Study and Visualize DNA Replication Mechanisms at the Nanoscale

Force extension, manipulation, and visualization of DNA-protein interactions in DNA replication

Here, a single double stranded DNA (dsDNA) molecule is tethered between two beads and incubated with Sytox Orange, a dsDNA fluorescent marker, and fluorescently labeled replication protein A (RPA), an ssDNA-binding replication protein.

Figure 1 shows a dsDNA molecule being stretched with a constant velocity of 140 nm/s while at the same time force, distance and fluorescence signals are continuously recorded. By overlapping all data sets, we can see that as the end-to-end distance (grey) increases, Sytox molecules (green) bind between the basepairs of dsDNA. Stretching the DNA even further initiates DNA melting and the formation of single-stranded DNA (ssDNA), which can be seen both globally by a drop in the force (red), and locally by the visualization of the ssDNA binding protein RPA (blue).

As soon as RPA begins binding we can observe a drop in the force signal, indicating the stabilization of melted DNA, which – being coated by RPA – cannot be reannealed with its complementary strand to form dsDNA again. This keeps DNA unwound for the polymerase to replicate it in the subsequent stages of the replication process. Finally, relaxing the molecule back to less than 25 pN results in Sytox Orange dissociation while RPA remained bound.

1 Dual-color fluorescence kymograph corresponding to the extension and retraction of a single dsDNA. End-to-end distance (grey) and force (red) data sets are overlapped to the fluorescent image showing the true correlation of the data sets.

Investigation of the activity and states of DNA replication motor proteins

Here, a DNA molecule is caught and tethered between a bead and a single DNA polymerase (DNAp) replicating it. We can calculate the DNAp activity as it replicates the DNA, by stretching the DNA strand with a constant force and measuring the distance between the beads.

Figure 2 shows measured data of the activity of T7 DNA polymerase. This protein participates in DNA replication and has 3’ to 5’ exonuclease activity, which is enhanced in the presence of force. Optical tweezers hold the DNA construct (8.3 kbp), tethered between two beads (ø=1.86 μm) at a constant force of 45 pN to observe force-induced exonucleolysis at the single-molecule level. Short activity bursts ranging between 3 and 10 nucleotides are revealed, interspersed by frequent pauses of varying duration.

Single-molecule measurements of stepping behavior of biomolecular motors along nucleic acids will supply important new information about their enzymatic mechanisms. Next to this experiment, the multicolor fluorescence detection allows quantifying the FRET efficiency changes in time and enables correlating a specific conformational change with activity bursts of DNAp.

2 Activity bursts of DNA polymerase performing force-induced exonucleolysis on an ssDNA.

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 a 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 3 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.

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