<meta charset="utf8"></head><body>通过 ssDNA-RPA 束生长观察到的 CMG 介导的 DNA 解旋

<ol>
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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. 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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=Tunability+of+DNA+polymerase+stability+during+eukaryotic+DNA+replication.">Tunability of DNA polymerase stability during eukaryotic DNA replication.</a> Mol Cell. 77 (1), 17-25.e5 (2020).</li><li>Li, Z., Xu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-translational+modifications+of+the+mini-chromosome+maintenance+proteins+in+DNA+replication.">Post-translational modifications of the mini-chromosome maintenance proteins in DNA replication.</a> Genes. 10 (5), 331 (2019).</li><li>Lewis, J. 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>

<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>
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		<tr>
			<td>APS 10%&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A3678-25G</td>
			<td></td>
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			<td>ATP (200 mM)</td>
			<td>Sigma-Aldrich</td>
			<td>A7699-5G</td>
			<td></td>
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			<td>ATP-g-s (100 mM)</td>
			<td>Sigma-Aldrich</td>
			<td>11162306001</td>
			<td></td>
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			<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>
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			<td>Biotinylated anti-digoxigenin antibody&#160;</td>
			<td>Perkin Elmer</td>
			<td>N/A</td>
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			<td>Blu Tack</td>
			<td>Bostik</td>
			<td>N/A</td>
			<td>Adhesive putty</td>
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		<tr>
			<td>Boric acid</td>
			<td>Thermo Fisher Scientific</td>
			<td>B/3800/53</td>
			<td></td>
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		<tr>
			<td>BSA; 33 mg/mL</td>
			<td>SIGMA-ALDRICH</td>
			<td>A3858-10G</td>
			<td>Diluted from the stock</td>
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		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>C7902</td>
			<td></td>
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		<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>
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			<td>Casein (5% in water, 50 mg/mL)</td>
			<td>Sigma-Aldrich</td>
			<td>C4765-10ML</td>
			<td></td>
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		<tr>
			<td>ChemiDoc system</td>
			<td>Bio-Rad</td>
			<td>N/A</td>
			<td></td>
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		<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>
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		<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>
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			<td>EGTA</td>
			<td>Sigma-Aldrich</td>
			<td>03779-10G</td>
			<td></td>
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			<td>Epoxy - 5 minute</td>
			<td>Devcon</td>
			<td>20845</td>
			<td></td>
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			<td>Fujifilm FLA-5000&#160;</td>
			<td>Fujifilm</td>
			<td>&#160;FLA-5000&#160;</td>
			<td>Fluorescent image analyzer</td>
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			<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>
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			<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>
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		<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

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.

<meta charset="utf8"></head><body>作者聚焦：通过单分子可视化揭示真核 DNA 复制的动力学

通过 CMG 解旋酶对 DNA 解旋进行单分子实时可视化

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.

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

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

754 次观看.  The Francis Crick Institute. 在我们的实验室中，我们正在研究真核 DNA 复制的分子机制。我们想了解单分子水平基因组复制的动力学。我们使用多个系统可视化 DNA 复制，包括纯化的蛋白质和非洲爪蟾卵衍生的提取物。我们对 DNA 复制的大部分了解来自批量实验、细胞成像和结构生物学。这些方法确实很强大，但它们通常只能告诉我们有关单个蛋白质的动态或瞬态行为的信...