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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X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+of+eukaryotic+CMG+helicase+activation.">The mechanism of eukaryotic CMG helicase activation.</a> Nature. 555 (7695), 265-268 (2018).</li><li>Ilves, I., Petojevic, T., Pesavento, J. J., Botchan, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+the+MCM2-7+helicase+by+association+with+Cdc45+and+GINS+proteins.">Activation of the MCM2-7 helicase by association with Cdc45 and GINS proteins.</a> Mol Cell. 37 (2), 247-258 (2010).</li><li>Dewar, J. M., Walter, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+DNA+replication+termination.">Mechanisms of DNA replication termination.</a> Nat Rev Mol Cell Biol. 18 (8), 507-516 (2017).</li><li>Burnham, D. R., Kose, H. B., Hoyle, R. B., Yardimci, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+of+DNA+unwinding+by+the+eukaryotic+replicative+helicase.">The mechanism of DNA unwinding by the eukaryotic replicative helicase.</a> Nat Commun. 10 (1), 2159 (2019).</li><li>Kose, H. B., Larsen, N. B., Duxin, J. P., Yardimci, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+the+eukaryotic+replicative+helicase+at+lagging-strand+protein+barriers+support+the+steric+exclusion+model.">Dynamics of the eukaryotic replicative helicase at lagging-strand protein barriers support the steric exclusion model.</a> Cell Rep. 26 (8), 2113-2125.e6 (2019).</li><li>Kose, H. B., Xie, S., Cameron, G., Strycharska, M. S., Yardimci, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Duplex+DNA+engagement+and+RPA+oppositely+regulate+the+DNA-unwinding+rate+of+CMG+helicase.">Duplex DNA engagement and RPA oppositely regulate the DNA-unwinding rate of CMG helicase.</a> Nat Commun. 11 (1), 3713 (2020).</li><li>Tanner, N. A., Van Oijen, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+DNA+replication+at+the+single-molecule+level.">Visualizing DNA replication at the single-molecule level.</a> Methods Enzymol. 475, 259-278 (2010).</li><li>Swoboda, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+oxygen+scavenging+for+photostability+without+ph+drop+in+single-molecule+experiments.">Enzymatic oxygen scavenging for photostability without ph drop in single-molecule experiments.</a> ACS Nano. 6 (7), 6364-6369 (2012).</li><li>Baris, Y., Taylor, M. R. G., Aria, V., Yeeles, J. T. 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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+replication+origin+melting+nucleated+by+CMG+helicase+assembly.">Mechanism of replication origin melting nucleated by CMG helicase assembly.</a> Nature. 606 (7916), 1007-1014 (2022).</li><li>Zeman, M. K., Cimprich, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Causes+and+consequences+of+replication+stress.">Causes and consequences of replication stress.</a> Nat Cell Biol. 16 (1), 2-9 (2014).</li><li>Sparks, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+CMG+helicase+bypasses+DNA-protein+cross-links+to+facilitate+their+repair.">The CMG helicase bypasses DNA-protein cross-links to facilitate their repair.</a> Cell. 176 (1-2), 167-181.e21 (2019).</li><li>Taylor, M. R. G., Yeeles, J. T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+initial+response+of+a+eukaryotic+replisome+to+DNA+damage.">The initial response of a eukaryotic replisome to DNA damage.</a> Mol Cell. 70 (6), 1067-1080.e12 (2018).</li><li>Amunugama, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+fork+reversal+during+DNA+interstrand+crosslink+repair+requires+CMG+unloading.">Replication fork reversal during DNA interstrand crosslink repair requires CMG unloading.</a> Cell Rep. 23 (12), 3419-3428 (2018).</li><li>Wu, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRAIP+is+a+master+regulator+of+DNA+interstrand+crosslink+repair.">TRAIP is a master regulator of DNA interstrand crosslink repair.</a> Nature. 567 (7747), 267-272 (2019).</li><li>Vrtis, K. B., Dewar, J. M., Chistol, G., Wu, R. A., Graham, T. G. W., Walter, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-strand+DNA+breaks+cause+replisome+disassembly.">Single-strand DNA breaks cause replisome disassembly.</a> Mol Cell. 81 (6), 1309-1318.e6 (2021).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M3148-25ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylamide / Bis Solution 40%&#160;</td>
			<td>BioRad</td>
			<td>1610148</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose UltraPure&#160;</td>
			<td>Invitrogen</td>
			<td>16500-500</td>
			<td>Used to prepare Tris Buffer (pH 8)</td>
		</tr>
		<tr>
			<td>&#196;KTA Pure&#160;</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%&#160;</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/10.1016/j.ymeth.2012.03.033</td>
		</tr>
		<tr>
			<td>Biotinylated anti-digoxigenin antibody&#160;</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&#160;</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&#160;</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&#160;</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purified as desribed in &#8216;Single Molecule Analysis&#8217;, 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&#160;</td>
			<td>Fujifilm</td>
			<td>&#160;FLA-5000&#160;</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.&#160;</td>
		</tr>
		<tr>
			<td>Glass or quartz slide with holes for tubing&#160;</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&#160;</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>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>M2393</td>
			<td></td>
		</tr>
		<tr>
			<td>Millex-GP (Syringe filters) 0.22 &#181;m&#160;</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%&#8211;12% Bis-Tris Protein Gels</td>
			<td>Thermo Fisher Scientific</td>
			<td>10247002;</td>
			<td></td>
		</tr>
		<tr>
			<td>NuPAGE MOPS SDS Running Buffer&#160;</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 &#181;L), 10 mM dNTPs (40 &#181;L), 5x Phusion HF buffer (400 &#181;L) and water (540 &#181;L).</td>
		</tr>
		<tr>
			<td>Peptide NH2-CHHHHHHHHHHLPETGG-COOH, labelled with LD655-MAL&#160;</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/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&#8221;, outer diameter 0.043&#8221;)</td>
			<td>Becton Dickinson</td>
			<td>427406</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene tubing PE60 (inner diameter 0.03&#8221;, outer diameter 0.048&#8221;)</td>
			<td>Becton Dickinson</td>
			<td>427416</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-Prep Chromatography Columns; 10 ml and 20 ml&#160;</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/10.1073/pnas.1101046108</td>
		</tr>
		<tr>
			<td>Spin-X Centrifuge Tube Filters; 0.22 &#181;m cellulose acetate&#160;</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;&#160;</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 &#181;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 &#181;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&#160;</td>
			<td>NEB</td>
			<td>B0202S</td>
			<td></td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>Sigma-Aldrich</td>
			<td>T9281-25ML&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>TEV protease (1 mg/mL); EZCut&#160;</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.

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.

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/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/files/ftp_upload/67212/67212fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Research

JoVE Journal

Biochemistry

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.

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.

779 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.

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

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