preparation of a biotin-peg flow cell assembly for single molecule total internal reflection fluorescence microscopy

CMG-Mediated DNA Unwinding Observed by ssDNA-RPA Tract Growth

DNA replication is tightly regulated, as a cell must duplicate its genome accurately to prevent mutations, disease, or death. Eukaryotic DNA replication is carried out by the replisome complex, which unwinds parental DNA and uses single-stranded DNA (ssDNA) as a template to synthesize new DNA onto. In the G1 phase, catalytically inactive double hexamers of MCM2-7 are loaded onto double-stranded DNA (dsDNA) at replication origins1. In the S phase, MCM2-7 complexes are activated by the binding of CDC45 and GINS2 to form 11-subunit CMG complexes (CDC45, MCM2-7, GINS). Each CMG initiates DNA unwinding in opposite directions, forming the core unit that the replisome arranges itself around3.
Two decades ago the CMG helicase was first identified as an 11-subunit complex, essential for DNA replication4. Since then, our understanding of CMG has advanced considerably, from loading and activation5,6, to DNA unwinding and termination7. Traditional biochemical and structural biology techniques have been critical to many of these discoveries; however, these methods were often limited in their ability to study the more dynamic aspects of CMG. Single-molecule methods use physical manipulation of individual biomolecules to measure or visualize their activity one molecule at a time. This can be used to provide insight into the real-time dynamics of proteins that are often missed or undetectable by other techniques8,9.
Here, we describe a total internal reflection fluorescence (TIRF) microscopy assay to visualize DNA unwinding by CMG helicase in real time. Purified, fluorescently labeled CMG is loaded onto the free 3&#39; end of long DNA containing a pre-made DNA fork structure. Linear DNA is stretched on a biotin-PEG coverslip in a microfluidic flow cell by tethering each end of the DNA sequentially to the surface. This approach allows for more uniform DNA tethering, which significantly reduces the variation that must be accounted for during data analysis. In the presence of ATP-&#947;-s, CMG is loaded onto the single-stranded DNA at the 3&#39; end of the fork. ATP-&#947;-s is a slowly hydrolyzable ATP analog, which permits CMG binding onto DNA but not unwinding. Subsequent addition of ATP, along with purified, fluorescently tagged RPA, activates CMG and initiates extensive DNA unwinding. Visually, CMG translocates along the DNA, leaving a growing tract of RPA-bound ssDNA behind it. The untethered DNA end travels with CMG, forming a &#34;tight ball&#34; due to compaction caused by RPA binding. The flow cell design allows the buffer to be exchanged at any point during unwinding, giving great control during and over each experiment.
This protocol is divided into four methods, which can be performed independently of one another. Section 1 describes the preparation of a 20-kb linear forked DNA substrate for single-molecule assays. Section 2 outlines the purification and fluorescent labeling of Drosophila melanogaster CMG (DmCMG). Key information about the expression of DmCMG is included in the notes section. Section 3 covers the preparation of a microfluidic flow cell that can be used on a TIRF microscope. Section 4 describes how to carry out the single-molecule DNA unwinding assay.

Author Spotlight: Unraveling the Dynamics of Eukaryotic DNA Replication Through Single-Molecule Visualization

Single-Molecule Real-Time Visualization of DNA Unwinding by CMG Helicase

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 what 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.

Preparation of a Biotin-PEG Flow Cell Assembly for Single Molecule Total Internal Reflection Fluorescence Microscopy

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 by one 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 of 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 whole 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 and each glass piece. Allow the epoxy to cure for 30 to 60 minutes.

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Research

JoVE Journal

Biochemistry

105 Views. 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 by one 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 po...

Video: Preparation of a Biotin-PEG Flow Cell Assembly for Single Molecule Total Internal Reflection Fluorescence Microscopy

Faithful genome duplication is essential for preserving the genetic stability of dividing cells. DNA replication is carried out during the S phase by a dynamic complex of proteins termed the replisome. At the heart of the replisome is the&#160;CDC45-MCM2-7-GINS (CMG) helicase, which separates the two strands of the DNA double helix such that DNA polymerases can copy each strand. During genome duplication, replisomes must overcome a plethora of obstacles and challenges. Each of these threatens genome stability, as failure to replicate DNA completely and accurately can lead to mutations, diseases, or cell death. Therefore, it is of great interest to understand how CMG functions in the replisome during both normal replication and replication stress. Here, we describe a total internal reflection fluorescence (TIRF) microscopy assay using recombinant purified proteins, which allows for real-time visualization of surface-tethered stretched DNA molecules by individual CMG complexes. This assay provides a powerful platform to investigate CMG behavior at the single-molecule level, allowing helicase dynamics to be directly observed with real-time control over reaction conditions.

This protocol demonstrates performing a single-molecule assay for live visualization of DNA unwinding by CMG helicase. It describes (1) preparing a DNA substrate, (2) purifying fluorescently labeled Drosophila melanogaster CMG helicase, (3) preparing a microfluidic flow cell for total internal reflection fluorescence (TIRF) microscopy, and (4) the single-molecule DNA unwinding assay.

Faithful genome duplication is essential for preserving the genetic stability of dividing cells. DNA replication is carried out during the S phase by a ...

Single Molecule Total Internal Reflection Fluorescence Assay for Visualizing CMG-Mediated DNA Unwinding

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 five 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 and biotinylated anti-digoxigenin antibody diluted in blocking buffer containing 25 nanomolar 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 tracked 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.