In our lab, we are studying the molecular mechanisms of eukaryotic DNA replication. We want to understand the dynamics of genome duplication at the single molecule level. We visualize DNA replication using multiple systems, including purified proteins and extract derived from Xenopus eggs.
Much of what we know about DNA replication has come from bulk experiments, cell imaging, and structural biology. These methods are really powerful, but they're often limited in what they can tell us about the dynamics or transient behaviors of individual proteins. This is why single molecule techniques are really well suited to studying, and it's why their use is growing in the field.
Our protocol for live visualization of DNA unwinding outlines a platform that can be built upon and modified to investigate different aspects of DNA replication. It can provide a better understanding of the molecular mechanism of CMG activity in the presence of chosen ribosome components and replicative stress. To understand the complex systems at play during DNA replication, we isolate key proteins, and visualize their real-time behavior in vitro.
The insights we gain contribute to a more complete picture of DNA replication. To prepare the flow cell, cut the biotin-PEG cover slip in half. Prepare small glass pieces by etching and snapping a glass slide into approximately 2.4 centimeters X 1 centimeter pieces.
Using a 0.8 millimeter diamond coated drill bit, drill two holes, 1.4 millimeters apart in the glass pieces. Test the holes by inserting inlet or outlet tubing. Cut double-sided tape, the same shape as the glass pieces.
Align the slide on the tape, and stab a needle through each hole to mark their positions on the tape. Using a razor blade, cut a channel that encompasses both holes. Clean the glass piece with acetone.
Dry the glass piece with tissue paper and place it on a clean surface. Then peel off one side of the adhesive tape and stick it onto the glass piece so that both holes are fully inside the channel. Using a P1000 pipette tip, apply firm pressure on the tape to seal the tape to the glass piece.
Peel the second side of the tape off both glass pieces. Arrange the slides on a clean surface with the sticky side facing up. Using plastic tweezers pick up the half biotin-PEG cover slip by the edge and lower the PEG functionalized side onto the adhesive.
Press down the cover slip with a finger to secure it in place. Rub the cover slip surface using a pipette tip with moderate pressure to remove air bubbles. And then flip over the flow cell.
For each flow cell, cut approximately 10 centimeters of polyethylene 20 and 60 tubing. Cut the tubing tip at an angle less than 45 degrees and insert the tubing into the holes in the slide. If the hole diameter is correct, the tubing stands up in the hole on its own.
Mix the epoxy components well. Use a P200 pipette tip to dab epoxy around the tubing in each glass piece. Allow the epoxy to cure for 30 to 60 minutes.
To begin, arrange the freshly prepared filter-sterilized blocking buffer, 10x reaction buffer one and two on a working platform. Place open tubes containing 2.5 milliliters of blocking buffer and 5 milliliters of ultrapure water in a desiccator and degas under vacuum. After 15 minutes, release the pressure and remove the tubes.
Place the prepared biotin-PEG flow cell assembly on a microscope stage, and secure it using adhesive putty at each end. Connect the flow cell outlet tubing to the syringe pump via needle. Switch on the Objective Heater to 30 degrees Celsius.
Secure one to two milliliters of degassed water in a tube to a separate piece of adhesive putty near the flow cell. Insert the inlet tubing into the tube until it reaches the bottom, and flow water through the channel. Increase the flow rate to remove any trapped bubbles near the inlet tubing.
Add 100 microliters of degassed blocking buffer to a 20 microliter aliquot of one milligram per milliliter Streptavidin. Attach the open tube to adhesive putty. Transfer the inlet tubing from the water to the Streptavidin and start the flow at a rate of 40 microliters per minute for two minutes.
After five minutes, wash out excess Streptavidin with the blocking buffer. Flow in biotinylated DNA diluted in blocking buffer containing 25 nanomolar SYTOX Orange. Image using live view with the 532 nanometer laser to watch the DNA tethering to the surface in real time.
Once the desired density of DNA on the surface is achieved, flow in a blocking buffer containing 25 nanomolar SYTOX, to wash out free DNA. Now, flow in biotinylated anti digoxigenin antibody diluted in blocking buffer containing 25 nano molar SYTOX Orange. Wash out the biotinylated anti digoxigenin antibody and SYTOX Orange with blocking buffer.
Flow 50 microliters of ATP-gamma-S mix into the flow cell. Add purified CMG helicase to approximately 100 nanomolar final concentrations in 30 microliters of ATP-gamma-S mix, and flow at 20 microliters per minute for 20 microliters. Incubate for 15 minutes.
Next, flow in ATP RPA mix at 40 microliters per minute for 80 microliters. Using each laser with 50 to 100 milliseconds exposure, visualize and acquire eGFP-RPA with a 488 nanometer laser. Then, acquire CMG with a 640 nanometer laser.
Finally, visualize and acquire SYTOX Orange stained DNA with a 532 nanometer laser. CMG helicase unwinding of DNA was visualized by the linear growth of the yellow RPA tract over time, indicating the unwound DNA strands. Single stranded DNA damage reduced the number of successful unwinding events observed as demonstrated by fewer complete traces in the data.
