We thank Gheorghe Chistol for providing pGC261 plasmid and the Francis Crick Institute Chemical Biology Facility for peptide synthesis and labeling. This work was funded by The Francis Crick Institute, which receives core funding from Cancer Research UK, the UK Medical Research Council, and The Wellcome Trust (CC2133).

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

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The authors do not have competing financial interests or other conflicts of interest.

This assay provides a platform to observe and investigate the real-time dynamics of individual CMGs, both in isolation and in the context of the desired additional factors. However, as with many single-molecule fluorescence techniques, there are some common challenges that can require optimization to overcome. These usually relate to imaging fluorophores over long periods of time (photobleaching, brightness), DNA substrate preparation (DNA damage), the quality of the flow cell surface (background noise, non-specific interactions), or the quality of the purified protein preparation (nuclease contamination, labeling efficiency).
Each fluorophore varies in photostability and brightness, so it is important to choose an appropriate molecule. When imaging fluorescently labeled oligomeric proteins, like RPA, a lower laser power can be used as many fluorophores will be excited in close proximity, generating a visible signal. For imaging single fluorophores, for example, CMG labeled on a single subunit, a higher laser power is needed to observe the fluorophore clearly. Fluorophore lifetime can be extended by minimizing laser exposure, such as by reducing the frequency at which images are taken. Additionally, exciting a fluorophore generates reactive oxygen species (ROS), which can contribute to photobleaching. Including an oxygen scavenging system in the imaging buffer can extend the lifetime of fluorophores by eliminating ROS. However, some oxygen scavenging systems can affect pH12.
Regarding DNA substrate preparation, it is crucial to minimize DNA damage, such as nicks or single-stranded gaps. Excessive damage prevents extensive DNA unwinding, limiting how much data can be collected. Damage can arise from mechanical shearing, excessive heating, as a result of nuclease contamination, or ROS generated during imaging. Shearing can be minimized by handling the DNA sample with care by using wide-bore tips for pipetting, pipetting slowly, and&#160;avoiding flicking the sample. The effect of ROS can be minimized by either reducing laser exposure or including an oxygen scavenging system in the imaging buffer. After the preparation of the DNA substrate, it is possible to use commercial DNA repair kits to repair the damage before performing an unwinding reaction.
The efficiency of DNA unwinding also depends on the purity and activity of CMG. It is a good practice to assess sample purity after each purification step by SDS-PAGE electrophoresis to determine where optimization is necessary. If too many contaminants are observed after the final step, it may help to modify the salt gradient volumes used for the elution from CaptoHiRes Q (5/50) column. It is also highly important to remove any excess fluorescent peptide used for the protein labeling, as it can create undesirable background on the coverslip surface. It is also essential to avoid nuclease contamination, as this can degrade the DNA substrate. After an experiment, staining the remaining DNA with SYTOX orange can be a good way to check whether the DNA has been degraded significantly or not. A certain level of DNA damage is unavoidable over the course of an experiment, but significant damage often indicates problematic nuclease contamination.
The assay is also inherently limited by the resolution of diffraction-limited spots, requiring fluorescent proteins to be hundreds of base pairs away (if not more) to distinguish them as separate. This limits the detail in which CMG progression and interactions can be observed.
The number of unwinding events we observe for each analysis varies. For a successful experiment, we expect to see at least several RPA tracts of sufficient length per field of view 512x512 pixels (pixel size = 154.6 nm). Multiple fields of view can be imaged in the same experiment, allowing more data collection when necessary. The tracts do not need to be of the same length nor reach the end of the DNA to be useful. For example, the average tether distance for each experiment can be determined by measuring the length of SYTOX-stained DNA prior to adding CMG. This can be used to estimate how much DNA has been unwound for any RPA tract (as long as enough DNA is unwound to visibly move the fork) by converting distance from &#39;&#181;m traveled&#39; to &#39;kb unwound&#39;.
CMG exhibits unwinding activity on a variety of DNA substrates, but it is essential to provide a free 3&#39; DNA end on a polyT flap of at least 30 nt to accommodate the footprint of CMG10. Including multiple biotin moieties at the fork ensures robust surface tethering. The rest of the DNA substrate can be re-designed in a multitude of ways, such as to include different DNA sequences, lengths and chemical modifications. The conformation of the DNA can be altered by using different concentrations of magnesium acetate. At higher concentrations (&#8805;10 mM) of magnesium acetate, the RPA-coated ssDNA filament is compacted, leading to the DNA being pulled taught by RPA binding during unwinding. This can be useful as it prevents the DNA from moving excessively, allowing the position of CMG and of unwinding progression to be more accurately measured. At low concentrations (~3 mM) of magnesium acetate, the RPA-ssDNA remains relaxed throughout.
The described single-molecule assay represents a platform that can be built upon and modified to investigate further aspects of DNA replication. During DNA replication, CMG acts as a core that the replisome and its components assemble around. Therefore, additional purified proteins can be added to this assay, including accessory factors like TIMELESS, TIPIN, and CLASPIN, to study their effect on CMG dynamics. These proteins have been shown to affect the rate of replication forks13, but it is not clear how they affect the rate of CMG unwinding. Therefore, it would be interesting to investigate how different replisome proteins affect CMG using this assay. Addition of DNA polymerases may give better insight into DNA replication beyond DNA unwinding alone, as described previously with yeast proteins14. Furthermore, purification of modified CMG may provide a better understanding of how certain mutations or post-translational modifications affect helicase activity15,16. Additionally, designing different DNA substrates can allow DNA unwinding by CMG to be studied under a variety of conditions mimicking replication stress17. These modifications include DNA obstacles9,18, inter-strand crosslinks19,20,21, and discontinuities in the DNA strands22.

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.

<table><tbody><tr><td>2-Mercaptoethanol</td><td>Sigma-Aldrich</td><td>M3148-25ML</td><td></td></tr><tr><td>Acrylamide / Bis Solution 40%&amp;nbsp;</td><td>BioRad</td><td>1610148</td><td></td></tr><tr><td>Agarose UltraPure&amp;nbsp;</td><td>Invitrogen</td><td>16500-500</td><td>Used to prepare Tris Buffer (pH 8)</td></tr><tr><td>&amp;Auml;KTA Pure&amp;nbsp;</td><td>GE Healthcare</td><td>-</td><td>Used with Fraction Collector F9-C; protein purification system</td></tr><tr><td>ANTI-FLAG M2 affinity gel</td><td>Sigma-Aldrich</td><td>A2220</td><td></td></tr><tr><td>APS 10%&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A3678-25G</td><td></td></tr><tr><td>ATP (200 mM)</td><td>Sigma-Aldrich</td><td>A7699-5G</td><td></td></tr><tr><td>ATP-g-s (100 mM)</td><td>Sigma-Aldrich</td><td>11162306001</td><td></td></tr><tr><td>Biotin-PEG coverslip</td><td>N/A</td><td>N/A</td><td>Prepared as described: https://doi-org.bdigitaluss.remotexs.co/10.1016/j.ymeth.2012.03.033</td></tr><tr><td>Biotinylated anti-digoxigenin antibody&amp;nbsp;</td><td>Perkin Elmer</td><td>N/A</td><td></td></tr><tr><td>Blu Tack</td><td>Bostik</td><td>N/A</td><td>Adhesive putty</td></tr><tr><td>Boric acid</td><td>Thermo Fisher Scientific</td><td>B/3800/53</td><td></td></tr><tr><td>BSA; 33 mg/mL</td><td>SIGMA-ALDRICH</td><td>A3858-10G</td><td>Diluted from the stock</td></tr><tr><td>CaCl2</td><td>Sigma-Aldrich</td><td>C7902</td><td></td></tr><tr><td>CaptoHiRes Q (5/50)</td><td>Cytiva</td><td>29275878</td><td>Connected to the AKTA protein purification system; high-resolution ion exchange chromatography column</td></tr><tr><td>Casein (5% in water, 50 mg/mL)</td><td>Sigma-Aldrich</td><td>C4765-10ML</td><td></td></tr><tr><td>ChemiDoc system</td><td>Bio-Rad</td><td>N/A</td><td></td></tr><tr><td>Clips</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Cutting board</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Dialysis tubing (3.5 kDa)</td><td>Fisher Scientific</td><td>11425859</td><td></td></tr><tr><td>digoxigenin-11-dUTP</td><td>Roche</td><td>11209256910</td><td></td></tr><tr><td>DNA loading dye 6x&amp;nbsp;</td><td>NEB</td><td>B7024S</td><td></td></tr><tr><td>dNTP 10 mM</td><td>NEB</td><td>N0447S</td><td></td></tr><tr><td>Double-sided tape (0.14 mm thick)</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>DTT</td><td>Fluorochem Limited</td><td>M02712-10G</td><td></td></tr><tr><td>DYKDDDDK peptide&amp;nbsp;</td><td>N/A</td><td>N/A</td><td>Synthetised by chemical biology STP of The Francis Crick Institute</td></tr><tr><td>EDTA</td><td>Thermo Fisher Scientific</td><td>D/0700/68</td><td></td></tr><tr><td>EGFP-RPA&amp;nbsp;</td><td>N/A</td><td>N/A</td><td>Purified as desribed in &amp;lsquo;Single Molecule Analysis&amp;rsquo;, Series Methods in Molecular Biology, Vol. 783 (2011), Peterman, E. J. G. and Wuite G. J. L. editors, Humana Press. Protein information: 2 mM, human, expressed in E. coli.</td></tr><tr><td>EGTA</td><td>Sigma-Aldrich</td><td>03779-10G</td><td></td></tr><tr><td>Epoxy - 5 minute</td><td>Devcon</td><td>20845</td><td></td></tr><tr><td>Fujifilm FLA-5000&amp;nbsp;</td><td>Fujifilm</td><td>&amp;nbsp;FLA-5000&amp;nbsp;</td><td>Fluorescent image analyzer</td></tr><tr><td>GelRed Nucleic Acid Stain; 10000x</td><td>Cambridge Bioscience</td><td>41003-BT</td><td>Example of nucleic acid stain which is a safer alternative to ethidium bromide.&amp;nbsp;</td></tr><tr><td>Glass or quartz slide with holes for tubing&amp;nbsp;</td><td>Dremel Model 395</td><td></td><td>1 mm thick slide, cut to 2.4 cm x 1cm, two holes drilled 1.4 mm apart. The holes should be drilled to different sizes to accommodate different tubing at each end.</td></tr><tr><td>Glycerol</td><td>Fisher Scientific</td><td>G/0650/17</td><td></td></tr><tr><td>Glycine HCl, pH 3.5</td><td>Sigma-Aldrich</td><td>G8898</td><td></td></tr><tr><td>Hamilton syringe, 1000 series GASTIGHT, PTFE luer lock 1010TLL, PTFE Luer lock (with slots), volume 10 mL</td><td>Merck</td><td>26211-U</td><td></td></tr><tr><td>HEPES pH 7.5</td><td>Sigma-Aldrich</td><td>H4034</td><td></td></tr><tr><td>I-CeuI</td><td>NEB</td><td>R0699L</td><td></td></tr><tr><td>KCl</td><td>Fisher Scientific</td><td>P/4280/53</td><td></td></tr><tr><td>Magnesium acetate</td><td>Sigma-Aldrich</td><td>M2545</td><td></td></tr><tr><td>Maxi GeBaFlex-tube Dialysis Kit; MWCO 14 kDa&amp;nbsp;</td><td>Generon</td><td>D055</td><td></td></tr><tr><td>Metal spatula</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Metal tweezers</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>M2393</td><td></td></tr><tr><td>Millex-GP (Syringe filters) 0.22 &amp;micro;m&amp;nbsp;</td><td>Merck</td><td>SLGP033RS</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S7653</td><td></td></tr><tr><td>Needle</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>NotI-HF</td><td>NEB</td><td>R3189L</td><td></td></tr><tr><td>NuPAGE 4%&amp;ndash;12% Bis-Tris Protein Gels</td><td>Thermo Fisher Scientific</td><td>10247002;</td><td></td></tr><tr><td>NuPAGE MOPS SDS Running Buffer&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>NP0001</td><td></td></tr><tr><td>Objective heater</td><td>Okolab</td><td>N/A</td><td></td></tr><tr><td>PCR mix - 2x</td><td></td><td></td><td>Prepared from Phusion DNA polymerase (20 &amp;micro;L), 10 mM dNTPs (40 &amp;micro;L), 5x Phusion HF buffer (400 &amp;micro;L) and water (540 &amp;micro;L).</td></tr><tr><td>Peptide NH2-CHHHHHHHHHHLPETGG-COOH, labelled with LD655-MAL&amp;nbsp;</td><td>N/A</td><td>N/A</td><td>peptide NH2-CHHHHHHHHHHLPETGG-COOH, labeled with LD655-MAL (Lumidyne Technologies) on the cysteine residue, was synthetised and purified by the peptide chemistry STP of The Francis Crick Institute</td></tr><tr><td>pGC261 plasmid</td><td>N/A</td><td>N/A</td><td>https://doi-org.bdigitaluss.remotexs.co/10.1016/j.cell.2018.10.053</td></tr><tr><td>Phusion High-Fidelity DNA Polymerase</td><td>NEB</td><td>M0530L</td><td></td></tr><tr><td>Plastic tweezers</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Polyethylene tubing PE20 (inner diameter 0.015&amp;rdquo;, outer diameter 0.043&amp;rdquo;)</td><td>Becton Dickinson</td><td>427406</td><td></td></tr><tr><td>Polyethylene tubing PE60 (inner diameter 0.03&amp;rdquo;, outer diameter 0.048&amp;rdquo;)</td><td>Becton Dickinson</td><td>427416</td><td></td></tr><tr><td>Poly-Prep Chromatography Columns; 10 ml and 20 ml&amp;nbsp;</td><td>Bio-Rad</td><td>7311550</td><td></td></tr><tr><td>Potassium Glutamate (L-glutamic acid potassium salt monohydrate)</td><td>Sigma</td><td>G1501-1KG</td><td></td></tr><tr><td>Protease inhibitor cocktail (cOmplete, EDTA free)</td><td>Roche</td><td>5056489001</td><td></td></tr><tr><td>Pump 11 Elite Infusion/Withdrawal Programmable Single Syringe</td><td>Harvard Apparatus</td><td>70-4504</td><td></td></tr><tr><td>QIAquick PCR Purification Kit</td><td>QIAGEN</td><td>28104</td><td></td></tr><tr><td>Razor blade</td><td>VWR International Ltd</td><td>233-0156</td><td></td></tr><tr><td>rCutSmart Buffer; 10x</td><td>NEB</td><td>B6004S</td><td></td></tr><tr><td>Sodium acetate</td><td>Thermo Fisher Scientific</td><td>S/2120/53</td><td></td></tr><tr><td>Sortase (pentamutant)</td><td>N/A</td><td>N/A</td><td>Purified based on: https://doi-org.bdigitaluss.remotexs.co/10.1073/pnas.1101046108</td></tr><tr><td>Spin-X Centrifuge Tube Filters; 0.22 &amp;micro;m cellulose acetate&amp;nbsp;</td><td>Fisher Scientific</td><td>10310361</td><td></td></tr><tr><td>Sterile scalpel</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Steritop Vacuum Driven Disposable Filtration System; 0.22 um; PES;&amp;nbsp;</td><td>Millipore</td><td>S2GPT05RE</td><td></td></tr><tr><td>Streptavidin (1 mg/mL) in 1x PBS buffer</td><td>Sigma</td><td>S4762-10MG</td><td>20 &amp;micro;L aliquotes</td></tr><tr><td>SYBR Gold</td><td>Thermo Fisher Scientific</td><td>S11494</td><td>Highly sensitive nucleic acid stain we used to visualise DNA fork substrate.</td></tr><tr><td>SYTOX orange; 5 &amp;micro;M in DMSO</td><td>Thermo Fisher Scientific</td><td>S11368</td><td></td></tr><tr><td>T4 DNA ligase</td><td>NEB</td><td>M0202M</td><td></td></tr><tr><td>T4 DNA Ligase Reaction Buffer&amp;nbsp;</td><td>NEB</td><td>B0202S</td><td></td></tr><tr><td>TEMED</td><td>Sigma-Aldrich</td><td>T9281-25ML&amp;nbsp;</td><td></td></tr><tr><td>TEV protease (1 mg/mL); EZCut&amp;nbsp;</td><td>Biovision</td><td>7847-10000</td><td></td></tr><tr><td>Tissue grinders, Dounce type (40 mL, Wheaton)</td><td>DWK Life Sciences</td><td>432-1273</td><td></td></tr><tr><td>Tris base</td><td>Sigma-Aldrich</td><td>T1503</td><td>Used to prepare Tris Buffer (pH 8)</td></tr><tr><td>Tris HCl</td><td>Sigma-Aldrich</td><td>T3253</td><td></td></tr><tr><td>Tween-20</td><td>Promega UK Ltd</td><td>H5152</td><td></td></tr></tbody></table>

single-molecule real-time visualization of dna unwinding by cmg helicase

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.

When CMG unwinds DNA, a characteristic RPA tract will grow over time (Figure 5). The 5&#39; end of the unwound DNA is tethered to the surface; hence, it is seen as a linear stretch of RPA signal between the tether and the fork. The 3&#39; end is not tethered and, therefore, moves with the fork and is observed as a compact EGFP-RPA signal. The position of the compacted unwound translocation strand corresponds approximately to the position of the replication fork, which moves together with LD655-CMG visualized via a 640 nm laser.
It is important to minimize damage to the DNA substrate, as damage such as single-stranded DNA nicks reduces the number of observable unwinding events, limiting the amount of data that can be collected (Figure 6).
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/67212/67212fig05.jpg" /> 
Figure 5:&#160;Single-molecule DNA unwinding assay. The DNA substrate is tethered to a coverslip surface. Purified CMG labeled with LD655 is incubated with the DNA for 15 min in ATP-g-s. ATP and purified EGFP-labelled RPA are added, initiating extensive DNA unwinding by CMG. A cartoon schematic (left) and kymograph of representative data (right) are shown. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/67212/67212fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/67212/67212fig06.jpg" /> 
Figure 6:&#160;DNA damage reduces assay throughput. (A) CMG cannot unwind DNA past a break in the DNA backbone (DNA nick). A nick on the leading strand template causes CMG to slide off the DNA, and both CMG and leading strand template are lost. A nick on the lagging strand template causes the lagging strand template to separate from the rest of the DNA, and each DNA piece retracts to their respective tether. This is illustrated by (i) cartoon schematics and (ii) kymographs of these events (ii). Representative data with (B) a minimally damaged DNA substrate versus (C) a more damaged DNA substrate at (i) 5 min, (ii) 15 min, and (iii) 60 min in a single field of view. The more damaged DNA substrate does not generate long tracts of unwinding, as the CMGs encounter nicks earlier on, despite similar levels of unwinding activity (similar density of growing RPA spots at 5 min, indicating similar CMG loading/unwinding efficiency). The field of view is 512 x 512 pixels (pixel size = 154.6 nm). 1% laser power (488 nm) imaging EGFP-RPA. Scale bar showing 10 &#181;m. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/67212/67212fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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.

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

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-real-time-visualization-dna-unwinding-cmg

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

1.4K Views.  The Francis Crick Institute. 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.

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

A Simple, Robust, and High Throughput Single Molecule Flow Stretching Assay Implementation for Studying Transport of Molecules Along DNA

We describe a simple, robust and high throughput single molecule flow-stretching assay for studying 1D diffusion of molecules along DNA. In this assay, glass coverslips are functionalized in a one-step reaction with silane-PEG-biotin. Flow cells are constructed by sandwiching an adhesive tape with pre-cut channels between a functionalized coverslip and a PDMS slab containing inlet and outlet holes. Multiple channels are integrated into one flow cell and the flow of reagents into each channel can be fully automated, which significantly increases the assay throughput and reduces hands-on time per assay. Inside each channel, biotin-&#955;-DNAs are immobilized on the surface and a laminar flow is applied to flow-stretch the DNAs. The DNA molecules are stretched to &#62;80% of their contour length and serve as spatially extended templates for studying the binding and transport activity of fluorescently labeled molecules. The trajectories of single molecules are tracked by time-lapse Total Internal Reflection Fluorescence (TIRF) imaging. Raw images are analyzed using streamlined custom single particle tracking software to automatically identify trajectories of single molecules diffusing along DNA and estimate their 1D diffusion constants.

This protocol demonstrates a simple, robust and high throughput single molecule flow-stretching assay for studying one-dimensional (1D) diffusion of molecules along DNA.

We describe a simple, robust and high throughput single molecule flow-stretching assay for studying 1D diffusion of molecules along DNA. In this assay, ...

Real-time Tracking of DNA Fragment Separation by Smartphone

Slab gel electrophoresis (SGE) is the most common method for the separation of DNA fragments; thus, it is broadly applied to the field of biology and others. However, the traditional SGE protocol is quite tedious, and the experiment takes a long time. Moreover, the chemical consumption in SGE experiments is very high. This work proposes a simple method for the separation of DNA fragments based on an SGE chip. The chip is made by an engraving machine. Two plastic sheets are used for the excitation and emission wavelengths of the optical signal. The fluorescence signal of the DNA bands is collected by smartphone. To validate this method, 50, 100, and 1,000 bp DNA ladders were separated. The results demonstrate that a DNA ladder smaller than 5,000 bp can be resolved within 12 min and with high resolution when using this method, indicating that it is an ideal substitute for the traditional SGE method.

Traditional slab gel electrophoresis (SGE) experiments require a complicated apparatus and high chemical consumption. This work presents a protocol that describes a low-cost method to separate DNA fragments within a short timeframe.

Slab gel electrophoresis (SGE) is the most common method for the separation of DNA fragments; thus, it is broadly applied to the field of biology and ...

Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA processing, and telomere elongation. During these processes, various proteins bind and resolve G4 structures to perform their function. As the function of G4 often depends on the stability of its folded structure, it is important to investigate how G4 binding proteins regulate the stability of G4. This work presents a method to manipulate single G4 molecules using magnetic tweezers, which enables studies of the regulation of G4 binding proteins on a single G4 molecule in real time. In general, this method is suitable for a wide scope of applications in studies for proteins/ligands interactions and regulations on various DNA or RNA secondary structures.

A single-molecule magnetic tweezers platform to manipulate G-quadruplexes is reported, which allows for the study of G4 stability and regulation by various proteins.

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA ...

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and functions. It gave us the opportunity to explore a multiplicity of biophysical mechanisms, e.g., how bacterial adhesins bind to host surface receptors in more detail. Among other factors, the success of SMFS experiments depends on the functional and native immobilization of the biomolecules of interest on solid surfaces and AFM tips. Here, we describe a straightforward protocol for the covalent coupling of proteins to silicon surfaces using silane-PEG-carboxyls and the well-established N-hydroxysuccinimid/1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimid (EDC/NHS) chemistry in order to explore the interaction of pilus-1 adhesin RrgA from the Gram-positive bacterium Streptococcus pneumoniae (S. pneumoniae) with the extracellular matrix protein fibronectin (Fn). Our results show that the surface functionalization leads to a homogenous distribution of Fn on the glass surface and to an appropriate concentration of RrgA on the AFM cantilever tip, apparent by the target value of up to 20% of interaction events during SMFS measurements and revealed that RrgA binds to Fn with a mean force of 52 pN. The protocol can be adjusted to couple via site specific free thiol groups. This results in a predefined protein or molecule orientation and is suitable for other biophysical applications besides the SMFS.

This protocol describes the covalent immobilization of proteins with a heterobifunctional silane coupling agent to silicon-oxide surfaces designed for the atomic force microscopy based single molecule force spectroscopy which is exemplified by the interaction of RrgA (pilus-1 tip adhesin of S. pneumoniae) with fibronectin.

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

Real-time Observation of the DNA Strand Exchange Reaction Mediated by Rad51

The DNA strand exchange reaction mediated by Rad51 is a critical step of homologous recombination. In this reaction, Rad51 forms a nucleoprotein filament on single-stranded DNA (ssDNA) and captures double-stranded DNA (dsDNA) non-specifically to interrogate it for a homologous sequence. After encountering homology, Rad51 catalyzes DNA strand exchange to mediate pairing of the ssDNA with the complementary strand of the dsDNA. This reaction is highly regulated by numerous accessary proteins in vivo. Although conventional biochemical assays have been successfully employed to examine the role of such accessory protein in vitro, kinetic analysis of intermediate formation and its progression into a final product has proven challenging due to the unstable and transient nature of the reaction intermediates. To observe these reaction steps directly in solution, fluorescence resonance energy transfer (FRET)-based real-time observation systems of this reaction were established. Kinetic analysis of real-time observations shows that the DNA strand exchange reaction mediated by Rad51 obeys a three-step reaction model involving the formation of a three-strand DNA intermediate, maturation of this intermediate, and the release of ssDNA from the mature intermediate. The Swi5-Sfr1 complex, an accessary protein conserved in eukaryotes, strongly enhances the second and third steps of this reaction. The FRET-based assays presented here enable us to uncover the molecular mechanisms through which recombination accessary proteins stimulate the DNA strand exchange activity of Rad51. The primary goal of this protocol is to enhance the repertoire of techniques available to researchers in the field of homologous recombination, particularly those working with proteins from species other than Schizosaccharomyces pombe, so that the evolutionary conservation of the findings presented herein can be determined.

Fluorescence resonance energy transfer-based real-time observation systems of the DNA strand exchange reaction mediated by Rad51 were developed. Using the protocols presented here, we are able to detect the formation of reaction intermediates and their conversion into products, while also analyzing the enzymatic kinetics of the reaction.

The DNA strand exchange reaction mediated by Rad51 is a critical step of homologous recombination. In this reaction, Rad51 forms a nucleoprotein filament ...

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a fluorescent dye and are spotted by a photodetector following their separation. Single molecule fluorescence imaging can provide ultrasensitive protein detection with its ability for visualizing individual fluorescent molecules. However, the application of this powerful imaging method to electrophoresis assays is hampered by the lack of ways to characterize the homogeneity of fluorescent labeling of each protein species across the proteome. Here, we developed a method to evaluate the labeling homogeneity across the proteome based on a single molecule fluorescence imaging assay. In our measurement using a HeLa cell sample, the proportion of proteins labeled with at least one dye, which we termed &#8216;labeling occupancy&#8217; (LO), was determined to range from 50% to 90%, supporting the high potential of the application of single molecule imaging to sensitive and precise proteome analysis.

Here, we present a protocol to assess the labeling homogeneity for each protein species in a complex protein sample at the single molecule level.

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

Biology

Imaging the Motion of Fully Reconstituted Cdc45/Mcm2-7/GINS (CMG)

Hybrid Ensemble and Single-molecule Assay to Image the Motion of Fully Reconstituted CMG

Eukaryotes have one replicative helicase known as CMG, which centrally organizes and drives the replisome, and leads the way at the front of replication forks. Obtaining a deep mechanistic understanding of the dynamics of CMG is critical to elucidating how cells achieve the enormous task of efficiently and accurately replicating their entire genome once per cell cycle. Single-molecule techniques are uniquely suited to quantify the dynamics of CMG due to their unparalleled temporal and spatial resolution. Nevertheless, single-molecule studies of CMG motion have thus far relied on pre-formed CMG purified from cells as a complex, which precludes the study of the steps leading up to its activation. Here, we describe a hybrid ensemble and single-molecule assay that allowed imaging at the single-molecule level of the motion of fluorescently labeled CMG after fully reconstituting its assembly and activation from 36 different purified S. cerevisiae polypeptides. This assay relies on the double functionalization of the ends of a linear DNA substrate with two orthogonal attachment moieties, and can be adapted to study similarly complex DNA-processing mechanisms at the single-molecule level.

Author Spotlight: Investigating the Motion Dynamics of the Eukaryotic Replisome Components at the Single-Molecule Level

We report a hybrid ensemble and single-molecule assay to directly image and quantify the motion of fluorescently labeled, fully reconstituted Cdc45/Mcm2-7/GINS (CMG) helicase on linear DNA molecules held in place in an optical trap.

Eukaryotes have one replicative helicase known as CMG, which centrally organizes and drives the replisome, and leads the way at the front of replication ...

Single-Molecule Fluorescence Visualization of DNA Polymerase Dynamics at G-Quadruplexes

The ability of proteins&#160;involved in eukaryotic DNA replication to&#160;overcome obstacles &#8212; such as protein and DNA &#39;roadblocks&#39;&#160;&#8212; is critical for ensuring faithful&#160;genome duplication.&#160;G-quadruplexes are higher-order nucleic acid structures that form in guanine-rich regions of DNA and have been shown to act as obstacles, interfering with genomic maintenance pathways. This study introduces a real-time, fluorescence microscopy-based method to observe DNA polymerase interactions with G-quadruplex structures. Short, primed DNA oligonucleotides containing a G-quadruplex were immobilized on functionalized glass coverslips within a microfluidic flow cell. Fluorescently labeled DNA polymerases were introduced, allowing their behavior and stoichiometry to be monitored over time. This approach enabled the observation of polymerase behavior as it was stalled by a G-quadruplex. Specifically, using fluorescently labeled yeast polymerase &#948;, it was found that upon encountering a G-quadruplex, the polymerase undergoes a continuous cycle of binding and unbinding. This single-molecule assay can be adapted to study interactions between various DNA-maintenance proteins and obstacles on the DNA substrate.

This protocol outlines a fluorescence microscopy-based single-molecule DNA replication assay, enabling real-time visualization of interactions between DNA polymerases and obstacles such as G-quadruplex structures.

The ability of proteins&#160;involved in eukaryotic DNA replication to&#160;overcome obstacles &#8212; such as protein and DNA ...

Single-Molecule Imaging to Visualize Fusion Protein Condensates Assembly on DNA Curtains

To image the EWS-FLI1 condensation formation on DNA curtains, open the imaging software and find and mark the positions of the nine zig-zag patterns under bright-field. Then, turn on the flow at 0.2 milliliters per minute to stain the DNA with double-stranded DNA dye for 10 minutes.
Next, dilute the mCherry-EWS-FLI1 protein with the imaging buffer at a concentration of 100 nanomoles and 100 microliters. Then, load the protein sample through the valve with a 100-microliter glass syringe, and change the flow rate to 0.4 milliliters per minute.
After turning on the 488-nanometer laser, pre-scan each region to check the DNA distribution state, and select the region in which the DNA molecules distribute evenly. Then, set the laser power to 10% for the 488-nanometer laser and 20% for the 561-nanometer laser, and using the power meter, measure the real laser power near the prism.
Start acquiring images at two-second intervals with both 488 and 561-nanometer lasers simultaneously. Then, change the valve from the manual mode to injection mode to let the imaging buffer flush the protein sample into the flow cell after 60 seconds.
To remove the free EWS-FLI1, keep washing the flow cell with the imaging buffer for five minutes with only the 561-nanometer laser switched on. Then, stop the flow and incubate at 37 degrees Celsius for 10 minutes.
After 10 minutes, turn on the flow at 0.4 milliliters per minute to let the DNA extend, and acquire images at two-second intervals between different frames to obtain high-throughput data of EWS-FLI1 condensate formation.