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Study and visualize DNA organization mechanisms at the nanoscale

Part of: DNA organization

Study and visualize DNA organization mechanisms at the nanoscale

C-Trap DNA Organization

Kinetochore-mediated chromosome segregation

In this experiment, a microtubule was immobilized to a biotinylated coverslip, and Ndc80 complexes, a key protein complex in the kinetochore structure, were attached to streptavidin-coated beads. By moving the bead with the C-Trap, we generated a rupture force between the microtubule and the kinetochore structure.

Researchers from David Barford’s team quantitatively assessed the outer kinetochore’s force resistance of the Dam1 and Ndc80 complexes. By comparing rupture forces of various mutations, they get a better understanding of which protein domains influence the kinetochore’s ability to withstand microtubule forces during cell division.

Read more: Our blogpost
Read more: K. Muir et. al. (2023) Science

Observing mechano-chemical cycles of DNA packaging by φ29 motor with base pair resolution

Here, we tethered a φ29 DNA packaging motor and its dsDNA substrate between two optically trapped beads. Using optical tweezers we can perform pulling and equilibrium experiments to probe the structural dynamics and conformational changes during genome packaging.

As packaging proceeds, the DNA contour length remaining outside the capsid decreases over time. By measuring the end-to-end distance between the two traps we can observe individual mechanochemical cycles of this molecular machine (Figures 1 and 2).

The duration of each cycle, as well as the amount of DNA packaged during each of them, provide meaningful information regarding the operation of the motor. Further data analysis provides mechanistic information on the underlying biological function.

Sample courtesy of Prof. Dr. Carlos Bustamante at UC Berkeley.
Read more: Christol et al. (2012) Cell

1 Trace of DNA packaging trajectory recorded using a C-Trap. Raw data was collected at 78125 Hz (grey) and decimated to 250 Hz (black dots). The staircase is color-coded to indicate the different phases of the mechanochemical cycle (dwells in red, translocation events in blue).

2 Pairwise distribution function of the packaging trajectory. The analysis reveals the size of the φ29 motor power strokes, corresponding to ~2.5 bp, which can be clearly resolved when the molecule is under high tension (> 35 pN).

Measurement of conformational changes

Here, we use optical tweezers to catch and tether a DNA molecule, organized in nucleosomes via DNA-histone complexes. Using optical tweezers we can perform pulling and equilibrium experiments to probe the structural dynamics and conformational changes of DNA packaging complexes.

First, the DNA-histone complex is gradually extended while simultaneously measuring the force and extension. This results in the generation of a force-distance curve which provides information about the winding and unwinding of DNA around the histones with basepair resolution. Figure 3 shows that the force drops as the DNA unwinds from the histone complexes and that the DNA unwinds from the histone complexes consecutively.

In addition, we can follow and calculate the activity of DNA organization proteins over time. This is done by applying a constant force on the DNA while simultaneously measuring the distance between the two beads. Figure 4 shows the (un)winding of DNA around histones, measured by the shortening and lengthening bursts of the end-to-end distance between the two beads.

3 Force-distance curve showing DNA unwinding from histone complexes as it is being stretched using optical tweezers.

4 Winding and unwinding of DNA around histones, measured at a fixed trap distance.

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 6 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 7). In the figure, the length increases shown are the result of disrupting DNA bridges by pulling on one side of the beads.

6 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).

7 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.
Read more:
Brouwer et al. (2016) Nature


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