Study and Visualize DNA Repair Mechanisms at the Nanoscale

Part of: DNA Repair

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.

C-Trap DNA Repair Puller

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.

C-Trap DNA Replication Full

1 Force-distance curves of DNA in the absence (left) and presence (right) of RecA.

Brouwer et al. (2017)
Cell Reports

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.

C-Trap DNA Repair

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.

C-Trap STED Multi-color

Kymograph showing the dynamics of XRCC4 (green), XLF (red), and XRCC4-XLF complexes (yellow) on DNA.

Data courtesy of Prof. Erwin Peterman and Prof. Gijs Wuite at the VU University Amsterdam.

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.

C-Trap Q-Trap

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.

C-Trap Confocal Q-Trap

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).

C-Trap Example Data Q-Trap

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.

Data courtesy of Prof. Erwin Peterman and Prof. Gijs Wuite at the VU University Amsterdam.

Brouwer et al. (2016)
Nature

Solutions

C-Trap™

Optical Tweezers and Fluorescence Microscopy

The C-Trap™ is the world’s first instrument that allows simultaneous manipulation and visualization of molecular interactions in real-time. It combines high-resolution optical tweezers, confocal microscopy or STED nanoscopy with an advanced microfluidics system in a truly integrated and correlated solution.

m-Trap™

Optical Tweezers and Fluorescence Microscopy

The m-Trap™ is the first entry-level optical tweezers instrument specifically developed for high resolution single-molecule research. Ultra-high force resolution and stability, with incredible throughput, ease of use and modularity ‒ all at an unprecedented price level.

Key Product FeaturesC-Trap™m-Trap™
Visualization of binding position and diffusion of proteins
co-Localization studies
DNA-protein-DNA interactions using two DNA strands
Enzymatic activity that alters DNA/RNA mechanics (e.g. exonucleases, DNAp, helicases, SSB)
Rapid buffer exchange for fast experimental workflow
Single-molecule FRET studies

Want to learn more?

Would you like to receive exclusive news on the latest products, single-molecule events and breakthrough science from us?


You can unsubscribe at any time from our marketing emails. By submitting the form you agree to LUMICKS' privacy policy.

Take your research to the next level.

Get exclusive news on the latest products, single-molecule events and breakthrough science.

Newsletter pop up
By clicking the subscribe button you agree to LUMICKS’ privacy policy.