. Simultaneous force and extension measurements allow correlating the protein activity and binding kinetics with the mechanical properties of the protein-DNA complex.
2 Kymograph showing the dynamics of XRCC4 (green), XLF (red), and XRCC4-XLF complexes (yellow) on DNA.
Force extension, manipulation and visualization of DNA-protein-DNA interactions
Here we use a quadruple trap configuration to trap beads and catch two DNA molecules in between. The two DNA molecules are held in close proximity in the presence of DNA bridging proteins. This allows for the study of complex DNA interactions involving multiple DNA molecules.
Figure 3 shows an example in which two DNA molecules are trapped using four optical traps and incubated with 200 nM of XRCC4 and 200 nM of XLF. As we increase the distance between the two trap pairs, we can observe the formation of protein bridges (orange), consisting of both XRCC4 (green) and XLF (red).
We can further manipulate the beads with force to further validate bridge stability and study the behavior of proteins under tension. In addition, by pulling on one bead, we can disrupt the bridges in a controlled manner resulting in a stepwise length (L) increase between the upper and lower beads (figure 4). In the figure, the length increases shown are the result of disrupting DNA bridges by pulling on one side of the beads.
3 Two DNA molecules trapped using four optical traps. DNA bridging proteins XRCC4 (green) and XLF (red) can be seen both individually and as a DNA bridging complex (orange).
4 Stepwise length increases between the upper and lower beads in a quadruple trap configuration, comprising of two DNA molecules and multiple DNA-bridging proteins.